Marie‐Josée Hébert

Background. Listeriosis is a foodborne disease of significant public health concern that primarily affects persons with recognized underlying conditions or diseases that impair cell-mediated immunity. The degree of risk posed by the different underlying conditions is crucial to prioritize prevention programs that target the highest risk populations.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Acute vascular rejection (AVR) is characterized by immune-mediated vascular injury and heightened endothelial cell (EC) apoptosis. We reported previously that apoptotic ECs release a bioactive C-terminal fragment of perlecan referred to as LG3. Here, we tested the possibility that LG3 behaves as a neoantigen, fuelling the production of anti-LG3 antibodies of potential importance in regulating allograft vascular injury. We performed a case-control study in which we compared anti-LG3 IgG titers in kidney transplant recipients with AVR (n=15) versus those with acute tubulo-interstitial rejection (ATIR) (n=15) or stable graft function (n=30). Patients who experienced AVR had elevated anti-LG3 titers pre and posttransplantation compared to subjects with ATIR or stable graft function (p<0.05 for both mediators). Elevated pretransplant anti-LG3 titers (OR: 4.62, 95% CI: 1.08-19.72) and pretransplant donor-specific antibodies (DSA) (OR 4.79, 95% CI: 1.03-22.19) were both independently associated with AVR. To address the functional role of anti-LG3 antibodies in AVR, we turned to passive transfer of anti-LG3 antibodies in an animal model of vascular rejection based on orthotopic aortic transplantation between fully MHC-mismatched mice. Neointima formation, C4d deposition and allograft inflammation were significantly increased in recipients of an ischemic aortic allograft passively transferred with anti-LG3 antibodies. Collectively, these data identify anti-LG3 antibodies as novel accelerators of immune-mediated vascular injury and obliterative remodeling.

Chronic renal failure (CRF) leads to decreased drug renal clearance due to a reduction in the glomerular filtration rate. However, little is known about how renal failure affects renal metabolism and elimination of drugs. Because both depend on the activity of uptake and efflux by renal transporters as well as enzymes in tubular cells, the purpose of this study was to investigate the effects of CRF on the expression and activity of select renal drug transporters and cytochrome P450. Two groups of rats were studied: control and CRF (induced by 5/6 nephrectomy). Compared with control rats, we observed reductions in the expression of both protein and mRNA of Cyp1a, sodium-dependent phosphate transport protein 1, organic anion transporter (Oat)1, 2, and 3, OatK1/K2, organic anion-transporting polypeptide (Oatp)1 and 4c1, P-glycoprotein, and urate transporter 1, whereas an induction in the protein and mRNA expression of Mrp2, 3, and 4 and Oatp2 and 3 was observed. Cyp3a expression remained unchanged. Similar results were obtained by incubating a human proximal tubule cell line (human kidney-2) with sera from CRF rats, suggesting the presence of uremic modulators. Finally, the renal elimination of [(3)H]digoxin and [(14)C]benzylpenicillin was decreased in CRF rats, compared with controls, as shown by a 4- and 9-fold accumulation, respectively, of these drugs in kidneys of rats in CRF. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs and reduced renal clearance that could, at least partially, explain the tubular toxicity of many drugs.

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations.Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4ʹ,6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H2O2: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2′‐[4‐ethoxyphenyl]‐5‐[4‐methyl‐1‐piperazinyl]‐2.5′‐bi‐1H‐benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.

Background Ischemia-reperfusion injury (IRI) is a major risk factor for chronic renal failure. Here, we characterize the different modes of programmed cell death in the tubular and microvascular compartments during the various stages of IRI-induced AKI, and their relative importance to renal fibrogenesis. Methods We performed unilateral renal artery clamping for 30 minutes and contralateral nephrectomy in wild-type mice (C57BL/6) or caspase-3 −/− mice. Results Compared with their wild-type counterparts, caspase-3 −/− mice in the early stage of AKI had high urine cystatin C levels, tubular injury scores, and serum creatinine levels. Electron microscopy revealed evidence of tubular epithelial cell necrosis in caspase-3 −/− mice, and immunohistochemistry showed upregulation of the necroptosis marker receptor-interacting serine/threonine-protein kinase 3 (RIPK3) in renal cortical sections. Western blot analysis further demonstrated enhanced levels of phosphorylated RIPK3 in the kidneys of caspase-3 −/− mice. In contrast, caspase-3 −/− mice had less microvascular congestion and activation in the early and extension phases of AKI. In the long term (3 weeks after IRI), caspase-3 −/− mice had reduced microvascular rarefaction and renal fibrosis, as well as decreased expression of α -smooth muscle actin and reduced collagen deposition within peritubular capillaries. Moreover, caspase-3 −/− mice exhibited signs of reduced tubular ischemia, including lower tubular expression of hypoxia-inducible factor-1 α and improved tubular injury scores. Conclusions These results establish the pivotal importance of caspase-3 in regulating microvascular endothelial cell apoptosis and renal fibrosis after IRI. These findings also demonstrate the predominant role of microvascular over tubular injury as a driver of progressive renal damage and fibrosis after IRI.

Antibodies that are specific to organ donor HLA have been involved in the majority of cases of antibody-mediated rejection in solid organ transplant recipients. However, recent data show that production of non-HLA autoantibodies can occur before transplant in the form of natural autoantibodies. In contrast to HLAs, which are constitutively expressed on the cell surface of the allograft endothelium, autoantigens are usually cryptic. Tissue damage associated with ischemia-reperfusion, vascular injury, and/or rejection creates permissive conditions for the expression of cryptic autoantigens, allowing these autoantibodies to bind antigenic targets and further enhance vascular inflammation and renal dysfunction. Antiperlecan/LG3 antibodies and antiangiotensin II type 1 receptor antibodies have been found before transplant in patients with de novo transplants and portend negative long–term outcome in patients with renal transplants. Here, we review mounting evidence suggesting an important role for autoantibodies to cryptic antigens as novel accelerators of kidney dysfunction and acute or chronic allograft rejection.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components. Proteolysis of extracellular matrix components and the production of cryptic bioactive factors play key roles in vascular remodeling. We showed previously that extracellular matrix proteolysis is triggered by the apoptosis of endothelial cells (EC), resulting in the release of an anti-apoptotic C-terminal fragment of endorepellin (LG3). Here, we characterize the endorepellin-cleaving proteases released by apoptotic EC using a multifaceted proteomics strategy. Cathepsin L (CathL), a cysteine protease known to be associated with cardiovascular disease progression in animal models and humans, was isolated from medium conditioned by apoptotic EC. CathL cleaved recombinant endorepellin in vitro, leading to LG3 release. Inhibition of CathL activity in EC exposed to pro-apoptotic stimuli prevented LG3 release without modulating the development of apoptosis in EC. Inhibition of caspase-3 activation in EC with the biochemical inhibitor DEVD-fluoromethyl ketone or small interfering RNAs concomitantly prevented CathL release by EC, LG3 production, and the development of paracrine anti-apoptotic activity. These data demonstrate that caspase-3 activation is a novel pathway of importance for triggering extracellular CathL release and the cleavage of extracellular matrix components. Apoptosis of endothelial cells (EC) 4The abbreviations used are: EC, endothelial cell(s); LDL, low density lipoprotein; SMC, smooth muscle cells; ECM, extracellular matrix; t-PA, tissue plasminogen activator; MMC, mitomycin C; siRNA, small interfering; mTOR, mammalian target of rapamycin; CathL, cathepsin L; HUVEC, human umbilical vein endothelial cells; FMK, fluoromethyl ketone; BMP-1, bone morphogenetic protein-1; LC, liquid chromatography; MS/MS, tandem mass spectroscopy. is increasingly recognized as an important component of the “response to injury” process, as most clinical risk factors of atherosclerosis (such as hypertension (1.Hamet P. deBlois D. Can J. Cardiol. 2001; 17: 26-28Google Scholar, 2.Vogt C.J. Schmid-Schonbein G.W. Microcirculation. 2001; 8: 129-139Crossref PubMed Google Scholar), hyperglycemia (3.Baumgartner-Parzer S.M. Wagner L. Pettermann M. Grillari J. Gessl A. Waldhausl W. Diabetes. 1995; 44: 1323-1327Crossref PubMed Google Scholar, 4.Du X.L. Sui G.Z. 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During vascular remodeling, EC injury and apoptosis are followed by migration of α-smooth muscle actin-positive cells (smooth muscle cells (SMC) and myofibroblasts) that accumulate within the intima through a state of resistance to apoptosis largely dependent on Bcl-xl overexpression (14.Pollman M. Hall J. Mann M. Zhang L. Gibbons G. Nat. Med. 1998; 4: 222-227Crossref PubMed Scopus (262) Google Scholar, 15.Suzuki J. Isobe M. Morishita R. Nishikawa T. Amano J. Kaneda Y. Cardiovasc. Res. 2000; 45: 783-787Crossref PubMed Scopus (44) Google Scholar, 16.Saxena A. McMeekin J.D. Thomson D.J. J. Pathol. 2002; 196: 335-342Crossref PubMed Scopus (44) Google Scholar). Recent evidence from our group and others suggests that apoptotic EC favor neointima formation through the release of paracrine mediators, which in turn, increase Bcl-xl expression and inhibit the apoptosis of vascular SMC and fibroblasts (17.Raymond M.A. Vigneault N. Luyckx V. Hebert M.J. Biochem. Biophys. Res. Commun. 2002; 291: 261-269Crossref PubMed Scopus (26) Google Scholar, 18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The production of biologically active mediators by apoptotic EC is at least partially dependent on pericellular proteolysis, leading to basement membrane and extracellular matrix (ECM) degradation with the release of cryptic anti-apoptotic factors (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A C-terminal fragment of endorepellin (perlecan domain V) released in association with EC apoptosis was found to heighten Bcl-xl expression in SMC and fibroblasts (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Perlecan is a basement membrane modular proteoglycan composed of five structural domains (21.Iozzo R.V. Nat. Rev. Mol. Cell Biol. 2005; 6: 646-656Crossref PubMed Scopus (402) Google Scholar). The C-terminal domain, also called endorepellin, comprises three laminin-like globular (LG1-LG3) modules interspaced by four epidermal growth factor-like repeats. The C-terminal LG3 motif mediates most of the anti-apoptotic and fibrogenic activity of endorepellin (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 22.Mongiat M. Sweeney S.M. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). LG3 levels were recently observed to be increased in patients with chronic renal allograft rejection (23.Goligorsky M.S. Addabbo F. O'Riordan E. J. Am. Soc. Nephrol. 2007; 18: 2233-2239Crossref PubMed Scopus (42) Google Scholar), further supporting the contention that EC apoptosis and ECM proteolysis play key roles in vascular remodeling. In the last two decades a convincing body of evidence has demonstrated that the ECM is an extremely dynamic structure of crucial importance for the regulation of cell adhesion, migration, survival, and differentiation. Proteolysis of basement membrane and/or ECM components (such as collagen IV, XV, XVIII, and perlecan) and liberation of split products with new functions (such as tumstatin, arrestin, endostatin, and endorepellin LG3 motif) have central roles in angiogenesis, wound-healing, tissue-remodeling, and atherosclerosis (24.Lutgens S.P. Cleutjens K.B. Daemen M.J. Heeneman S. FASEB J. 2007; 21: 3029-3041Crossref PubMed Scopus (266) Google Scholar). We reported previously that LG3 production by apoptotic EC occurs downstream of caspase activation (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Yet endorepellin does not harbor caspase-cleavage sites (25.Gonzalez E.M. Reed C.C. Bix G. Fu J. Zhang Y. Gopalakrishnan B. Greenspan D.S. Iozzo R.V. J. Biol. Chem. 2005; 280: 7080-7087Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). This suggested that caspase activation leads to the release/activation of an as yet uncharacterized protease(s) which could then cleave endorepellin and release LG3. Hence, we used a multifaceted proteomics strategy to characterize the endorepellin-cleaving proteases released by apoptotic EC. Here, we describe the importance of cathepsin L (CathL), a lysosomal endoprotease associated with the development of atherosclerotic diseases in animal models and humans, as a key enzyme responsible for LG3 production by apoptotic EC. Cell Culture and Reagents—Human umbilical vein endothelial cells (HUVEC, Clonetics), WI-38 human fibroblasts (ATCC), and A7R5 vascular SMC (ATCC) were cultured as described elsewhere (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Caspase inhibitors (ZVAD-FMK, DEVD-FMK, and LEHD-FMK) were purchased from R&D Systems. The CathL inhibitors ZFF-FMK and ZFA-FMK were from Calbiochem and MP Biomedicals, respectively. t-PA-STOP was obtained from American Diagnostica, Inc. Human recombinant endorepellin was purified as documented previously (22.Mongiat M. Sweeney S.M. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Tissue plasminogen activator (t-PA) protein levels were measured with commercially available enzyme-linked immunosorbent assay kits (MedSystems) according to the supplier's protocol. Human CathL, recombinant t-PA, and human α2-macroglobulin, a bone morphogenetic protein-1 (BMP-1) inhibitor, were obtained from Sigma-Aldrich, Roche, and R&D Systems, respectively. Conditioned Media—Serum-free media conditioned by apoptotic or non-apoptotic EC were generated as described previously (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In brief, to generate medium conditioned by non-apoptotic EC (SSC-no-apo), confluent HUVEC were exposed to the pan-caspase inhibitor ZVAD-FMK (100 μm) for 2 h, washed, and then serum-starved for 4 h in RPMI 1640 (Invitrogen), whereas in medium conditioned by apoptotic EC (SSC-apo), HUVEC were instead exposed to DMSO for 2 h, washed, and then serum-starved for 4 h. For CathL inhibition, HUVEC were pretreated with ZFF-FMK (15 μm), ZFA-FMK (100 μm) or DMSO (control), washed, and serum-starved for 4 h. To inhibit t-PA proteolytic activity, HUVEC were exposed to t-PA-STOP (1.23 nmol/ml) or vehicle for 4 h during serum starvation. Conditioned media were collected and stored at -20 °C. Mitomycin C (MMC, Sigma-Aldrich) served as another pro-apoptotic stimulus (26.Raymond M.A. Mollica L. Vigneault N. Desormeaux A. Chan J.S. Filep J.G. Hebert M.J. FASEB J. 2003; 17: 515-517Crossref PubMed Scopus (43) Google Scholar). In brief, confluent HUVEC were grown to confluence in endothelial cell basal medium (Clonetics) and exposed to MMC at 0.01 mg/ml or vehicle for 24 h, and the conditioned media were harvested. To generate medium conditioned by autophagic EC, HUVEC grown to confluence in endothelial cell basal medium were exposed to rapamycin (Wyeth) (1 μg/ml) or vehicle for 4 h, and the conditioned media were harvested. For BMP-1/Tolloid-like metalloprotease inhibition, HUVEC were incubated with α2-macroglobulin (27 nm) or vehicle (phosphate-buffered saline/glycerol) during serum starvation for 4 h. Characterization of the Proteases Produced by Apoptotic and Non-apoptotic EC—To characterize the complete set of proteases secreted by apoptotic EC downstream of caspase activation, we tested two comparative proteomic approaches: SDS-PAGE-LC-MS/MS and two-dimensional LC-MS/MS (27.Pshezhetsky A.V. Fedjaev M. Ashmarina L. Mazur A. Budman L. Sinnett D. Labuda D. Beaulieu J.F. Menard D. Nifant'ev I. Levy E. Proteomics. 2007; 7: 2201-2215Crossref PubMed Scopus (47) Google Scholar). SSC-apo and SSC-no-apo were centrifuged to eliminate cell debris and apoptotic bodies (28.Hristov M. Erl W. Linder S. Weber P.C. Blood. 2004; 104: 2761-2766Crossref PubMed Scopus (355) Google Scholar). Proteins in supernatants were concentrated, denatured, and digested with trypsin. Tryptic peptides were fractionated by weak anion exchange high performance liquid chromatography and analyzed by LC-MS/MS or resolved by SDS-PAGE, digested “in gel,” and analyzed by LC-MS/MS. In addition, to specifically characterize proteins with anti-apoptotic activity released during EC apoptosis, we conducted a functional analysis of SSC-apo, where the medium was fractionated with fast protein liquid chromatography and tested for anti-apoptotic activity on SMC. The biologically active fraction was resolved by SDS-PAGE, and bands present only in the bioactive fraction were sequenced with LC-MS/MS. Immunoblotting and in Vitro Digestion Assay—For all experiments, equal volumes of all conditioned media were concentrated by centrifugation through a 10-kDa Vivaspin concentrator according to the manufacturer's specifications (Vivascience). For in vitro digestion assays, recombinant endorepellin (148.5 ng) (22.Mongiat M. Sweeney S.M. San Antonio J.D. Fu J. Iozzo R.V. J. Biol. Chem. 2003; 278: 4238-4249Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) was incubated with t-PA (51 ng) or CathL (37.8 ng) at 37 °C for 48 h in RPMI 1640 (total volume of 40 μl). A fixed volume for all conditions was loaded onto the gel. Proteins were separated on 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Western blotting was performed as described elsewhere (17.Raymond M.A. Vigneault N. Luyckx V. Hebert M.J. Biochem. Biophys. Res. Commun. 2002; 291: 261-269Crossref PubMed Scopus (26) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and the membranes were probed with a polyclonal rabbit antibody against the LG3 fragment of perlecan (17.Raymond M.A. Vigneault N. Luyckx V. Hebert M.J. Biochem. Biophys. Res. Commun. 2002; 291: 261-269Crossref PubMed Scopus (26) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) or monoclonal anti-CathL antibody (BD Transduction Laboratories). As a loading control, proteins from the same digestion sample were loaded on a gel and stained with Coomassie Blue. Detection of Intracellular CathL Activity—CathL activity in EC was measured with the CathL detect kits (Calbiochem) as specified by the supplier. The subcellular localization of active CathL, defined by the presence of the fluorescent product, was evaluated by confocal microscopy (Leica SP5 confocal microscope). Fluorescence Microscopy for the Assessment of Apoptosis and Necrosis—Fluorescence microscopy of unfixed/unpermeabilized adherent cells stained with Hoechst 33342 (1 μg/ml) and propidium iodide (5 μg/ml) was undertaken as described in our previous work (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Subcellular Localization of Proteases during Apoptosis—HUVEC were grown on glass-bottom Petri dishes (Mat-Tek Corp.) coated with 1% gelatin and serum-starved for 4 h. The cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% Triton X-100. They were then blocked and stained with anti-t-PA antibody (American Diagnostica Inc.) and a biotinylated anti-mouse antibody (Dako) followed by a streptavidin-Alexa-488 antibody (Invitrogen). Nuclei were stained with To-Pro3/DNA (Molecular Probes) for confocal microscopy. Evaluation of Lysosomal Destabilization—Lysosomal permeabilization was assessed by fluorescence microscopy with acridine orange (Calbiochem) staining (5 μg/ml for 15 min at 37 °C) 26). Caspase-3 Silencing—HUVEC were grown on gelatin 1% until 50% confluence. The cells were then transfected with Oligofectamine (Invitrogen) employing ON-TARGETplus SMARTpool caspase-3 small interfering (si)RNA (Dharmacon RNAi Technologies, Thermo Scientific) or control siRNAs at a final concentration of 100 nm annealed oligo. After 72 h the cells were serum-starved for 4 h. To assess caspase-3 silencing, protein extracts from HUVEC were harvested and immunoblotted for caspase-3 (Abcam). Statistical Analysis—The results are expressed as the mean ± S.E. The data were analyzed by Student's t test (with Bonferroni corrections when appropriate) or analysis of variance. p < 0.05 was deemed significant for all tests. Characterization of Proteases Released during Apoptosis of EC—Using a multifaceted proteomic strategy, five proteases were identified in SSC-apo and were not found in SSC-no-apo (Table 1). CathL and t-PA were observed to have putative cleaving activity for endorepellin of potential relevance to LG3 production, according to computer simulation (ACD Protein Manager software) (Fig. 1A). To confirm this activity, human recombinant endorepellin was incubated in the presence of CathL or t-PA, and fragmentation was evaluated by immunoblotting with an antibody specific for the C-terminal LG3 motif of endorepellin. Base-line degradation of endorepellin in vitro was evident, as reported previously (25.Gonzalez E.M. Reed C.C. Bix G. Fu J. Zhang Y. Gopalakrishnan B. Greenspan D.S. Iozzo R.V. J. Biol. Chem. 2005; 280: 7080-7087Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), yet the production of a C-terminal ∼23-kDa fragment characteristic of LG3 was enhanced in the presence of CathL or t-PA (Fig. 1B) (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar).TABLE 1List of proteases found exclusively in SSC-apo or in the bioactive fractionProtein nameType of LC-MS/MS approach# of peptides identifiedPeptide sequenceFunctionalTwo-dimensional LCSDS-PAGEADAM 17×2(R) ADPDPMKNTCK (L)Cathepsin L×3(K) VFQEPLFYEAPR (S)(R) LYGMNEEGWR (R)(R) NHCGIASAASYPTVADAMTS 4××2(R) QVRPQSAPQAMHCTILR (S)(R) YSFFVPRPTPSTPRPTPQDWLHR (R)SPUVE×1(R) LPVVLPQSTLNLAKPDFGAEAK (L)t-PA×1(R) DEKTQMIYQQHQSWLRPVLR (S) Open table in a new tab Because we reported previously that in normal non-apoptotic cell systems, BMP-1/Tolloid-like metalloprotease is a key protease implicated in endorepellin cleavage (25.Gonzalez E.M. Reed C.C. Bix G. Fu J. Zhang Y. Gopalakrishnan B. Greenspan D.S. Iozzo R.V. J. Biol. Chem. 2005; 280: 7080-7087Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), we also tested the possibility that BMP-1 could participate in apoptosis-induced LG3 production. α2-Macroglobulin, a BMP-1 inhibitor (29.Zhang Y. Ge G. Greenspan D.S. J. Biol. Chem. 2006; 281: 39096-39104Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), did not modulate LG3 release, suggesting that BMP-1 is not involved in our system (Fig. 1C). CathL Plays a Central Role in the Generation of LG3 by Apoptotic EC—Having confirmed that CathL and t-PA cleave recombinant endorepellin, we then investigated whether inhibiting their activity during the development of EC apoptosis decreases production of the anti-apoptotic LG3 fragment. HUVEC were exposed to ZFF-FMK (15 μm) and ZFA-FMK (100 μm), two irreversible and cell-permeable CathL inhibitors, or to vehicle for 2 h and serum-starved for 4 h. 5 ml of media conditioned by HUVEC in the presence of ZFF-FMK, ZFA-FMK, or vehicle were concentrated, the same volumes were loaded, and proteins were separated by SDS-PAGE and membranes probed for LG3. The amount of proteins released by HUVEC during serum starvation was not altered by CathL inhibition, as evaluated by Ponceau red staining (Fig. 2A). Yet both inhibitors significantly reduced LG3 release (Fig. 2A). Also, to evaluate whether inhibition of CathL activity during conditioning altered the anti-apoptotic activity of conditioned medium, the development of apoptosis was estimated in SMC and fibroblasts exposed to these conditioned media. Consistent with our previous results, serum starvation of SMC and fibroblasts for 24 h induced a significant apoptotic response (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar, 19.Laplante P. Raymond M.A. Gagnon G. Vigneault N. Sasseville A.M. Langelier Y. Bernard M. Raymond Y. Hebert M.J. J. Immunol. 2005; 174: 5740-5749Crossref PubMed Scopus (99) Google Scholar, 20.Laplante P. Raymond M.A. Labelle A. Abe J. Iozzo R.V. Hebert M.J. J. Biol. Chem. 2006; 281: 30383-30392Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Apoptosis of SMC and fibroblasts was inhibited by medium conditioned by apoptotic HUVEC (SSC Control) (Fig. 2, B and C). Inhibition of CathL activity in HUVEC during conditioning significantly decreased the anti-apoptotic activity of conditioned medium on SMC and fibroblasts (Fig. 2, B and C). We then evaluated whether inhibiting CathL activity in HUVEC during serum starvation could reduce LG3 release through an indirect inhibitory effect on EC apoptosis. CathL inhibition in HUVEC during serum starvation did not inhibit EC apoptosis nor did it modulate necrosis, whereas the pancaspase inhibitor, ZVAD-FMK, significantly reduced apoptosis compared with the control (Fig. 2D). Increased levels of the active form of CathL (25 kDa) were found in 5 ml of SSC control compared with an equal volume of SSC produced by HUVEC in which development of apoptosis was blocked by pan-caspase inhibition (ZVAD-FMK) (Fig. 3A). Collectively, these results indicate that EC apoptosis leads to increased LG3 production at least in part through CathL-dependent proteolysis. Increased CathL levels were also found in medium conditioned by apoptotic HUVEC exposed to the DNA-damaging agent MMC compared with an equal volume of medium conditioned by non-apoptotic HUVEC (Fig. 3A) (18.Raymond M.A. Desormeaux A. Laplante P. Vigneault N. Filep J.G. Landry K. Pshezhetsky A.V. Hebert M.J. FASEB J. 2004; 18: 705-707Crossref PubMed Scopus (70) Google Scholar). Because serum starvation may also activate autophagy, a non-apoptotic form of programmed cell death, we tested the possibility that autophagy induction per se could activate CathL release. HUVEC were exposed to rapamycin (1 μg/ml for 4 h), a pharmacological inducer of autophagy acting through inhibition of the mammalian target of rapamycin (mTOR) (30.Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5647) Google Scholar). HUVEC exposed to rapamycin developed an autophagic response, as assessed by acridine orange staining (data not shown) in the absence of apoptosis. However, CathL levels were not increased in medium conditioned by autophagic EC (Fig. 3A). Because necrosis can result in the release of proteases (31.Zong W.X. Thompson C.B. Genes Dev. 2006; 20: 1-15Crossref PubMed Scopus (704) Google Scholar), we questioned whether EC necrosis also enhanced CathL release. HUVEC were heated for 30 min at 65 °C, a method that induced primary EC necrosis in previous work (26.Raymond M.A. Mollica L. Vigneault N. Desormeaux A. Chan J.S. Filep J.G. Hebert M.J. FASEB J. 2003; 17: 515-517Crossref PubMed Scopus (43) Google Scholar). Active CathL levels were not increased in medium conditioned by necrotic HUVEC compared with an equal volume of medium conditioned by normal HUVEC (data not shown). These results indicate that CathL is translocated extracellularly during EC apoptosis and plays a key role in pericellular proteolysis and LG3 release but not in the regulation of EC apoptosis per se. Because proteases may directly activate receptors and signaling pathways of importance for inhibition of apoptosis, we evaluated whether CathL released by apoptotic EC exerted direct anti-apoptotic activity on target cells (32.Isermann B. Vinnikov I.A. Madhusudhan T. Herzog S. Kashif M. Blautzik J. Corat M.A. Zeier M. Blessing E. Oh J. Gerlitz B. Berg D.T. Grinnell B.W. Chavakis T. Esmon C.T. Weiler H. Bierhaus A. Nawroth P.P. Nat. Med. 2007; 13: 1349-1358Crossref PubMed Scopus (329) Google Scholar). SMC were exposed to SSC-apo concomitantly with ZFF-FMK or vehicle. Inhibition of CathL proteolytic activity in SMC exposed to SSC failed to blunt the anti-apoptotic response of SMC (Fig. 3B). Of note, ZFF-FMK concentrations used on SMC proved to be effective in inhibiting endorepellin cleavage during apoptosis of EC and in suppressing the anti-apoptotic activity of SSC. These results suggest that the proteolytic activity of CathL is required for the production of anti-apoptotic ECM fragments during EC apoptosis but not for the direct activation of anti-apoptotic pathways in SMC. Inhibition of t-PA activity in serum-starved EC did not prevent the development of apoptosis but attenuated LG3 production (data not shown and Fig. 4A). However, no differences were apparent in the levels of t-PA present in medium conditioned by apoptotic HUVEC (SSC control) compared with medium conditioned by non-apoptotic HUVEC (SSC ZVAD-FMK), as evaluated by enzyme-linked immunosorbent assay (Fig. 4B), indicating that t-PA in soluble form is not incr

BK polyomavirus is ubiquitous, with a seropositivity rate of over 75% in the adult population. Primary infection is thought to occur in the respiratory tract, but asymptomatic BK virus latency is established in the urothelium. In immunocompromised host, the virus can reactivate but rarely compromises kidney function except in renal grafts, where it causes a tubulointerstitial inflammatory response similar to acute rejection. Restoring host immunity against the virus is the cornerstone of treatment. This review covers the virus-intrinsic features, the posttransplant microenvironment as well as the host immune factors that underlie the pathophysiology of polyomavirus-associated nephropathy. Current and promising therapeutic approaches to treat or prevent this complication are discussed in relation to the complex immunopathology of this condition.

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.

Rationale: Endothelial apoptosis is increased in association with acute and chronic vascular rejection (VR) of solid allografts. Apoptotic endothelial cells (EC) release LG3, a C-terminal fragment of perlecan of potential importance in vascular remodeling and neointima formation. Objective: Our 2 goals were to determine whether circulating levels of LG3 are increased in association with acute VR of renal allografts and to evaluate the impact of LG3 on vascular remodeling. Methods and Results: We conducted a case-control study to compare serum LG3 levels in human renal transplant patients with acute VR, tubulo-interstitial rejection (ATIR) and normal graft function. Aorta transplantation between fully MHC-mismatched mice in association with intravenous LG3 injection was used to characterize the impact of LG3 on vascular remodeling. Scratch assays evaluated the promigratory activity of LG3 on vascular smooth muscle cells (VSMC) in vitro. Serum LG3 levels were significantly elevated in human renal transplant patients with acute VR ( n =16) compared to ATIR ( n =16) and normal graft function ( n =32, P =0.004). In patients with acute VR, graft loss was associated with elevated LG3 levels. Increasing LG3 serum levels in aortic allograft recipients significantly increased neointima formation. LG3 injection fostered accumulation of α-smooth muscle actin–positive cells and decreased the number of CD31 positive EC. LG3 increased the migration of VSMC through extracellular signal-regulated kinases 1/2-dependent pathways. Conclusion: These results indicate that LG3 is a novel regulator of obliterative vascular remodeling during rejection.

Pneumocystis pneumonia (PCP) is associated with morbidity and mortality in solid organ transplant (SOT) recipients. In this case-control study, we determined the association between posttransplant PCP and 3 variables: cytomegalovirus (CMV) infection, allograft rejection, and prophylaxis. Eight transplant centers participated. For each case (SOT recipient with PCP), 3–5 controls (SOT recipients without PCP) were included. Controls were matched to the cases based on transplant center, type of allograft, and date of transplantation (±6 months). We enrolled 53 cases and 209 controls. Transplant types included kidney (n = 198), heart (n = 30), liver (n = 15), kidney-pancreas (n = 14), and lung (n = 5). PCP occurred beyond 12 months after transplantation in 43 (81.1%) cases. Thirty-four cases (64.1%) required admission to the intensive care unit, and 28 (52.8%) had mechanical ventilation. Allograft failure occurred in 20 (37.7%) cases, and 14 (26.9%) died. No patient developed PCP prophylaxis breakthrough. The proportion of female sex (P = .009), kidney dysfunction (P = .001), cardiac diseases (P = .005), diabetes mellitus (P = .03), allograft rejection (P = .001), CMV infection (P = .001), and severe lymphopenia (P = .001) were significantly higher in cases. In the logistic regression model, CMV infection (adjusted odds ratio [aOR], 4.6 [95% confidence interval {CI}, 2.0–10.5]) and allograft rejection (aOR, 3.0 [95% CI, 1.5–6.1]) significantly increased the likelihood of PCP. PCP was mostly a late-onset disease occurring after complete course of prophylaxis, particularly among patients with CMV infection or allograft rejection. PCP is associated with significant allograft loss. Extended prophylaxis targeting recipients with allograft rejection or CMV infection may reduce the risk of PCP.

Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses. In vivo, apoptotic endothelial cells release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles (ApoExos). Only ApoExos are immunogenic: their injection causes inflammation and autoimmunity in mice. Based on deep sequencing of total RNA, we report that apoptotic bodies and ApoExos are loaded with divergent RNA cargos that are not released by healthy endothelial cells. Apoptotic bodies, like endothelial cells, contain mainly ribosomal RNA whereas ApoExos essentially contain non-ribosomal non-coding RNAs. Endogenous retroelements, bearing viral-like features, represented half of total ApoExos RNA content. ApoExos also contained several copies of unedited Alu repeats and large amounts of non-coding RNAs with a demonstrated role in autoimmunity such as U1 RNA and Y RNA. Moreover, ApoExos RNAs had a unique nucleotide composition and secondary structure characterized by strong enrichment in U-rich motifs and unstably folded RNAs. Globally, ApoExos were therefore loaded with RNAs that can stimulate a variety of RIG-I-like receptors and endosomal TLRs. Hence, apoptotic endothelial cells selectively sort in ApoExos a diversified repertoire of immunostimulatory "self RNAs" that are tailor-made for initiation of innate immune responses and autoimmunity.

Abstract Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.

Tissue repair and fibrosis, an abnormal form of repair, occur in most human organs in response to injury or inflammation. Fibroblasts play a major role in the normal repair process by differentiating into myofibroblasts that synthesize extracellular matrix (ECM) components and favor tissue remodeling to reestablish normal function and integrity. However, their persistent accumulation at the site of injury is a hallmark of fibrosis. Autophagy is a catabolic process that occurs in eukaryotic cells as a stress response to allow cell survival and maintenance of cellular homeostasis by degrading and recycling intracellular components. Recent advances identify autophagy as an important regulator of myofibroblast differentiation, tissue remodeling, and fibrogenesis. In this mini-review, we provide an overview of the interactions between autophagy, ECM, and fibrosis, and emphasize the molecular mechanisms involved in myofibroblast differentiation. We also describe the emerging concept of secretory autophagy as a new avenue for intercellular communication at the site of tissue injury and repair.

A single-center cohort study of kidney and kidney-pancreas recipients was conducted to evaluate the association between new immunosuppressive regimens and risk of thrombotic microangiopathy (TMA). From January 1st,1996 to December 31, 2002, 368 patients received a kidney or kidney-pancreas transplant at our center. Four immunosuppressive regimens were evaluated as potential risk factors of TMA: cyclosporin + mycophenolate mofetil (CsA + MMF), cyclosporin + sirolimus (CsA + SRL), tacrolimus + myophenolate mofetil (FK + MMF), and tacrolimus + sirolimus (FK + SRL). Thirteen patients developed biopsy-proven TMA in the absence of vascular rejection. The incidence of TMA was significantly different in the four immunosuppressive regimens studied (p < 0.001). The incidence of TMA was highest in the CsA + SRL group (20.7%). The relative risk of TMA was 16.1 [95% confidence interval (CI): 4.3-60.8] for patients in the CsA + SRL group as compared with those in the FK + MMF group. We also investigated in vitro the pathophysiological basis of this association. The CsA-SRL combination was found to be the only regimen that concomitantly displayed pro-necrotic and anti-angiogenic activities on arterial endothelial cells. We propose that this combination concurs to development of TMA through dual activities on endothelial cell death and repair.

Abstract Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-xL overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-xL protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of α-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.

Physiological adaptation to the hyperosmolar milieu of the renal medulla involves a complex series of signaling and gene expression events in which NaCl and urea activate different cellular processes. In the present study, we evaluated the effects of NaCl and urea, individually and in combination, on the viability of murine inner medullary collecting duct (mIMCD3) cells. Exposure to hyperosmolar NaCl or urea caused comparable dose- and time-dependent decreases in cell viability, such that 700 mosmol/kgH2O killed >90% of the cells within 24 h. In both cases, cell death was an apoptotic event. For NaCl, loss of viability at 24 h paralleled decreases in RNA and protein synthesis at 4h, whereas lethal doses of urea had little or no effect on these biosynthetic processes. Cell cycle analysis showed both solutes caused a slowing of the G2/M phase. Surprisingly, cells exposed to a combination of NaCl + urea were significantly more osmotolerant such that 40% survived 900 mosmol/kgH2O. Madin-Darby canine kidney cells but not human umbilical vein endothelial cells also exhibited a similar osmotolerance response. Enhanced survival was not associated with a restoration of normal biosynthetic rates or cell cycle progression. However, the combination of NaCl + urea resulted in a shift in Hsp70 expression that appeared to correlate with survival. In conclusion, hyperosmolar NaCl and urea activate independent and complementary cellular programs that confer enhanced osmotolerance to renal medullary epithelial cells.

In order to ensure transplantation's long-term success, transplant recipients need to comply with a strict regimen of immunosuppressant medication on a daily basis for the rest of their lives. Nonadherence is one of the major causes of organ rejection. Because compliance is voluntary, it is likely to be influenced by an individual's beliefs and feelings. This study examined the impact on compliance of the following factors: (1) transplant-related stress; (2) general perceived stress; (3) psychosocial distress and (4) feelings of indebtedness and guilt towards the donor. Fifty kidney recipients (34 men, 16 women) filled out self-report questionnaires. The results indicate that 46% acknowledged sub-optimal compliance in the last month; patients more often reported not taking the medication exactly as prescribed than forgetting to take it. The results also suggest that psychological distress and general perceived stress affect compliance negatively, whereas feelings of indebtedness improve it. These results have implications for the understanding and management of compliance following organ transplantation.

Kidney transplantation entails a high likelihood of endothelial injury. The endothelium is a target of choice for injury by ischemia-reperfusion, alloantibodies, and autoantibodies. A certain degree of ischemia-reperfusion injury inevitably occurs in the immediate posttransplant setting and can manifest as delayed graft function. Acute rejection episodes, whether T-cell or antibody-mediated, can involve the graft micro- and macrovasculature, leading to endothelial injury and adverse long-term consequences on graft function and survival. In turn, caspase-3 activation in injured and dying endothelial cells favors the release of extracellular vesicles (apoptotic bodies and apoptotic exosome-like vesicles) that further enhance autoantibody production, complement deposition, and microvascular rarefaction. In this review, we present the evidence for endothelial injury, its causes and long-term consequences on graft outcomes in the field of kidney transplantation.

Abstract Background In solid organ transplant (SOT) recipients, the primary vaccination series against Coronavirus Disease 2019 is 3 doses followed by boosters. We determined whether a fourth dose booster induced Omicron BA.4/5 neutralizing antibodies (nAbs) and T cells in a large multicenter cohort study. Methods Serum was collected 4–6 weeks post-third and post-fourth doses of messenger RNA vaccine in 222 SOT recipients. nAbs were measured using a pseudovirus neutralization assay that targeted the Omicron BA.4/5 spike protein. A subset underwent T-cell testing. Results The median age of the cohort was 63 years (interquartile range [IQR], 50–68) with 61.7% men. BA.4/5 nAb detection increased from 26.6% (59 of 222) post-third dose to 53.6% (119 of 222) post-fourth dose (P &amp;lt; .0001). In patients with breakthrough infection prior to the fourth dose (n = 27), nAbs were detected in 77.8% and median nAb titers were significantly higher compared with those with 4 vaccine doses alone (P &amp;lt; .0001). Factors associated with a low BA.4/5 neutralization response after the fourth dose were older age (odds ratio [OR], 0.96; 95% confidence interval [CI], .94–.99), mycophenolate use (OR, 0.39; 95% CI, .20–.77) and prednisone use (OR, 0.34; 95% CI, .18–.63), and vaccine type (OR, 0.72; 95% CI, .51–.99), while breakthrough infection prior to the fourth dose (OR, 3.6; 95% CI, 1.3–9.9) was associated with a greater nAb response. Polyfunctional BA.4/5-specific CD4+ T cells significantly increased after 4 doses and were identified in 76.9% of patients at a median frequency of 213/106 cells (IQR, 98–650). Conclusions In summary, a booster significantly increases BA.4/5-specific neutralization and polyfunctional CD4+ T-cell responses, suggesting protection from severe disease even with new Omicron variants. However, SOT recipients who are older and on mycophenolate and prednisone need additional preventative strategies.

Increased endothelial apoptosis and decreased apoptosis of vascular smooth muscle cells (VSMC) are central to initiation of myo-intimal thickening. We hypothesized that apoptosis of endothelial cells (EC) induces the release of anti-apoptotic mediator(s) active on VSMC. We found that serum-free medium conditioned by apoptotic EC decreases apoptosis of VSMC compared with fresh serum-free medium. Inhibition of endothelial apoptosis during conditioning with a pan-caspase inhibitor ZVAD-FMK blocked the release of the anti-apoptotic factor(s) active on VSMC. VSMC exposed to serum-free medium conditioned by apoptotic EC showed increased ERK 1/2 phosphorylation, enhanced Bcl-xl expression, and inhibition of p53 expression. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as a C-terminal fragment of the domain V of perlecan. Serum-free medium supplemented with either a synthetic peptide containing the EGF motif of the domain V of perlecan or chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, increased ERK 1/2 phosphorylation and Bcl-xl protein levels while inhibiting apoptosis of VSMC. These results suggest that a proteolytic activity developing downstream of activated caspases in apoptotic EC initiates degradation of pericellular proteoglycans and liberation of bioactive fragments with a robust impact on inhibition of VSMC apoptosis.

Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with beta1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering beta1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response.

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Little is known about the risk of venous thrombosis following kidney transplant. To determine this we estimated the risk of thromboembolic events (TEs) in a cohort of consecutive patients who underwent kidney transplantation at a single tertiary care center over an 11-year period and calculated standardized incidence ratios (SIRs) for a first TE in kidney transplant recipients compared with the general population. We then performed a nested case–control study and compared patients with and without TEs to identify risk factors for thrombosis. Among 913 kidney transplant recipients (KTRs), 68 patients developed these events. The SIR for TEs in KTRs compared with the general population was 7.9 over the duration of follow-up. The risk was particularly higher in the first post-transplant year (SIR 26.1) but remained elevated afterward (SIR 5.2). Hospitalization, use of sirolimus, low hemoglobin level, and use of renin–angiotensin system inhibitors were independently associated with these events. When cases of TEs that occurred during hospitalization were excluded, the risk of these events remained elevated. The risk of TEs in KTRs was eightfold higher than in the general population but not fully explained by the increased risk associated with hospitalization. Our results underscore the important risk of thrombosis in patients who received a kidney transplant, making vigilance mandatory especially during hospitalization. Little is known about the risk of venous thrombosis following kidney transplant. To determine this we estimated the risk of thromboembolic events (TEs) in a cohort of consecutive patients who underwent kidney transplantation at a single tertiary care center over an 11-year period and calculated standardized incidence ratios (SIRs) for a first TE in kidney transplant recipients compared with the general population. We then performed a nested case–control study and compared patients with and without TEs to identify risk factors for thrombosis. Among 913 kidney transplant recipients (KTRs), 68 patients developed these events. The SIR for TEs in KTRs compared with the general population was 7.9 over the duration of follow-up. The risk was particularly higher in the first post-transplant year (SIR 26.1) but remained elevated afterward (SIR 5.2). Hospitalization, use of sirolimus, low hemoglobin level, and use of renin–angiotensin system inhibitors were independently associated with these events. When cases of TEs that occurred during hospitalization were excluded, the risk of these events remained elevated. The risk of TEs in KTRs was eightfold higher than in the general population but not fully explained by the increased risk associated with hospitalization. Our results underscore the important risk of thrombosis in patients who received a kidney transplant, making vigilance mandatory especially during hospitalization. Deep venous thrombosis (DVT) and pulmonary embolism (PE) are two phenotypes of the same thromboembolic disease. If untreated, this disease has a high mortality.1.Horlander K.T. Mannino D.M. Leeper K.V. Pulmonary embolism mortality in the United States, 1979-1998: an analysis using multiple-cause mortality data.Arch Intern Med. 2003; 163: 1711-1717Crossref PubMed Scopus (488) Google Scholar Earlier studies have documented an increased risk of thromboembolic events (TEs) after kidney transplantation (KT).2.Humar A. Johnson E.M. Gillingham K.J. et al.Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.Transplantation. 1998; 65: 229-234Crossref PubMed Scopus (92) Google Scholar,3.Poli D. Zanazzi M. Antonucci E. et al.Renal transplant recipients are at high risk for both symptomatic and asymptomatic deep vein thrombosis.J Thromb Haemost. 2006; 4: 988-992Crossref PubMed Scopus (48) Google Scholar Although in the first months after transplantation the risk of TE can be related to the transplant surgery, there seems to be an increased long-term TE risk in kidney transplant recipients (KTRs).4.Allen R.D. Michie C.A. Murie J.A. et al.Deep venous thrombosis after renal transplantation.Surg Gynecol Obstet. 1987; 164: 137-142PubMed Google Scholar The magnitude and the determinants of the increased TE risk after KT are poorly defined. Moreover, in the general population, a major risk factor for TE is hospitalization.5.Herner S.J. Paulson D.C. Delate T. et al.Evaluation of venous thromboembolism risk following hospitalization.J Thromb Thrombolysis. 2011; 32: 32-39Crossref PubMed Scopus (8) Google Scholar It remains unknown whether the relatively high frequency of hospitalization in KTRs may explain the persistently elevated TE incidence in this patient population. Although traditional risk factors for TE such as age, history of TE, and malignancy2.Humar A. Johnson E.M. Gillingham K.J. et al.Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.Transplantation. 1998; 65: 229-234Crossref PubMed Scopus (92) Google Scholar,3.Poli D. Zanazzi M. Antonucci E. et al.Renal transplant recipients are at high risk for both symptomatic and asymptomatic deep vein thrombosis.J Thromb Haemost. 2006; 4: 988-992Crossref PubMed Scopus (48) Google Scholar have been linked to TE in KTRs, little is known about the effect of transplant-specific factors on TE risk. Although various immunosuppressive agents have been shown to possess procoagulant effects,6.Kazory A. Ducloux D. Acquired hypercoagulable state in renal transplant recipients.Thromb Haemost. 2004; 91: 646-654PubMed Google Scholar their effect on TE risk in KTR is unknown. For example, although sirolimus, which is increasingly being used for maintenance immunosuppression in KTRs, has been linked to an increased TE risk in cardiac and lung transplant recipients, an association with TE in KTRs has never been shown.7.Thibodeau J.T. Mishkin J.D. Patel P.C. et al.Sirolimus use and incidence of venous thromboembolism in cardiac transplant recipients.Clin Transplant. 2012; 26: 953-959Crossref PubMed Scopus (18) Google Scholar,8.Ahya V.N. McShane P.J. Baz M.A. et al.Increased risk of venous thromboembolism with a sirolimus-based immunosuppression regimen in lung transplantation.J Heart Lung Transplant. 2011; 30: 175-181Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar A recent study reported that TE was more common in KTRs with low estimated glomerular filtration rate (eGFR <30ml/min per 1.73m2) 1 year after transplantation compared with patients with better graft function.9.Abbott K.C. Cruess D.F. Agodoa L.Y. et al.Early renal insufficiency and late venous thromboembolism after renal transplantation in the United States.Am J Kidney Dis. 2004; 43: 120-130Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar In another report, the use of a vitamin D receptor activator in combination with dual renin–angiotensin system (RAS) inhibitor seemed to be protective for TE.10.Moscarelli L. Zanazzi M. Bertoni E. et al.Renin angiotensin system blockade and activated vitamin D as a means of preventing deep vein thrombosis in renal transplant recipients.Clin Nephrol. 2011; 75: 440-450Crossref PubMed Scopus (14) Google Scholar As most studies measured putative risk factors at the time of transplantation instead of measuring them at or close to the time of TE, a pathophysiologically relevant exposure time window is lacking from previous reports.2.Humar A. Johnson E.M. Gillingham K.J. et al.Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.Transplantation. 1998; 65: 229-234Crossref PubMed Scopus (92) Google Scholar,9.Abbott K.C. Cruess D.F. Agodoa L.Y. et al.Early renal insufficiency and late venous thromboembolism after renal transplantation in the United States.Am J Kidney Dis. 2004; 43: 120-130Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Hence, we undertook the present study to determine the magnitude and the secular trends of the increased TE risk observed after renal transplantation as compared with the risk of TE in the general population. We also aimed to determine whether the increased risk was explained by an increased probability of hospitalization in KTRs, and we identified risk factors for TE in this patient population. The cohort consisted of 913 patients who had 979 renal transplantations. The median follow-up was 5.9 years (range 0–22), with a total of 6760 person–years (p–y). The mean age at transplantation was 47 (± 12) years, and 63% of patients were men. In all, 68 patients developed a TE (1.0/100 per year), among whom 42 were diagnosed with DVT alone, 21 with PE alone, and 5 with both DVT and PE. The thrombus was located in the proximal veins in 85% of the subjects with DVT. Nine patients had recurrent TE events. Among the first TE events, 84% were initially treated with unfractionated heparin followed by warfarin, and 9% were treated with unfractionated heparin alone. The mean duration of therapy was 5 months for DVT and 6 months for PE for patients treated with warfarin. The six patients undergoing heparin treatment had a treatment duration varying from 1 month to life long. Loss to follow-up occurred in 26 patients (2.8% of the cohort). Although only one subject died of his TE, patients who experienced a TE had an increased mortality risk (hazard ratio 2.1 (1.2–3.8)). The cumulative incidence for TE at 1, 5, and 10 years after renal transplantation was 3.0, 5.8, and 8.4% (Figure 1). The standardized incidence ratio (SIR) was calculated based on age- and gender-stratified incidences. The SIR in KTRs compared with the general population was 7.9 (95% confidence interval (CI): 6.2–10.0) when averaged for the whole duration of post-transplant follow-up (Table 1).Table 1Thromboembolic events in kidney transplantation recipients and in the general populationKidney transplant recipientsReference general populationSIR95% CIEventsP–yRate per 1000 p–yEventsP–yRate per 1000 p-yOverall67aOne case that occurred at the age of >70 was excluded because of the low number of patients in that age group.6760.029.9152,69949,794,3871.067.926.19–9.99Women262359.4411.4427,44525,092,2141.098.895.93–12.83Men414400.589.3225,25424,702,1731.027.415.39–9.95Within first year post transplantation27903.5229.8852,69949,794,3871.0626.1417.58–37.50After first year post transplantation39bOne case was classified in the >70 age group after 1 year of follow-up because of aging one year. This case was excluded.5541.147.0452,69949,794,3871.065.233.77–7.08Age 20–39 years161289.1112.41926819,751,8720.4727.3016.16–43.3940–49131739.087.481027412,421,7960.839.155.09–15.2550–59241992.7212.001504710,585,3831.428.465.55–12.4060–69141394.9510.04181107,035,3362.573.872.20–6.34Abbreviations: CI, confidence interval; p-y, person–years; SIR, standardized incidence ratio.a One case that occurred at the age of >70 was excluded because of the low number of patients in that age group.b One case was classified in the >70 age group after 1 year of follow-up because of aging one year. This case was excluded. Open table in a new tab Abbreviations: CI, confidence interval; p-y, person–years; SIR, standardized incidence ratio. To determine whether this increased risk was due to the transplantation itself, we calculated SIR for the first post-transplant year, and SIR for later time periods. When only the events and person–time of the first post-transplant year were considered, the SIR for TE in KTRs was 26.1 (95% CI: 17.6–37.5) compared with the general population. After the first post-transplant year, the SIR for TE in KTRs was 5.2 (95% CI: 3.8–7.1) compared with the general population, whereas this ratio was 4.3 (95% CI: 2.6–6.7) after 5 years and 3.9 (95% CI: 1.6–8.1) after 10 years (Figure 2). Our results suggest that the elevated TE risk observed in KTRs is mediated by the initial surgery. However, other factors likely contribute to the increased TE risk observed in our patient population, as the risk remains persistently elevated even after 5–10 years of follow-up. Compared with the general population, KT conferred a similar increase in risk in both men and women. However, there was a disproportionate increase in TE risk associated with KT in younger compared with older patients (Table 1). We questioned whether the augmented TE risk in transplant patients was due to their frequent hospitalizations. When the cases and corresponding person–time that occurred during a hospitalization were excluded (n=20), the SIR decreased to 5.6 (95% CI: 4.2–7.4). Hence, the risk of TE after KT is partly, but not fully, explained by the increased likelihood of KTRs to be hospitalized compared with the general population. In an attempt to verify whether the increased TE risk was due to factors known to provoke thrombosis, we excluded patients with TE during hospitalization, patients with recent trauma or surgery, and patients with a malignancy history. The SIR decreased but remained high (4.6, 95% CI (3.3–6.3)), indicating that the incidence of ‘unprovoked’ TE is increased after KT. To determine other potential risk factors for TE in our patient population, we turned to a nested case–control study design within our cohort of KTRs. We identified 68 cases who were matched to 260 controls. Controls were cohort members who did not have TE and were alive at the time of the corresponding TE of the case. Table 2 shows the population characteristics of the case–control study.Table 2Patient characteristicsTotal cohort (n=328)TE (n=68)Control (n=260)P-valueMen (%)203 (62)41 (60)162 (62)NSMean age in years (s.d.)50 (11)50 (11)49 (12)NSMedian cold ischemia (min) (IQR)810 (624–1020)750 (538–960)840 (653–1035)NSLiving donor (%)42 (13)6 (9)36 (14)NSInduction (ATG or anti-IL2 receptor blockade) (%)164 (50)30 (44)134 (52)NSSmoking (past or present) at transplantation (%)77 (23)17 (25)60 (23)NSTE before transplantation (%)8 (2)3 (4)5 (2)NSMalignancy before transplantation (%)17 (5)5 (7)12 (5)NSMalignancy after transplantation (%)18 (6)8 (12)10 (4)<0.05Mean hemoglobin (mmol/l) (s.d.)7.7 (1.2)7.3 (1.2)7.8 (1.2)<0.01Mean hemoglobin (g/dl) (s.d.)12.4 (19.9)11.8 (19.6)12.6 (19.8)<0.01Mean eGFR (ml/min per 1.73m2) (s.d.)57 (22)50 (22)58 (21)<0.05Median proteinuria (g/l) (IQR)0 (0–0.2)0.05 (0–0.9)0 (0–0.2)<0.05Aspirin use (%)61 (19)8 (12)53 (20)NSRAS blocker use (%)109 (33)15 (22)94 (36)<0.05CNI use (%)301 (92)56 (82)245 (94)<0.05Sirolimus use (%)35 (11)13 (19)22 (9)<0.05Steroids use (%)252 (77)60 (88)192 (74)<0.05Mycophenolate mofetil use (%)232 (71)42 (62)190 (73)NSAzathioprine use (%)27 (8)8 (12)19 (7)NSAbbreviations: ATG, anti-thymocyte globulin; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; IL2, interleukin 2; IQR, interquartile range; NS, nonsignificant; RAS, renin–angiotensin system; TE, thromboembolic events. Open table in a new tab Abbreviations: ATG, anti-thymocyte globulin; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; IL2, interleukin 2; IQR, interquartile range; NS, nonsignificant; RAS, renin–angiotensin system; TE, thromboembolic events. Cases and controls were similar in patient and transplantation characteristics. Approximately 92% were Caucasian. Both groups had a similar history in terms of TE before transplantation and smoking. The occurrence of malignancy after transplantation was more frequent in patients with TE than in control patients, although malignancy before transplantation occurred similarly in both groups. Among the patients who were hospitalized at the time of the index date, the proportion who received prophylactic subcutaneous heparin at any time during hospitalization was 42% in cases and 58% in controls (P=0.3). We did not observe a lower incidence of TE after 2003, the year when a thrombosis prophylaxis protocol was introduced for the transplant hospitalization. eGFR was lower and proteinuria more frequent among patients with TE compared with controls. In addition, anemia, use of sirolimus, and hospitalization at the index date were more frequent, whereas the use of RAS inhibitors was lower in patients with TE compared with controls. In univariate analyses, malignancy after transplantation (odds ratios (OR) 3.9 (95% CI: 1.3–11.7)), lower hemoglobin level (OR 3.8 (95% CI: 1.9–7.7)), sirolimus use (OR 2.7 (95% CI: 1.3–5.7)), steroid use (OR 3.7 (95% CI: 1.5–9.0)), and hospitalization at the index date (OR 8.7 (95% CI: 3.4–22.4)) were associated with TE (Table 3). The use of RAS inhibitors was inversely associated with TE (OR 0.4 (95% CI: 0.2–0.9)) as was calcineurin inhibitor use (OR 0.3 (95% CI: 0.1–0.7)). Aspirin use and renal function parameters, such as eGFR or proteinuria, were not associated with TE. The use of induction therapy, biopsy-proven rejection, and donor type (living vs. deceased) were not associated with TE. In multivariate analyses, risk factors for TE included low hemoglobin levels (OR 2.3 (95% CI: 1.0–5.0)), sirolimus use (OR 3.0 (95% CI: 1.3–7.0)), and hospitalization (OR 10.3 (95% CI: 3.5–30.5)). The use of RAS inhibitors was associated with a lower TE risk (OR 0.45 (95% CI: 0.21–0.98)).Table 3Risk of thromboembolic events among renal transplant recipientsUnivariate modelMultivariate modelTE before transplantation2.4 (0.6–10.0)—Induction therapy0.6 (0.3–1.4)—Malignancy after transplantation3.9 (1.3–11.7)2.5 (0.7–8.4)eGFR<60ml/min per 1.73m21.7 (0.9–3.0)—Proteinuria >1g/l2.0 (0.8–5.0)—Hemoglobin <6.7mmol/l3.8 (1.9–7.7)2.3 (1.0–5.0)CNI0.3 (0.1–0.7)0.5 (0.2–1.3)Sirolimus2.7 (1.3–5.7)3.0 (1.3–7.0)Steroids3.7 (1.5–9.0)2.5 (0.9–7.3)Aspirin0.5 (0.2–1.1)—RAS inhibitor0.4 (0.2–0.9)0.5 (0.2–0.9)Hospitalization8.7 (3.4–22.4)10.3 (3.5–30.5)Abbreviations: CNI, calcineurin inhibitor; RAS, renin–angiotensin system; TE, thromboembolic event.Conditional logistic regression odds ratios and 95% confidence intervals. Open table in a new tab Abbreviations: CNI, calcineurin inhibitor; RAS, renin–angiotensin system; TE, thromboembolic event. Conditional logistic regression odds ratios and 95% confidence intervals. To our knowledge, this is the first detailed study to date to describe TE risk after KT. We observed that KTR patients have a risk of TE that is eightfold higher than that of the general population. The risk was highest in the first year after transplantation but remained elevated in the following years. Although a greater probability to be hospitalized partly explained the higher incidence of TE in KTR compared with the general population, these patients remained at greater risk even when hospital-acquired TE cases were excluded. Hospitalization was the strongest risk factor for TE in our cohort and was associated with a 10-fold increased risk for TE. Other risk factors were sirolimus use and low hemoglobin levels. RAS inhibition seemed to have a protective effect for TE. Although not related to the thrombotic event per se, we observed an increase in mortality among patients with TE, suggesting that this group had a greater comorbidity burden. Over the course of follow-up, 7.4% of patients who received a KT at out center experienced a TE. Previous studies have reported on TE risk following KT, with incidence estimates ranging between 0.6 and 25%.11.Venkateswara K. Smith E.J. Alexander J.W. et al.Thromboembolic disease in renal allograft recipients. What is its clinical significance?.Arch Surg. 1976; 111: 1086-1092Crossref PubMed Scopus (33) Google Scholar, 12.Arnadottir M. Bergentz S.E. Bergqvist D. et al.Thromboembolic complications after renal transplantation: a retrospective analysis.World J Surg. 1983; 7: 757-761Crossref PubMed Scopus (25) Google Scholar, 13.Bergqvist D. Bergentz S.E. Bornmyr S. et al.Deep vein thrombosis after renal transplantation: a prospective analysis of frequency and risk factors.Eur Surg Res. 1985; 17: 69-74Crossref PubMed Scopus (40) Google Scholar Comparison between incidence rates are of limited value because of the differences in follow-up and mortality rates between different cohorts. There is also variability in the use of TE prophylaxis between the studies. In 1998, an incidence of 6.4% was reported in a retrospective study by using graduated compression stockings as TE prophylaxis.2.Humar A. Johnson E.M. Gillingham K.J. et al.Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.Transplantation. 1998; 65: 229-234Crossref PubMed Scopus (92) Google Scholar In another study that actively screened asymptomatic KTR for TE, the incidence was 9.1% using 3 months of low-dose subcutaneous unfractionated heparin or low-molecular-weight heparin.3.Poli D. Zanazzi M. Antonucci E. et al.Renal transplant recipients are at high risk for both symptomatic and asymptomatic deep vein thrombosis.J Thromb Haemost. 2006; 4: 988-992Crossref PubMed Scopus (48) Google Scholar However, the clinical importance of asymptomatic TE remains uncertain. The major limitation of the previous studies is that they did not report follow-up times for their patients or cumulative incidence rates after KT, nor did they compare the risk with that of the general population. We observed a cumulative incidence at 5 years of 5.8% and an eightfold risk compared with the general population. The highest number of TEs were previously described in the first month after transplantation, and these studies have suggested that the incidence remains high until the fifth month after transplantation.2.Humar A. Johnson E.M. Gillingham K.J. et al.Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis.Transplantation. 1998; 65: 229-234Crossref PubMed Scopus (92) Google Scholar,4.Allen R.D. Michie C.A. Murie J.A. et al.Deep venous thrombosis after renal transplantation.Surg Gynecol Obstet. 1987; 164: 137-142PubMed Google Scholar Our results suggest that the risk is high in the first year after transplantation, and although it decreases in subsequent years, it remains significantly elevated. In the general population, the increase in TE incidence with age is well known; however, in KTRs, the incidence in younger and older patients is similar. Moreover, young transplanted patients had an impressive increased TE risk compared with the low incidence of TE in this age group in the general population (Table 1). Our results underline the importance of hospitalization in TE risk. In the general population, in the year following hospitalization, the risk of TE was fivefold compared with that of nonhospitalized subjects.14.Holmqvist M.E. Neovius M. Eriksson J. et al.Risk of venous thromboembolism in patients with rheumatoid arthritis and association with disease duration and hospitalization.JAMA. 2012; 308: 1350-1356Crossref PubMed Scopus (102) Google Scholar Our data indicate that hospitalization has an impressive impact in KTRs, as they experienced a 10-fold increase in risk during hospitalizations. We validated these results by taking hospitalization within the 2 months before the index date (compared with TE during hospitalization). Hospitalization increased the risk of TE sixfold by enlarging the time frame of hospitalization (OR 6.2, 95% CI 2.4–15.8). In our cohort, among the patients who were hospitalized at the time of the index date, the proportion of prophylactic heparin use was lower in those who developed TE versus those who did not. Although this suggests that subcutaneous heparin may have exerted a protective role on the development of TE during hospitalization, the difference did not reach statistical significance. This could be due to the relatively low number of subjects who were hospitalized. We show that an increased probability of hospitalization in KTRs does not solely explain the increased TE risk observed in KTRs compared with the general population. Other factors have a role in the development of thrombosis. The prothrombotic effect of different immunosuppressive agents has been extensively studied. Cyclosporine is associated with a hypercoagulable state in vitro,15.Irish A.B. Green F.R. Environmental and genetic determinants of the hypercoagulable state and cardiovascular disease in renal transplant recipients.Nephrol Dial Transplant. 1997; 12: 167-173Crossref PubMed Scopus (49) Google Scholar although this was not confirmed in vivo.16.Gruber S.A. Chavers B. Payne W.D. et al.Allograft renal vascular thrombosis—lack of increase with cyclosporine immunosuppression.Transplantation. 1989; 47: 475-478Crossref PubMed Scopus (62) Google Scholar Cyclosporine use was not associated with TE in the present study. Recently, an association between steroid use and TE risk in the general population was reported.17.Johannesdottir S.A. Horvath-Puho E. Dekkers O.M. et al.Use of glucocorticoids and risk of venous thromboembolism: A Nationwide Population-Based Case-Control Study.JAMA Intern Med. 2013; 1: 1-10Google Scholar This relationship is possibly because of a steroid-related increase in different clotting factors.18.van Zaane B. Nur E. Squizzato A. et al.Systematic review on the effect of glucocorticoid use on procoagulant, anti-coagulant and fibrinolytic factors.J Thromb Haemost. 2010; 8: 2483-2493Crossref PubMed Scopus (139) Google Scholar Our univariate analyses showed an increased TE risk with steroid use. However, this association lost its significance in the multivariate model. Sirolimus has previously been linked to TE in other clinical contexts. An increased incidence of hepatic arterial thrombosis was observed in liver transplant patients treated with sirolimus in combination with cyclosporine.19.Trotter J.F. Sirolimus in liver transplantation.Transplant Proc. 2003; 35: 193S-200SAbstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar In vitro, sirolimus is associated with collagen-induced platelet aggregation.20.Babinska A. Markell M.S. Salifu M.O. et al.Enhancement of human platelet aggregation and secretion induced by rapamycin.Nephrol Dial Transplant. 1998; 13: 3153-3159Crossref PubMed Scopus (108) Google Scholar Sirolimus also increases prothrombotic tissue factor expression in endothelial cells.21.Steffel J. Latini R.A. Akhmedov A. et al.Rapamycin, but not FK-506, increases endothelial tissue factor expression: implications for drug-eluting stent design.Circulation. 2005; 112: 2002-2011Crossref PubMed Scopus (159) Google Scholar,22.Eisenreich A. Celebi O. Goldin-Lang P. et al.Upregulation of tissue factor expression and thrombogenic activity in human aortic smooth muscle cells by irradiation, rapamycin and paclitaxel.Int Immunopharmacol. 2008; 8: 307-311Crossref PubMed Scopus (26) Google Scholar Recent studies in lung and cardiac transplantation reported an increased TE risk associated with sirolimus use.7.Thibodeau J.T. Mishkin J.D. Patel P.C. et al.Sirolimus use and incidence of venous thromboembolism in cardiac transplant recipients.Clin Transplant. 2012; 26: 953-959Crossref PubMed Scopus (18) Google Scholar,8.Ahya V.N. McShane P.J. Baz M.A. et al.Increased risk of venous thromboembolism with a sirolimus-based immunosuppression regimen in lung transplantation.J Heart Lung Transplant. 2011; 30: 175-181Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In renal transplantation, one study looked at the risk in relation to sirolimus. No increase in TE risk with a sirolimus–cyclosporine-based regimen compared with an azathioprine–cyclosporine combination was documented.23.Langer R.M. Kahan B.D. Sirolimus does not increase the risk for postoperative thromboembolic events among renal transplant recipients.Transplantation. 2003; 76: 318-323Crossref PubMed Scopus (32) Google Scholar However, the conclusions of this retrospective study are limited by its small sample size. Given the low rate of sirolimus use in our study, more studies are needed to draw a definitive conclusion on the relationship between sirolimus and thrombosis in KTRs. We observed an inverse association between hemoglobin and TE risk. This was an unexpected finding, given the relationship between erythrocytosis and TE in polycythemia vera and in post-transplant erythrocytosis.24.Wickre C.G. Norman D.J. Bennison A. et al.Postrenal transplant erythrocytosis: a review of 53 patients.Kidney Int. 1983; 23: 731-737Abstract Full Text PDF PubMed Scopus (120) Google Scholar We hypothesize that the relationship is probably a reflection of the greater comorbidity burden and/or inflammation that are associated with having lower hemoglobin, which may in turn increase the TE risk. However, given the retrospective nature of the study, we had no biological material to study the link between anemia, inflammation, and TE. The association between low hemoglobin and TE risk may also be explained by a greater probability of blood transfusions in patients with anemia, although we had no information on the latter. Blood transfusion is a risk factor for TE25.Rogers M.A. Levine D.A. Blumberg N. et al.Triggers of hospitalization for venous thromboembolism.Circulation. 2012; 125: 2092-2099Crossref PubMed Scopus (132) Google Scholar possibly because stored transfused red blood cells exhibit greater adhesion to endothelial cells with sequestration in the lung.26.Zhu H. Zennadi R. Xu B.X. et al.Impaired adenosine-5'-triphosphate release from red blood cells promotes their adhesion to endothelial cells: a mechanism of hypoxemia after transfusion.Crit Care Med. 2011; 39: 2478-2486Crossref PubMed Scopus (53) Google Scholar Chronic kidney disease increases the TE risk possibly by a combination of an increase in proteins implicated in the development of thrombosis,27.Mahmoodi B.K. ten Kate M.K. Waanders F. et al.High absolute risks and predictors of venous and arterial thromboembolic events in patients with nephrotic syndrome: results from a large retrospective cohort study.Circulation. 2008; 117: 224-230Crossref PubMed Scopus (255) Google Scholar and the increased state of inflammation and endothelial damage.28.Wattanakit K. Cushman M. Stehman-Breen C. et al.Chronic kidney disease increases risk for venous thromboembolism.J Am Soc Nephrol. 2008; 19: 135-140Crossref PubMed Scopus (234) Google Scholar,29.Parikh A.M. Spencer F.A. Lessard D. et al.Venous thromboembolism in patients with reduced estimated GFR: a population-based perspective.Am J Kidney Dis. 2011; 58: 746-755Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Some reports have previously suggested that albuminuria is associated with TE.30.Ocak G. Verduijn M. Vossen C.Y. et al.Chronic kidney disease stages 1-3 increase the risk of venous thrombosis.J Thromb Haemost. 2010; 8: 2428-2435Crossref PubMed Scopus (35) Google Scholar,31.Kato S. Chernyavsky S. Tokita J.E. et al.Relationship between proteinuria and venous thromboembolism.J Thromb Thrombolysis. 2010; 30: 281-285Crossref PubMed Scopus (25) Google Scholar In the present study, neither impaired renal function nor the presence of proteinuria increased the TE risk. However, our observations are limited by an imprecise, semiquantitative measurement of proteinuria (dipstick analyses), which may have precluded a difference to be observed. We observed a protective effect of RAS inhibition on TE. The RAS contributes to a prothrombotic state. Angiotensin II stimulates the production of adhesion factors and plasminogen activator inhibitor-1 and induces thrombosis. RAS is associated with plasminogen activator inhibitor-1 increase, and ACE inhibition improves the fibrinolytic balance.32.Brown N.J. Agirbasli M.A. Williams G.H. et al.Effect of activation and inhibition of the renin-angiotensin system on plasma PAI-1.Hypertension. 1998; 32: 965-971Crossref PubMed Scopus (260) Google Scholar,33.Remkova A. Kratochvil'ova H. Durina J. Impact of the therapy by renin-angiotensin system targeting antihypertensive agents perindopril versus telmisartan on prothrombotic state in essential hypertension.J Hum Hypertens. 2008; 22: 338-345Crossref PubMed Scopus (49) Google Scholar In the PERTINENT study, ACE inhibition reduced d-dimer in patients with stable coronary artery disease over a 1-year follow-up.34.Ceconi C. Fox K.M. Remme W.J. et al.ACE inhibition with perindopril and biomarkers of atherosclerosis and thrombosis: results from the PERTINENT study.Atherosclerosis. 2009; 204: 273-275Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar We believe that in our cohort RAS blockade exerted its protective effect independent of reduction in proteinuria, as forcing proteinuria in the regression model did not modify the point estimates for the relationship between RAS inhibition and TE risk. However, this apparent protective effect may be due to immeasurable time bias resulting from imprecisions in information on in-hospital medication use in the database.35.Suissa S. Immeasurable time bias in observational studies of drug effects on mortality.Am J Epidemiol. 2008; 168: 329-335Crossref PubMed Scopus (143) Google Scholar Prospective studies with more precise determination of proteinuria are needed to clarify this issue. In conclusion, we have shown that the risk of TE events is eight times higher in KTRs compared with the general population. The risk is particularly strong in the first year after transplantation, remains elevated even after many years of follow-up, and is not fully explained by the greater probability of KTRs to be hospitalized. Given that hospitalization increased the risk of TE by a factor of 10, clinicians should consider anticoagulant prophylaxis in renal transplant patients. First, we performed a retrospective cohort study of KTRs who received a kidney allograft between 1 January 1990 and 31 December 2010 at the Centre Hospitalier de l'Université de Montréal. Subjects who had received dual organ transplantation (combined liver–kidney or pancreas–kidney) were excluded. Patients entered the cohort on the date of transplantation and were followed up until TE, death, graft loss, retransplant, or 31 March 2012, whichever occurred first. We then performed a case–control study nested within our cohort to determine the risk factors for TE. The choice of a nested case–control study design allowed us to take into account the biologically relevant time windows of exposure for transient risk factors while providing an unbiased estimate of the hazard ratio when risk set sampling is performed. To obtain information on TE, we used the electronic clinical database of the Centre Hospitalier de l'Université de Montréal that includes all KTRs who received a graft at our center since 1980. In our center, ∼80 KTs are performed each year. The data on patient history, transplantation characteristics, medication use, and clinical outcomes are entered prospectively by a dedicated medical technologist. It includes data on hospitalization and from the outpatient clinic. To obtain information on TE in the general population, we used the Q-VTE cohort formed by linking two provincial administrative health-care databases in the province of Québec: the Maintenance et exploitation des données pour l’étude de la clientèle hospitalière database, which comprises summary information on the diagnoses for all hospital admissions, collected on summary sheets at the time of hospital discharge, and the health-care services database of Régie de l’Assurance Maladie du Québec (RAMQ), which contains information on all physician reimbursement claims for services provided to Québec residents.36.Tagalakis V.P.V. Kahn S.R. Suissa S. Incidence of and mortality from venous thromboembolism in a real-world population: The Q-VTE Study Cohort.Am J Med. 2013; 126: 832.e13-832.e21Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar The RAMQ databases were previously validated for the diagnosis TE.37.Tagalakis V. Kahn S.R. Determining the test characteristics of claims-based diagnostic codes for the diagnosis of venous thromboembolism in a medical service claims database.Pharmacoepidemiol Drug Saf. 2011; 20: 304-307Crossref PubMed Scopus (26) Google Scholar The main outcome was first TE after KT. Second TE events were not considered in our analyses. TE was defined as symptomatic DVT and/or PE after renal transplantation. The diagnosis of DVT was based on objective confirmation by contrast venography, Doppler ultrasound, or magnetic resonance venography. The diagnosis of PE required objective confirmation by pulmonary angiography, spiral (helical) CT scanning with intravenous contrast, or ventilation perfusion scan with a high probability for PE. For the nested case–control study, cases were defined as patients having a first TE event during follow-up. Controls were selected randomly from the cohort who were free from TE and at risk for developing TE at the time of case diagnosis (index date). Individual matching was used. For each case, up to four controls were randomly selected from the cohort who were of the same age (±5 years) and gender, had the same date of transplantation (±1 year) and duration of follow-up after transplantation (±1 day), and who were alive and without TE on the date of TE diagnosis of the corresponding case (or index date). We chose to match on age and gender because these are important risk factors for TE in the general population, and as a result we considered them as potentially strong confounders.38.White R.H. The epidemiology of venous thromboembolism.Circulation. 2003; 107: I4-I8Crossref PubMed Scopus (1778) Google Scholar,39.Naess I.A. Christiansen S.C. Romundstad P. et al.Incidence and mortality of venous thrombosis: a population-based study.J Thromb Haemost. 2007; 5: 692-699Crossref PubMed Scopus (976) Google Scholar Independent variables collected at the time of transplantation included age, gender, dialyses modality and duration of dialysis, cold ischemic time, type of donor, induction therapy, smoking (current or past), history of malignancy or TE events, and diabetes. In addition, to collect data for transient risk factors in biologically pertinent time windows of exposure, we attributed an index date for all patients as described above (Figure 3). Independent variables collected in the days/weeks preceding the index date were body mass index, blood pressure, laboratory values (hemoglobin the lowest quartile <6.7mmol/l), hematocrit, and serum creatinine to calculate eGFR using the CKD-epi formula40.Levey A.S. Stevens L.A. Schmid C.H. et al.A new equation to estimate glomerular filtration rate.Ann Intern Med. 2009; 150: 604-612Crossref PubMed Scopus (15959) Google Scholar), proteinuria (measured by semiquantitative measurement (dipstick analyses)), medication use (including anticoagulant and antiplatelet agents), malignancy since transplantation date, and hospitalization at the index date. We validated the association of TE and hospitalization with information on hospitalization in the 2 months before the TE. Since 2003, all KTR patients are prescribed unfractionated heparin thromboprophylaxis following transplantation (twice daily 5000U subcutaneous heparin), which is usually discontinued at the time of hospital discharge. Before 2003, the decision to prescribe and/or discontinue TE thromboprophylaxis following transplantation was made by treating physicians. For hospital admissions that were unrelated to the initial transplant procedure, the prescription of thrombosis prophylaxis was left to the discretion of the treating physician for the entire study duration. Normally distributed variables are presented as the mean and s.d., and non-normally distributed variables as the median with interquartile range (25th and 75th percentile). Categorical variables are summarized using proportions. We calculated cumulative incidence rates for TE at 1, 5, and 10 years after transplantation in our cohort. As the mortality in renal transplant recipients is relatively high, we corrected the cumulative incidence for the competing risk of death. For both the cohorts of KTRs and the general population, age- and gender-specific incidence rates and associated 95% CI were calculated using achieved age during follow-up, and as a result patients contributed person–time in different age categories while aging during follow-up.35.Suissa S. Immeasurable time bias in observational studies of drug effects on mortality.Am J Epidemiol. 2008; 168: 329-335Crossref PubMed Scopus (143) Google Scholar Second, SIRs were computed. For SIR calculation, we performed an indirect standardization (age- and gender-adjusted). Ninety-five percent CIs are provided. Risk factors for TE were identified using a multivariate conditional logistic regression model. The statistical software of SPSS (IBM statistics 19) was used and the significance level was set at 5%.

Tertiary lymphoid structures (TLS) accumulate at sites of chronic injury where they function as an ectopic germinal center, fostering local autoimmune responses. Vascular injury leads to the release of endothelial-derived apoptotic exosome-like vesicles (ApoExo) that contribute to rejection in transplanted organs. The purpose of the study was to evaluate the impact of ApoExo on TLS formation in a model of vascular allograft rejection. Mice transplanted with an allogeneic aortic transplant were injected with ApoExo. The formation of TLS was significantly increased by ApoExo injection along with vascular remodeling and increased levels of antinuclear antibodies and anti-perlecan/LG3 autoantibodies. ApoExo also enhanced allograft infiltration by γδT17 cells. Recipients deficient in γδT cells showed reduced TLS formation and lower autoantibodies levels following ApoExo injection. ApoExo are characterized by proteasome activity, which can be blocked by bortezomib. Bortezomib treated ApoExo reduced the recruitment of γδT17 cells to the allograft, lowered TLS formation, and reduced autoantibody production. This study identifies vascular injury-derived extracellular vesicles (ApoExo), as initiators of TLS formation and demonstrates the pivotal role of γδT17 in coordinating TLS formation and autoantibody production. Finally, our results suggest proteasome inhibition with bortezomib as a potential option for controlling TLS formation in rejected allografts.

Patients with kidney transplant failure have a high risk of hospitalization and death due to infection. The optimal use of immunosuppressants after transplant failure remains uncertain and clinical practice varies widely.This prospective cohort study enrolled patients within 21 days of starting dialysis after transplant failure in 16 Canadian centers. Immunosuppressant medication use, death, hospitalized infection, rejection of the failed allograft, and anti-HLA panel reactive antibodies were determined at 1, 3, 6, and 12 months and and then twice yearly until death, repeat transplantation, or loss to follow-up.The 269 study patients were followed for a median of 558 days. There were 33 deaths, 143 patients hospitalized for infection, and 21 rejections. Most patients (65%) continued immunosuppressants, 20% continued prednisone only, and 15% discontinued all immunosuppressants. In multivariable models, patients who continued immunosuppressants had a lower risk of death (hazard ratio [HR], 0.40; 95% confidence interval [CI], 0.17 to 0.93) and were not at increased risk of hospitalized infection (HR, 1.81; 95% CI, 0.82 to 4.0) compared with patients who discontinued all immunosuppressants or continued prednisone only. The mean class I and class II panel reactive antibodies increased from 11% to 27% and from 25% to 47%, respectively, but did not differ by immunosuppressant use. Continuation of immunosuppressants was not protective of rejection of the failed allograft (HR, 0.81; 95% CI, 0.22 to 2.94).Prolonged use of immunosuppressants >1 year after transplant failure was not associated with a higher risk of death or hospitalized infection but was insufficient to prevent higher anti-HLA antibodies or rejection of the failed allograft.

Solid organ transplant (SOT) recipients are at risk for severe coronavirus disease 2019 (COVID-19), despite vaccination. Our study aimed to elucidate COVID-19 vaccine immunogenicity and evaluate adverse events such as hospitalization, rejection, and breakthrough infection in a SOT cohort.We performed a prospective, observational study on 539 adult SOT recipients (age ≥18 years old) recruited from 7 Canadian transplant centers. Demographics including transplant characteristics, vaccine types, and immunosuppression and events such as hospitalization, infection, and rejection were recorded. Follow ups occurred every 4-6 weeks postvaccination and at 6 and 12 months from first dose. Serum was processed from whole blood to measure anti-receptor binding domain (RBD) antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to assess immunogenicity.The COVID-19 vaccines were found to be safe in SOT recipients with low rates of rejection requiring therapy (0.7%). Immunogenicity improved after the third vaccine dose, yet 21% developed no anti-RBD response. Factors such as older age, lung transplantation, chronic kidney disease, and shorter duration from transplant were associated with decreased immunogenicity. Patients with at least 3 doses were protected from hospitalization when experiencing breakthrough infections. Significantly increased anti-RBD levels were observed in patients who received 3 doses and had breakthrough infection.Three or four doses of COVID-19 vaccines were safe, increased immunogenicity, and protected against severe disease requiring hospitalization. Infection paired with multiple vaccinations significantly increased anti-RBD response. However, SOT populations should continue to practice infection prevention measures, and they should be prioritized for SARS-CoV-2 pre-exposure prophylactics and early therapeutics.

Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1α,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.

Complications following endovascular aneurysm repair (EVAR) are related to deficient healing around the stent-graft (SG). New generations of SG with surface properties that foster vascular repair could overcome this limitation. Our goal was to evaluate the potential of a new nitrogen-rich plasma-polymerised biomaterial, designated PPE:N, as an external coating for polyethylene terephtalate (PET)- or polytetrafluoro-ethylene (PTFE)-based SGs, to promote healing around the implant. Thin PPE:N coatings were deposited on PET and PTFE films. Then, adhesion, growth, migration and resistance to apoptosis of vascular smooth muscle cells (VSMCs) and fibroblasts, as well as myofibroblast differentiation, were assessed in vitro. In another experimental group, chondroitin sulphate (CS), a newly described mediator of vascular repair, was added to normal culture medium, to search for possible additional benefit. PPE:N-coatings, especially on PET, increased and accelerated cell adhesion and growth, compared with control PET and with standard polystyrene culture plates (PCP). PPE:N was also found to increase the resistance to apoptosis in VSMC, an important finding as aneurysms are characterised by VMSC depletion caused by a pro-apoptotic phenotype. Addition of CS in solution further increased migration and resistance to apoptosis. In conclusion, PPE:N-coating and/or CS could promote vascular repair around SGs following EVAR.

Abstract Gene expression profiling of human donor T cells before allogeneic hematopoietic cell transplantation revealed that expression of selected genes correlated with the occurrence of graft-versus-host disease (GVHD) in recipients. The gene with the best GVHD predictive accuracy was SMAD3, a core component of the transforming growth factor-β signaling pathway, whose expression levels vary more than a 6-fold range in humans. The putative role of SMAD3 in the establishment of graft-host tolerance remained elusive. We report that SMAD3-KO mice present ostensibly normal lymphoid and myeloid cell subsets. However, the lack of SMAD3 dramatically increased the frequency and severity of GVHD after allogeneic hematopoietic cell transplantation into major histocompatibility complex-identical recipients. Lethal GVHD induced by SMAD3-KO donors affected mainly the intestine and resulted from massive tissue infiltration by T-bet+ CD4 T cells and granulocytes that caused tissue damage by in situ release of Th1 cytokines and oxidative-nitrosative mediators, respectively. Our report reveals the nonredundant roles of SMAD3 in the development of tolerance to the host. Furthermore, our data support the concept that SMAD3 levels in donor cells dictate the risk of GVHD and that SMAD3 agonists would be attractive for prevention of GVHD.

New-onsetdiabetes after transplant (NODAT) is a major contributor to cardiovascular disease after transplantation. Kidney transplantation (KT) recipients have low levels of exercise capacity. Resistance training (RT) might be of special benefit in this population because underlying disease and immunosuppressive drugs favour muscle loss and insulin resistance. The aim of this study was to assess the feasibility of implementing an RT program within a population of KT recipients and its impact on the incidence of NODAT and cardiometabolic risk factors.This pilot study was an open-randomized study. We randomized 24 patients with a 1:1 allocation to 2 parallel groups, the exercise group (E) or the control group (C). The E group was submitted to RT 3 times a week for 16 weeks. Anthropometric, body composition, cardiometabolic risk factors, muscle strength, cardiorespiratory fitness and well-being were measured before and after 16 weeks.Of the 24 recruited participants, 20 completed the study (10 in the E group and 10 in the C group). No injuries were reported. The intervention was associated with a significant increase in muscle strength (p=0.003). A significant group effect, in favour of the E group, was detected for the well-being score (p=0.03). However, no changes in body composition, cardiometabolic risk factors or cardiorespiratory fitness were noted for either group after the intervention.This pilot study suggests that RT appears to be secure and feasible and improves strength and well-being in patients after KT. However, it does not improve cardiometabolic risk factors.

Abstract Conversion from a calcineurin‐inhibitor‐based immunosuppression to a rapamycin‐based immunosuppression may preserve kidney graft function. The side effects of rapamycin can limit its usefulness, but their management and evolution are rarely reported in clinical trials. We performed a retrospective cohort study in patients transplanted before December 31, 2008 and who received rapamycin to replace calcineurin inhibitors. In 219 patients studied, 98% presented ≥1 side effects after starting rapamycin. Side effects occurring in ≥10% of patients were dyslipidemia (52%, 95% confidence interval ( CI ): 45–59%), peripheral edema (37%, 95% CI : 31–43%), cytopenia (36%, 95% CI : 30–42%), acne (29%, 95% CI : 23–35%), proteinuria (23%, 95% CI : 17–29%), and oral ulcers 14% (95% CI : 10–18%). Proteinuria, ulcers, and edema were difficult to manage and were more likely to cause cessation of rapamycin. Rapamycin was discontinued in 46% of patients (95% CI : 40–52%). Age (odds ratio [OR] per 10‐yr increase: 1.29, 95% CI : 1.05–1.59) and obesity ( OR : 2.57, 95% CI : 1.10–6.01) were independently associated with cessation of rapamycin. We conclude that successful control of dyslipidemia and cytopenia can be achieved without discontinuing rapamycin. Most other side effects are harder to manage. Leaner and younger patients are less likely to discontinue rapamycin due to side effects.

Apoptotic exosome-like vesicles (ApoExos) are a novel type of extracellular vesicle that contribute to the propagation of inflammation at sites of vascular injury when released by dying cells. ApoExos are characterized by the presence of the C-terminal perlecan LG3 fragment and 20S proteasome, and they are produced downstream of caspase-3 activation. In the present study, we assessed the relative roles of autophagy and caspase-3-mediated pathways in controlling the biogenesis and secretion of immunogenic ApoExos. Using electron microscopy and confocal immunofluorescence microscopy in serum-starved endothelial cells, we identified large autolysosomes resulting from the fusion of lysosomes, multivesicular bodies, and autophagosomes as a site of ApoExo biogenesis. Inhibition of autophagy with ATG7 siRNA or biochemical inhibitors (wortmannin and bafilomycin) coupled with proteomics analysis showed that autophagy regulated the processing of perlecan into LG3 and its loading onto ApoExos but was dispensable for ApoExo biogenesis. Caspase-3 activation was identified using caspase-3-deficient endothelial cells or caspase inhibitors as a pivotal regulator of fusion events between autolysosomes and the cell membrane, therefore regulating the release of immunogenic ApoExos. Collectively, these findings identified autolysosomes as a site of ApoExo biogenesis and caspase-3 as a crucial regulator of autolysosome cell membrane interactions involved in the secretion of immunogenic ApoExos.

Although SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTRs) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung, and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCWs), the second dose induced weak humoral responses in SOTRs, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, although the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.

Blockade of the mitochondrial permeability transition pore (mPTP) by cyclosporin A (CsA) inhibits apoptosis in various cell types. However, use of CsA in humans is associated with damage to the arterial endothelium. We evaluated whether inhibition of the apoptotic machinery by CsA promotes other forms of cell death in arterial endothelial cells (EC). Exposure of human umbilical artery EC (HUAEC) to clinically relevant concentrations of CsA for up to 24 h was associated with a significant increase in necrotic features. We detected inhibition of apoptosis and a significant increase in necrosis in HUAEC exposed concomitantly to CsA and mitomycin C, a proapoptotic DNA damaging agent. We found that CsA-induced cell death is independent of caspase activation, p53 induction, and calcineurin inhibition. However, bongkrekic acid, another mPTP blocker, also increased necrosis in HUAEC. Dihydroethidium and acridine orange staining revealed increased intracellular production of reactive oxygen species (ROS) followed by lysosomal damage in HUAEC exposed to CsA. Hydroxyl radical and superoxide scavengers and inhibition of cathepsin D activity significantly attenuated CsA-induced EC death. These results suggest that inhibition of the apoptotic machinery by CsA in arterial EC favors development of a necrotic form of cell death regulated by ROS and secondary lysosomal damage.

Elderly transplant candidates represent an increasingly important group on the waiting list for kidney transplantation. Yet the factors that determine posttransplantation outcomes in this population remain poorly defined.We performed a population-based retrospective cohort study involving all patients aged 60 years or older who received a first cadaveric kidney transplantation between 1985 and 2000 in the province of Quebec. The main outcomes were patient survival, overall graft survival, and treatment failure (patient death or graft loss within the first posttransplant year). Survival analyses were performed using a Cox proportional hazard model. Logistic regression identified factors predicting treatment failure.On multivariate analysis, the modifiable factors associated with patient survival were active smoking at transplantation [hazard ratio (HR) 2.09, 95% confidence interval (CI) 1.22-3.60)], body mass index (BMI) (HR 1.34 for a 5-point increase, 95% CI 1.05-1.67), and time on dialysis before transplantation (HR 1.10 for a 1-year increase, 95% CI 1.02-1.18). The only modifiable factor associated with graft survival was active smoking at transplantation (HR 2.04, 95% CI 1.24-3.30). Treatment failure was associated with time on dialysis before transplantation (odds ratio for dialysis >/=2 years 3.28, 95% CI 1.34-7.9).Our results show that active smoking, obesity, and time on dialysis before transplantation are modifiable risk factors associated with an increased risk of mortality after transplantation in elderly recipients. They represent potential targets for interventions aimed at improving patient and graft survival in elderly patients.

Deficient healing after endovascular aneurysm repair is thought to be related to the pro-apoptotic environment in abdominal aortic aneurysms and inertness of the graft materials. A bioactive coating containing both chondroitin sulfate (CS) and epidermal growth factor (EGF) was developed in order to increase the growth and resistance to apoptosis of vascular smooth muscle cells (VSMC) on biomaterials surfaces. CS and EGF were covalently grafted using carbodiimide chemistry and the coating was characterized and optimized using ellipsometry, static contact angle and ToF-SIMS. Its potential to improve cell adhesion, growth and resistance to apoptosis was assessed in vitro with rat aortic VSMC. Results showed that CS and EGF immobilization allowed for the creation of a uniform coating that increased cell adhesion, growth and resistance to apoptosis in serum-free medium. Overall, CS and EGF possess great potential as bioactive anti-apoptotic mediators for vascular repair.

Abstract Persistent endothelial injury promotes maladaptive responses by favoring the release of factors leading to perturbation in vascular homeostasis and tissue architecture. Caspase-3 dependent death of microvascular endothelial cells leads to the release of unique apoptotic exosome-like vesicles (ApoExo). Here, we evaluate the impact of ApoExo on endothelial gene expression and function in the context of a pro-apoptotic stimulus. Endothelial cells exposed to ApoExo differentially express genes involved in cell death, inflammation, differentiation, and cell movement. Endothelial cells exposed to ApoExo showed inhibition of apoptosis, improved wound closure along with reduced angiogenic activity and reduced expression of endothelial markers consistent with the first phase of endothelial-to-mesenchymal transition (endoMT). ApoExo interaction with endothelial cells also led to NF-κB activation. NF-κB is known to participate in endothelial dysfunction in numerous diseases. Silencing NF-κB reversed the anti-apoptotic effect and the pro-migratory state and prevented angiostatic properties and CD31 downregulation in endothelial cells exposed to ApoExo. This study identifies vascular injury-derived extracellular vesicles (ApoExo) as novel drivers of NF-κB activation in endothelial cells and demonstrates the pivotal role of this signaling pathway in coordinating ApoExo-induced functional changes in endothelial cells. Hence, targeting ApoExo-mediated NF-κB activation in endothelial cells opens new avenues to prevent endothelial dysfunction.

We previously reported associations between autoantibodies to the LG3 fragment of perlecan, anti-LG3, and a higher risk of delayed graft function (DGF) in kidney transplant recipients. Here, we aimed to determine whether some factors that modulate ischemia-reperfusion injury (IRI) can modify this association. We performed a retrospective cohort study in kidney transplant recipients in 2 university-affiliated centers. In 687 patients, we show that high pre-transplant anti-LG3 are associated with DGF when the kidney is transported on ice (odds ratio (OR): 1.75, 95% confidence interval 1.02–3.00), but not when placed on hypothermic perfusion pump (OR: 0.78, 95% CI 0.43–1.37). In patients with DGF, high pre-transplant anti-LG3 are associated with a higher risk of graft failure (subdistribution hazard ratio (SHR): 4.07, 95% CI: 1.80, 9.22), while this was not the case in patients with immediate graft function (SHR: 0.50, 95% CI 0.19, 1.29). High anti-LG3 levels are associated with a higher risk of DGF in kidneys exposed to cold storage, but not when hypothermic pump perfusion is used. High anti-LG3 are also associated with a higher risk of graft failure in patients who experience DGF, a clinical manifestation of severe IRI.

Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Disregulation of apoptosis within the vessel wall and upregulation of the Fas/Fas-ligand (Fas-L) system contribute to the development of atherosclerosis. Cross-sectional studies have suggested that elevated plasma levels of the soluble form of Fas (sFas) are associated with CVD. However, the role of sFas and sFas-L in predicting future cardiovascular events has yet to be defined.We evaluated the role of plasma sFas and sFas-L levels as predictors of CVD in a prospective cohort of 107 chronic hemodialysis patients.During the study period (27 months), 53 patients (49.5%) presented with at least one cardiovascular end point. On univariate analysis, baseline sFas levels were significantly associated with the occurrence of cardiovascular end points, whereas sFas-L levels were not. Using Cox proportional hazards, increased sFas levels were associated with a significantly greater risk for cardiovascular end points (P = 0.03). This effect was independent of baseline CVD history, classic risk factors for atherosclerosis (diabetes, hypercholesterolemia, hypertension, and smoking), and markers of inflammation (C-reactive protein [CRP], soluble intercellular adhesion molecule-1). Increased CRP levels also were associated with cardiovascular end points (P = 0.04). In addition, increased cardiovascular mortality was found in patients in the highest sFas tertile compared with those in the lowest tertile (27.8% versus 8.6%; P = 0.04).Increased plasma sFas levels are predictive of future CVD. These results suggest that sFas is a novel and independent predictor of active atherosclerotic disease in patients with ESRD.

Abstract Bioactive coatings constitute an interesting approach to enhance healing around implants, such as stent‐grafts used in endovascular aneurysm repair. Three different plasma techniques, namely NH 3 plasma functionalization and atmospheric‐ or low‐pressure plasma polymerization, are compared to create amino groups and covalently bind CS and EGF bioactive molecules on PET. The latter presents the greatest potential. CS + EGF coating is shown to strongly decrease cell apoptosis and cell depletion in serum‐free medium, while increasing cell growth compared to unmodified PET. This versatile biomimetic coating holds promise in promoting vascular repair around stent‐grafts, where resistance to apoptosis is a key issue. magnified image

The immunosuppressive drug tacrolimus requires strict therapeutic monitoring due to its narrow therapeutic index and high interindividual variability. Organic anion transporting polypeptide 1B3 (OATP1B3) is a human hepatocyte transporter involved in the hepatobiliary elimination of diverse endogenous and exogenous substances. Genetic variations within the solute carrier (SLCO) 1B3 gene that encodes OATP1B3 may contribute to interindividual differences in tacrolimus disposition. The purpose of the present study is to investigate the association between SLCO1B3 polymorphisms and tacrolimus pharmacokinetics in renal transplant recipients. We found significant correlations between two linked coding nonsynomymous polymorphisms, T334G and G699A, and mean dose-adjusted tacrolimus trough blood concentrations during the first week post-transplantation (p = 0.04) and when the target dose (10-12 ng/ml) was obtained (p = 0.01). Patients carrying the homozygous mutant haplotype had 14.3-fold higher risk (95% confidence interval: 1.43-100; p = 0.02) of having blood tacrolimus concentrations above the median level, and thus being classified as poor OATP1B3 transporters, than carriers of one or two copies of the wild-type haplotype. This study shows, for the first time, that SLCO1B3 polymorphism is associated with tacrolimus exposure in the early post-transplant period.

Although altruism is a key principle in our current organ donation and transplantation system, the meanings and implications of the term have been widely debated. Recently, a new type of living organ donation--anonymous and non-directed, also called living altruistic donation (LAD)--has brought the issue into sharper focus. Transplant physicians' views on altruism might influence their attitudes and actions toward living altruistic donors. This study aimed to explore such views among transplant physicians in France and Quebec. A total of 27 French and 19 Quebec transplant physicians participated in individual, semi-structured interviews between October 2004 and December 2005. The majority of these participants associated altruism with gratuitousness and saw altruistic acts as multiple and varied, ranging from showing consideration to saving a person's life. The transplant physicians' discourses on altruism were quite diverse, leading us to question the relevance of the concept in organ transplantation and the appropriateness of the term "living altruistic donation."

Autoantibodies against perlecan/LG3 (anti-LG3) have been associated with increased risks of delayed graft function, acute rejection, and reduced long-term survival. High titers of anti-LG3 antibodies have been found in de novo renal transplants recipients in the absence of allosensitizing or autoimmune conditions. Here, we seek to understand the pathways controlling anti-LG3 production prior to transplantation. Mice immunized with recombinant LG3 produce concomitantly IgM and IgG anti-LG3 antibodies suggesting a memory response. ELISpot confirmed the presence of LG3-specific memory B cells in nonimmunized mice. Purification of B1 and B2 subtypes identified peritoneal B1 cells as the major source of memory B cells reactive to LG3. Although nonimmunized CD4-deficient mice were found to express LG3-specific memory B cells, depletion of CD4+ T cells in wild type mice during immunization significantly decreased anti-LG3 production. These results demonstrate that B cell memory to LG3 is T cell independent but that production of anti-LG3 antibodies requires T cell help. Further supporting an important role for T cells in controlling anti-LG3 levels, we found that human renal transplant recipients show a significant decrease in anti-LG3 titers upon the initiation of CNI-based immunosuppression. Collectively, these results identify T cell targeting interventions as a means of reducing anti-LG3 levels in renal transplant patients.

Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS) has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) through serum starvation (SS). After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE) mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC). The increased Ang II was suppressed using lisinopril (an ACE inhibitor) treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

Apoptosis of endothelial cells prompts the release of apoptotic exosome-like vesicles (ApoExos), subtype extracellular vesicles secreted by apoptotic cells after caspase-3 activation. ApoExos are different from both apoptotic bodies and classical exosomes in their protein and nucleic acid contents and functions. In contrast to classical apoptotic bodies, ApoExos induce immunogenic responses that can be maladaptive when not tightly regulated. In the present study, we elucidated the mechanisms by which ApoExos are internalized by endothelial cells, which leads to shared specific and functional mRNAs of importance to endothelial function. Using flow cytometry and confocal microscopy, we revealed that ApoExos were actively internalized by endothelial cells. SiRNA-induced inhibition of classical endocytosis pathways with pharmacological inhibitors showed that ApoExos were internalized via phosphatidylserine-dependent macropinocytosis independently of classical endocytosis pathways. An electron microscopy analysis revealed that ApoExos increased the macropinocytosis rate in endothelial cells, setting in motion a positive feedback loop that increased the amount of internalized ApoExos. Deep sequencing of total RNA revealed that ApoExos possessed a unique protein-coding RNA profile, with PCSK5 being the most abundant mRNA. Internalization of ApoExos by cells led to the transfer of this RNA content from the ApoExos to cells. Specifically, PCSK5 mRNA was transferred to cells that had taken up ApoExos, and these cells subsequently expressed PCSK5. Collectively, our findings suggest that macropinocytosis is an effective entry pathway for the delivery of RNAs carried by ApoExos and that these RNAs are functionally expressed by the endothelial cells that internalize them. As ApoExos express a specific mRNA signature, these results suggest new avenues to understand how ApoExos produced at sites of vascular injury impact vascular function.

The expansion of kidney transplantation by living donation has led to a disproportional increase in the women to men ratio among donors and this difference cannot be explained on the basis of medical exclusion. The present study was designed to test whether women donors are more likely to (i) display altruistic and gender-typed nurturing behaviour and (ii) be subtly influenced by family pressure to donate and less able to resist this pressure.All 71 (61% women) individuals who had donated a kidney at our centre between 1995 and 2005 were sent a survey. Thirty-nine individuals (71% response rate; 64% female participation) filled out and returned the survey, which included standardized measures of altruism, self-esteem, family dynamics and endorsement of gender-stereotyped roles, as well as sociodemographic questions and questions about donation.Findings show no difference between women and men in terms of the psychological attributes measured. One woman and two men reported having felt pressure to donate, and 92% of women compared with 54% of men reported having felt free to change their mind. Men took longer than women to make the decision to donate.Results suggest that among individuals who have already donated, there is no evidence that women may be more inclined to donate than man because of differences in their psychosocial profiles or because they may be more vulnerable to family pressure. Future research may gain from focusing on men and women donors and non-donors in families where transplantation is being considered.

The overwhelming scarcity of organs within renal transplantation forces researchers and transplantation teams to seek new ways to increase efficacy. One of the possibilities is the use of personalized medicine, an approach based on quantifiable and scientific factors that determine the global immunological risk of rejection for each patient. Although this approach can improve the efficacy of transplantations, it also poses a number of ethical questions.The qualitative research involved 22 semi-structured interviews with nephrologists involved in renal transplantation, with the goal of determining the professionals' views about calculating the global immunological risk and the attendant ethical issues.The results demonstrate a general acceptance of this approach amongst the participants in the study. Knowledge of each patient's immunological risk could improve treatment and the post-graft follow-up. On the other hand, the possibility that patients might be excluded from transplantation poses a significant ethical issue. This approach is not seen as something entirely new, given the fact that medicine is increasingly scientific and evidence-based. Although renal transplantation incorporates scientific data, these physicians believe that there should always be a place for clinical judgment and the physician-patient relationship.The participants see the benefits of including the calculation of the global immunological risk within transplantation. Such data, being more precise and rigorous, could be of help in their clinical work. However, in spite of the use of such scientific data, a place must be retained for the clinical judgment that allows a physician to make decisions based on medical data, professional expertise and knowledge of the patient. To act in the best interests of the patient is key to whether the calculation of the global immunological risk is employed.

Transplanted organs have to cope with diverse immunologic and metabolic stressors that augment the percentage of stressed and dying cells. Cell death, whether apoptotic or necrotic, is crucial in various transplantation-associated conditions. Necrosis, a proinflammatory type of cell death classically considered as accidental, is increasingly recognized as a highly controlled death program. Apoptosis, the classical programmed cell death mode program, is tightly orchestrated and culminates in the activation of caspases. Apoptosis was classically regarded as a silent form of cell death, but mounting evidence indicates that apoptotic cells "don't go silently" and leave a heritage to the local microenvironment. This apoptotic legacy, embedded within the effector phase of apoptosis, is aimed, at least in part, at controlling leukocyte trafficking and fostering tissue remodeling at sites of apoptotic cell deletion and can promote maladaptive remodeling pathways of importance for obliterative vascular remodeling. Moreover, apoptotic cells can transfer bioactive molecules by the release of apoptotic membrane vesicles that, in turn, shapes the phenotype and functions of immune cells. In this review, we summarize recent data highlighting the importance of apoptosis-associated intercellular communication networks in the regulation of allograft remodeling and immune responses in transplantation.

In Brief Antibody-mediated injury is a major cause of allograft dysfunction and loss. Antibodies to ABH(O) blood group antigens are classic mediators of ABO-incompatible graft rejection, whereas donor-specific anti-HLA antibodies and, more recently, autoantibodies are appreciated as important contributors to allograft inflammation and dysfunction. In August 2016, the International Summit of the Canadian National Transplant Research Program focused on recent advances in the field of antibody-mediated rejection. Here, we describe work presented and discussed at the meeting, with a focus on 3 major themes: the importance of (1) natural antibodies and autoantibodies, (2) tissue injury–derived exosomes and autoimmunity, (3) inflammasome activation and innate immune responses in regulating allograft inflammation and dysfunction. Finally, we explore novel areas of therapeutic intervention that have recently emerged from these 3 major and overlapping fields of transplantation research. A summary report on these cutting-edge topics, from basic to translational, that impact allograft outcomes as discussed at the recent Canadian National Transplant Research Program (CNTRP) International Summit.

Animal studies suggest that microvascular rarefaction is a key factor in the acute kidney disease to CKD transition. Hence, delayed graft function appears as a unique human model of AKI to further explore the role of microvascular rarefaction in kidney transplant recipients. Here, we assessed whether delayed graft function is associated with peritubular capillary loss and evaluated the association between this loss and long-term kidney graft function.This observational, retrospective cohort study included 61 participants who experienced delayed graft function and 130 who had immediate graft function. We used linear regression models to evaluate associations between delayed graft function and peritubular capillary density expressed as the percentage of efficient cortical area occupied by peritubular capillaries in pre- and post-transplant graft biopsies. eGFRs 1 and 3 years post-transplant were secondary outcomes.Post-transplant biopsies were performed at a median of 113 days (interquartile range, 101-128) after transplantation. Peritubular capillary density went from 15.4% to 11.5% in patients with delayed graft function (median change, -3.7%; interquartile range, -6.6% to -0.8%) and from 19.7% to 15.1% in those with immediate graft function (median change, -4.5%; interquartile range, -8.0% to -0.8%). Although the unadjusted change in peritubular capillary density was similar between patients with and without delayed graft function, delayed graft function was associated with more peritubular capillary loss in the multivariable analysis (adjusted difference in change, -2.9%; 95% confidence interval, -4.0 to -1.8). Pretransplant peritubular capillary density and change in peritubular capillary density were associated with eGFR 1 and 3 years post-transplantation.Perioperative AKI is associated with lower density in peritubular capillaries before transplantation and with loss of peritubular capillaries following transplantation. Lower peritubular capillary density is linked to lower long-term eGFR.

Tertiary lymphoid structures are clusters of lymphoid tissue that develop post-natally at sites of chronic inflammation. They have been described in association with infection, autoimmune disorders, cancer, and allograft rejection. In their mature stage, TLS function as ectopic germinal centers, favoring the local production of autoantibodies and cytokines. TLS formation tends to parallel the severity of tissue injury and they are usually indicative of locally active immune responses. The presence of TLS in patients with solid tumors is usually associated with a better prognosis whereas their presence predicts increased maladaptive immunologic activity in patients with autoimmune disorders or allograft transplantation. Recent data highlight a correlation between active cell death and TLS formation and maturation. Our group recently identified apoptotic exosome-like vesicles, released by apoptotic cells, as novel inducers of TLS formation. Here, we review mechanisms of TLS formation and maturation with a specific focus on the emerging importance of tissue injury, programmed cell death and extracellular vesicles in TLS biogenesis.

Coronary artery disease (CAD) is the leading cause of death in patients with end-stage renal disease (ESRD). Recent evidence suggests that the expression of Fas, a molecule implicated in the initiation of apoptosis in various cell types, is increased at sites of atherosclerotic plaques. However, the significance of plasma levels of the soluble form of Fas (sFas) and its ligand (sFas-L) as markers of atherosclerosis has yet to be defined. The present report is a cross-sectional analysis of baseline data from an ongoing prospective study designed to evaluate the role of sFas and sFas-L as markers of CAD in ESRD. We evaluated the association between plasma levels of sFas and sFas-L and evidence of CAD in a cohort of 107 chronic hemodialysis patients. Plasma levels of sFas were significantly greater (P = 0.04) among subjects with (n = 64) than without evidence of CAD (n = 43). Plasma levels of sFas-L were similar in both groups. Using multivariate analysis, sFas level was found to be independently associated with CAD (P = 0.01) after adjustment for classic risk factors for CAD (hyperlipidemia, diabetes, hypertension, and smoking), markers of inflammation (C-reactive protein [CRP], intercellular adhesion molecule 1), and other confounders. An increase of one quintile in plasma concentration of sFas was associated with an odds ratio for CAD of 1.64 (95% confidence interval, 1.11 to 2.41). Models that incorporated sFas were significantly better at identifying patients with CAD than models limited to classic risk factors for atherosclerosis, alone (P = 0.008) or in combination with CRP levels (P = 0.006). In summary, increased plasma levels of sFas are associated with CAD in stable patients with ESRD. These results suggest that sFas may represent a novel and independent marker of CAD.

The mechanisms of cytoprotection conferred by stress preconditioning remain largely uncharacterized in endothelial cells (EC). We report that stress preconditioning of EC with serum starvation induces the release of soluble mediator(s) that confer resistance to apoptosis, increase proliferation, and enhance angiogenesis in a second set of "non-preconditioned" EC. Preconditioning was found to target specifically the mitochondrial control of apoptosis in EC with increased protein levels of Bcl-2, decreased protein levels of Bax, and decreased cytosolic release of cytochrome c. Regulators of apoptosis acting upstream and downstream of the mitochondria such as p53, cIAP-1, cIAP-2, and XIAP were not altered. Mediators classically associated with preconditioning in other cell types such as adenosine, opioids, and nitric oxide are not implicated in this cytoprotective loop. Blockade of protein kinase C-dependent signaling inhibited cytoprotection of EC. Further characterization of this paracrine pathway should provide insights into the molecular regulation of preconditioning in endothelial cells.

Uraemia is associated with endothelial dysfunction, but the effect of uraemic plasma on the gene expression pattern of human coronary arterial endothelial cells (HCAEC) has never been defined.HCAECs were exposed for 48 h to a culture medium supplemented with 20% uraemic vs normal plasma. We extracted mRNA and hybridized it onto Affymetrix HG-U133 Plus2 microarrays. We validated our findings for five genes of interest by real-time PCR and performed evaluations of cell proliferation and apoptosis in HCAECs exposed to uraemic vs normal plasma.Six genes involved in the regulation of cell-cycle progression (CDK-1, topoisomerase II, PDZ-binding kinase, CDCA1, protein SDP35, E2F transcription factor 8) and two genes of the cholesterol efflux system (ABCA1 and ABCG1) were down-regulated in HCAECs exposed to uraemic plasma (>1.75-fold change vs normal). Real-time PCR confirmed the down-regulation observed in the microarray experiment. Cell proliferation was significantly decreased in HCAECs exposed to uraemic vs normal plasma for 48 h (86 vs 95% of serum-starved control, P = 0.006). Exposure to uraemic plasma for 48 h was associated with increased apoptosis of HCAEC as compared with normal plasma (7.7 vs 2.8%, P < 0.001), a phenomenon that was further enhanced when oxidized LDLs (150 microg protein/ml) were added to the medium containing uraemic plasma (16.9 vs 7.7%, P < 0.001).The down-regulation of genes involved in cell-cycle progression and cholesterol efflux from HCAECs exposed to uraemic conditions could contribute to enhancing endothelial dysfunction and atherosclerosis in patients with chronic renal failure.

BK polyomavirus-associated nephropathy (BKPVAN) is a major cause of renal failure early after kidney transplantation. The present study reports the preliminary results of prospective monitoring including a preemptive strategy for BKPVAN during the first year after kidney transplantation. We monitored BK virus DNA in blood at months 1, 2, 3, 6, 9, and 12 among 92 subjects who received induction therapy (basiliximab or antithymocyte globulin), and maintenance immunosuppression with prednisone, mycophenolate mofetil, and tacrolimus. Patients with two or more consecutive measurements of viral load >104 copies/mL were treated with a stepwise approach including dose reduction or discontinuation of mycophenolate mofetil eventually followed by reduction of tacrolimus and introduction of leflunomide. Within 1 year, seven (7%) patients displayed sustained BK viremia at a median of 92 days after transplantation. Among 68 patients who underwent a renal allograft biopsy, seven were diagnosed as BKPVAN at a median of 15 weeks after transplantation. The diagnosis was achieved by a surveillance biopsy in four patients with stable renal function. BKPVAN was preceded by asymptomatic viremia except for two cases in whom BK viremia occurred at 6 or 11 months, after the histological diagnosis. At 12 months, six patients had cleared their viremia. Serum creatinine levels had stabilized in six recipients with BKPVAN estimated renal function was 43.7 ± 16.3 mL/min in patients with viremia and/or BKPVAN versus 61.3 ± 20.1 mL/min among patients who never became viremic (P = .03). None of the patients with viremia and/or BKPVAN lost the allograft. BKPVAN may occur early after kidney transplantation, at a low or undetectable viremia or at some weeks after the first positive viremia. Intensive monitoring during the first 4 months after transplantation together with early protocol biopsies or interventions prompted by BK viremia may optimize BKPVAN diagnosis at a subclinical stage, thus avoiding renal dysfunction.

Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are human hepatocyte transporterS that mediate the uptake of various endogenous and exogenous substances. Genetic variations in solute carrier transporter 1B1 (SLCO1B1) and SLCO1B3 genes, which encode OATP1B1 and OATP1B3 proteins, could affect the pharmacokinetics of drugs leading to interindividual differences in drug responses. The full extent of SLCO1B1 and SLCO1B3 polymorphisms in white Canadians was analyzed using DNA sequencing procedures. We identified 49 and 41 nucleotide sequence variants leading to 10 and 9 major haplotypes in SLCO1B1 and SLCO1B3 genes, respectively. We report several novel mutations within regulatory and coding regions that could affect gene transcription, translation and function. Comparison with other studies revealed that the distribution of SLCO1B1 and SLCO1B3 polymorphisms and haplotypes differs widely across populations. Data from this survey will ultimately contribute to the design of pharmacogenetic studies in the Canadian population.

Abstract TGF-β is an ubiquitous cytokine that plays a pivotal role in the maintenance of self-tolerance and prevention of immunopathologies. Under steady-state conditions, TGF-β keeps naive T cells in a resting state and inhibits Th1 and Th2 cell differentiation. Because rapid generation of Th1 and Th2 effector cells is needed in response to pathogen invasion, how do naive T cells escape from the quiescent state maintained by TGF-β? We hypothesized that stimulation by strong TCR agonists might interfere with TGF-β signaling. Using both primary mouse CD4+ T cells and human Jurkat cells, we observed that strong TCR agonists swiftly suppress TGF-β signaling. TCR engagement leads to a rapid increase in SMAD7 levels and decreased SMAD3 phosphorylation. We present evidence that TCR signaling hinders SMAD3 activation by inducing recruitment of TGF-βRs in lipid rafts together with inhibitory SMAD7. This effect is dependent on protein kinase Cθ, a downstream TCR signaling intermediary, as revealed by both pharmacological inhibition and expression of dominant-negative and constitutively active protein kinase Cθ mutants. This work broadens our understanding of the cross-talk occurring between the TCR and TGF-β signaling pathways and reveals that strong TCR agonists can release CD4 T cells from constitutive TGF-β signaling. We propose that this process may be of vital importance upon confrontation with microbial pathogens.

Ischemic, immunologic or pharmacological stressors can induce vascular injury and endothelial apoptosis in organ donors, in transplant candidates due to the impact of end stage organ failure on the vasculature, and in association with peri-transplantation events. Vascular injury may shape innate and adaptive immune responses, leading to dysregulation in the balance between tolerance and immunoreactivity to vascular-derived antigens. Mounting evidence shows that the early stages of apoptosis, characterized by the absence of membrane permeabilization, are prone to trigger various modes of intercellular communication allowing neoantigen production, exposure, or both. In this review, we present the evidence for the release of LG3, an immunogenic fragment of perlecan, as a consequence of caspase-3 dependent vascular apoptosis leading to the genesis of anti-LG3 autoantibodies and the consequences of these autoantibodies in native and transplanted kidneys.

The increased usage of marginal grafts has triggered interest in perfused kidney preservation to minimize graft injury. We used a donation after circulatory death (DCD) porcine kidney autotransplantation model to compare 3 of the most frequently used ex vivo kidney perfusion techniques: nonoxygenated hypothermic machine perfusion (non-oxHMP), oxygenated hypothermic machine perfusion (oxHMP), and normothermic ex vivo kidney perfusion (NEVKP).Following 30 min of warm ischemia, grafts were retrieved and preserved with either 16 h of non-oxHMP, oxHMP, or NEVKP (n = 5 per group). After contralateral nephrectomy, grafts were autotransplanted and animals were followed for 8 d. Kidney function and injury markers were compared between groups.NEVKP demonstrated a significant reduction in preservation injury compared with either cold preservation method. Grafts preserved by NEVKP showed superior function with lower peak serum creatinine (NEVKP versus non-oxHMP versus oxHMP: 3.66 ± 1.33 mg/dL, 8.82 ± 3.17 mg/dL, and 9.02 ± 5.5 mg/dL) and more rapid recovery. The NEVKP group demonstrated significantly increased creatinine clearance on postoperative day 3 compared with the cold perfused groups. Tubular injury scores on postoperative day 8 were similar in all groups.Addition of oxygen during HMP did not reduce preservation injury of DCD kidney grafts. Grafts preserved with prolonged NEVKP demonstrated superior initial graft function compared with grafts preserved with non-oxHMP or oxHMP in a model of pig DCD kidney transplantation.

Antibody-mediated rejection (AMR) causes more than 50% of late kidney graft losses. In addition to anti-human leukocyte antigen (HLA) donor-specific antibodies, antibodies against non-HLA antigens are also linked to AMR. Identifying key non-HLA antibodies will improve our understanding of AMR.We analyzed non-HLA antibodies in sera from 80 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis. IgM and IgG antibodies against 134 non-HLA antigens were measured in serum samples collected pretransplant or at the time of diagnosis.Fifteen non-HLA antibodies were significantly increased (P < 0.05) in AMR and mixed rejection compared with ACR or acute tubular necrosis pretransplant, and 7 at diagnosis. AMR and mixed cases showed significantly increased pretransplant levels of IgG anti-Ro/Sjögren syndrome-antigen A (SS-A) and anti-major centromere autoantigen (CENP)-B, compared with ACR. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B, and IgM anti-La/SS-B were associated with the presence of microvascular lesions and class-II donor-specific antibodies (P < 0.05). Significant increases in IgG anti-Ro/SS-A and IgM anti-CENP-B antibodies in AMR/mixed rejection compared with ACR were reproduced in an external cohort of 60 kidney transplant patients.This is the first study implicating autoantibodies anti-Ro/SS-A and anti-CENP-B in AMR. These antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.

To better define the survival and quality of life of patients with major left ventricular systolic dysfunction and end-stage renal disease treated by continuous ambulatory peritoneal dialysis (CAPD), we reviewed all cases who started CAPD between May 1984 and March 1993 who had an isotopic left ventricular ejection fraction (LVEF) < or = 35%. Seventeen patients (12 men and five women with a mean age of 51.6 +/- 14.9 years) met the inclusion criteria. Mean isotopic LVEF before initiation of CAPD was 24.8% +/- 8.2%. All patients were symptomatic from congestive heart failure. Thirteen patients were classified as New York Heart Association grade III or IV. Continuous ambulatory peritoneal dialysis was associated with a significant improvement of isotopic LVEF, of functional status, and of blood pressure control. In 10 patients with a second measurement on CAPD, LVEF increased from a mean value of 23.2% +/- 9.1% to a mean value of 30.3% +/- 8.1% (P < 0.01). This represents a 30% increase of LVEF. After 6 months on CAPD, 94% of patients were classified as New York Heart Association grade I or II. Actuarial survival rates were 94%, 80%, and 64% at 12, 18, and 24 months, respectively. The mean duration of CAPD was 24 +/- 17 months. These results suggest that current CAPD treatment is an elective modality of treatment in patients with concomitant heart and renal failure.

Peripheral arterial occlusive disease (PAOD) including lower-extremity and cerebrovascular atherosclerosis is a leading cause of morbidity in haemodialysis patients. Recent evidence suggests that the expression of Fas, a molecule implicated in the initiation of apoptosis in various cell types, is increased at sites of atherosclerotic plaques. However, the significance of plasma levels of the soluble form of Fas (sFas) as a marker of peripheral arterial disease has yet to be defined.The present report is based on a cross-sectional analysis of baseline data from an ongoing prospective study designed to evaluate the role of sFas as marker of PAOD in end-stage renal disease (ESRD). We evaluated the association between sFas levels and evidence of PAOD in a cohort of 107 chronic haemodialysis patients.Compared with subjects without evidence of disease (n=56), subjects with PAOD (n=51) had significantly higher plasma levels of sFas (30.0+/-8.9 vs 26.4+/-9.5 ng/ml; P=0.04). Using multiple regression, sFas was found to be associated with PAOD independently of classical risk factors for atherosclerosis (hypercholesterolaemia, diabetes, hypertension, and smoking), markers of inflammation (e.g. C-reactive protein, intercellular cell adhesion molecule type 1), and other risk factors (e.g. age, gender). An increase of one quintile in the plasma concentration of sFas was associated with an odds ratio of PAOD of 1.69 (95% CI: 1.09--2.63, P=0.01). In addition, models that incorporated sFas were significantly better at predicting PAOD than models limited to classical risk factors for atherosclerosis, alone or in combination with CRP levels (P=0.01).Increased plasma levels of sFas are associated with established PAOD. These results suggest that sFas may represent a novel and independent marker of atherosclerosis.

Non-HLA antibodies against the angiotensin II type 1 receptor (AT1 R) and the C-terminal fragment of perlecan (i.e., LG3) are associated with the development of renal allograft rejection. It is currently unknown how humans develop anti-AT1 R or anti-LG3 antibodies. The aim of this study was to investigate whether pregnancy-as a model of sensitization to polymorphic proteins-induces anti-AT1 R and/or anti-LG3 antibodies. We included 104 samples from women obtained after physiologic full-term pregnancy and 80 samples from healthy nonsensitized controls (40 women and 40 men). Both anti-AT1 R and anti-LG3 antibody levels were lower in pregnancy samples than in controls (both P < 0.05). By multivariate analysis, male gender was an independent predictor for high anti-AT1 R antibody levels (OR 3.66, P = 0.04) and pregnancy was predictive for low anti-LG3 antibody levels (OR 6.53, P = 0.0001). There was no correlation of anti-AT1 R with anti-LG3 antibody levels, either in the pregnancy or in the control samples (r(2) ≤ 0.03, P ≥ 0.26). In conclusion, physiologic full-term pregnancy does not induce anti-AT1 R or anti-LG3 antibodies and may even lower their levels. Therefore, anti-AT1 R and anti-LG3 antibodies are likely not caused by allosensitization. The lack of correlation of anti-AT1 R with anti-LG3 antibodies suggests different mechanisms of generation, which remain to be elucidated.

Background It is vitally important to seek input from key stakeholders to increase the quality and relevance of health-related research and accelerate its adoption into practice. Patients and caregivers have rarely been involved in setting research priorities in the transplantation and donation field. The objectives of this explorative study are: (i) to discuss research priorities within the Canadian National Transplant Research Program during a priority-setting exercise with patients, caregivers, organ donors and researchers and (ii) to compare the identified priorities with research published in 2 prestigious transplantation journals. Methods A pilot workshop attended by 10 patients and caregivers and 5 researchers was held in Montréal (Quebec, Canada) in August 2014 to identify research priorities. Priorities were identified using a thematic analysis of the workshop transcription conducted by multiple coders. These priorities were compared with the topics of research articles published in 2 major transplantation journals between 2012 and 2014. Results The themes of the 10 research priorities identified by study participants were related to different research domains: social, cultural, and environmental health factors (4); biomedical or clinical (4); and research about health systems and services (2). 26.7% of the research articles published were related to the identified priorities. Thirteen percent looked at ways to improve graft survival and 8.5% looked at the development of tolerance, 2 priorities identified by participants. Fewer than 5% examined the other 8 research priorities identified as important by workshop participants. Conclusions This is the first study reporting patients' and researchers' priorities in the field of transplantation and donation in Canada. There is a discrepancy between topics that key stakeholders find important and research published in 2 major transplantation journals. The research priorities identified during our initial workshop will be validated through a national survey and workshop.

It can be argued that living altruistic donors should remain anonymous and should not express preferences in the selection of organ recipients. This study aimed to describe the views of transplant physicians in France and Québec regarding these issues. A total of 27 French and 19 Québec renal transplant physicians took part in individual, semi-directed interviews. Almost all of the physicians agreed that anonymity is mandatory in living altruistic donation (LAD). Regarding the issue of directed donation, most of the French physicians (78%) were opposed to any form of the practice, compared to only a third of their Québec colleagues (32%). We found that these positions were embedded in their respective cultural, legal and social contexts. These results afford a better understanding of these complex issues in two different cultural contexts, and will be useful in the development of international guidelines for LAD.

Although both inflammation and apoptosis occur in acute coronary syndromes (ACSs), previous studies have not tested the diagnostic and prognostic utility of an approach that measures circulating markers of these pathways. The aim of the present study was to assess whether measuring soluble Fas (sFas) and high-sensitivity C-reactive protein (hs-CRP), as markers of apoptosis and inflammation, improve ACS diagnostic and prognostic accuracy. In a prospective cohort of consecutive subjects admitted to the hospital for suspicion of ACS, we measured sFas, hs-CRP, and troponin T in those who had a final noncardiac chest pain diagnosis (n = 100), those who had an ACS diagnosis and experienced (n = 218) or did not experience (n = 170) recurrent cardiac events during 1 year of follow-up. sFas was strongly and independently associated with a discharge diagnosis of an ACS versus noncardiac chest pain during the index hospitalization (odds ratio 16.16 for the second vs first tertile, 95% confidence interval [CI] 7.07 to 36.91; and odds ratio 25.40 for the third vs first tertile, 95% CI 9.38 to 68.75). However, hs-CRP was not. sFas significantly improved the diagnostic accuracy for ACSs (C statistic increased from 0.85 to 0.93, difference +0.08, 95% CI for the difference 0.05 to 0.11). The sFas levels were high and did not vary with time in the subjects having early versus late measurements (beta 0.00 ln pg/ml/hour, 95% CI -0.01 to 0.01). In contrast, troponin increased with time since the beginning of the symptoms (beta 0.07 ln microg/L/hour, 95% CI 0.04 to 0.10). Baseline sFas and hs-CRP did not predict recurrent cardiac events. In conclusion, our results suggest that in suspected ACS cases, sFas, but not hs-CRP, helps to improve the diagnostic accuracy and timeliness over and above standard diagnostic criteria.

Abstract Background BK polyomavirus virus ( BKP yV) screening and immunosuppression reduction effectively prevent graft loss due to BKP yV‐associated nephropathy ( BKPVAN ) during the first year after transplantation. The aim of our study was to evaluate the impact of this infection during longer follow‐up periods. Methods We reviewed the outcome of our screening and immunosuppression reduction protocol in 305 patients who received a kidney transplant between March 2008 and January 2013. Quantitative BKP yV DNA surveillance in plasma was performed at 1, 2, 3, 6, 9, and 12 months after transplantation. Patients with significant viremia and/or biopsy‐proven BKPVAN were treated with immunosuppression reduction and leflunomide. Results During the first post‐transplant year, 24 patients (7.9%) developed significant viremia at a median time of 95 days, and 18 patients had BKPVAN ; 23 of the 24 (7.5%) were treated according to our protocol (group BKV +); 225 patients (73.8%) did not develop any BK viremia (group BKV −). Allograft function was similar in both groups at 1 month post transplantation ( P =.87), but significantly worse at 1 year in the BKV + group ( P =.002). Thereafter, kidney function stabilized in the BKV + group and no differences in patient and graft survival were seen between the groups after a median follow‐up of 4 years. Conclusions We confirm the early occurrence of BKP yV replication after transplantation and the short‐term decline in renal function. However, early detection of BKP yV replication, prompt diagnosis, and reduction in immunosuppression may offer long‐term benefits for graft function.

Ischemia-reperfusion injury (IRI) is a major risk factor for chronic renal failure. Caspase-3, an effector responsible for apoptosis execution, is activated within the peritubular capillary (PTC) in the early stage of IRI-induced acute kidney injury (AKI). Recently, we showed that caspase-3-dependent microvascular rarefaction plays a key role in fibrosis development after mild renal IRI. Here, we further characterized the role of caspase-3 in microvascular dysfunction and progressive renal failure in both mild and severe AKI, by performing unilateral renal artery clamping for 30/60 min with contralateral nephrectomy in wild-type (C57BL/6) or caspase-3-/- mice. In both forms of AKI, caspase-3-/- mice showed better long-term outcomes despite worse initial tubular injury. After 3 wk, they showed reduced PTC injury, decreased PTC collagen deposition and α-smooth muscle actin expression, and lower tubular injury scores compared with wild-type animals. Caspase-3-/- mice with severe IRI also showed better preservation of long-term renal function. Intravital imaging and microcomputed tomography revealed preserved PTC permeability and better terminal capillary density in caspase-3-/- mice. Collectively, these results demonstrate the pivotal importance of caspase-3 in regulating long-term renal function after IRI and establish the predominant role of PTC dysfunction as a major contributor to progressive renal dysfunction.NEW & NOTEWORTHY Our findings demonstrate the pivotal importance of caspase-3 in regulating renal microvascular dysfunction, fibrogenesis, and long-term renal impairment after acute kidney injury induced by ischemia-reperfusion injury. Furthermore, this study establishes the predominant role of peritubular capillary integrity as a major contributor to progressive renal dysfunction after ischemia-reperfusion injury.

The Canadian National Transplant Research Program, launched in 2013 with funding from the Canadian Institutes for Health Research and partners, bridges research in the fields of solid organ transplant, hematopoietic cell transplant, and organ donation. We describe the philosophy, structure, accomplishments, and challenges faced by the Canadian National Transplant Research Program to expand on facilitators and overcome roadblocks to successfully developing a transdisciplinary national research structure.

Background. Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry–based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients. Methods. Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies. Results. The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log 2 [fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus −0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus −0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors ( P &lt; 0.05). Conclusions. AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

Abstract Deficient healing after endovascular aneurysm repair with a stent‐graft is thought to be related to pro‐apoptotic environment in abdominal aortic aneurysms and inertness of graft materials. We developed a bioactive coating containing chondroitin‐4‐sulfate and assessed its potential to improve cell adhesion, viability and resistance to apoptosis on PET surfaces. Coatings of collagen type I and CS were prepared and characterized by DMMB, FT‐IR, DSC, SEM and contact angle goniometry. Preliminary cell culture experiments with vascular smooth muscle cells showed increased adhesion and viability in serum‐free medium on CS‐coated surfaces compared to control PET films. magnified image

A 48-year-old man received a kidney transplant in 2004 and a pancreas transplant in 2007. His medical historywas also significant for bicuspid aortic stenosis. Maintenance immunosuppression consisted of mycophenolate mofetil (MMF), tacrolimus, and prednisone. He presented in May 2011 with recurrentdiarrhea after an empirical short-course antimicrobial treatment. Three weeks before admission, nonbloody diarrhea associated with abdominal discomfort and night sweats occurred. Fever was not reported. Physical examination revealed anasarca, systolic heart murmur, and a soft and distended abdomen that was mildly tender to palpation in the right upper quadrant. Results of blood tests and microbiological stool examination are shown in Figure 1. Abdominal computed tomography (CT) scan showed ascites and mesenteric lymphadenopathy. Colonoscopy was normal. Esogastroduodenoscopy showed candidal esophagitis and abnormal duodenal mucosa (Fig. 1). Duodenal biopsy showed infiltration with histiocytes containing abundant Ziehl-Neelsen–positive bacilli (Fig. 1). Upper endoscopy was repeated to obtain tissue specimens for culture. Thoracic CT scan showed multiple mediastinal lymph nodes.FIGURE 1: A, laboratory tests on admission and 2 months before admission: laboratory results mainly revealed deterioration of renal function, leucopenia, and thrombopenia. B, upper endoscopy showed mild thickening of the duodenal folds, and the duodenal mucosa had fine nodules and superficial ulcers. C, duodenal biopsy showed infiltration with foamy histiocytes (hematein-eosin-safran). D, duodenal biopsy demonstrated numerous acid-fast bacilli in the foamy histiocytes (Ziehl).A provisional diagnosis of Mycobacterium avium complex (MAC) infection was made, and treatment was started with clarithromycin, ethambutol, and rifabutin; estimated glomerular filtration rate was then at 14 mL/min/1.73 m2. MMF had been stopped on admission. Tacrolimus was reduced because of high trough levels. After 12 weeks, the mycobacterial culture of the duodenal tissue specimen grew Mycobacterium genavense, as stools (which were positive for auramine staining), blood, and ascites fluid cultures for mycobacteria were negative. Ethambutol was stopped and moxifloxacin was started. After 1 month of antibiotherapy, diarrhea and abdominal pains improved. Because of progression of heart disease, the patient resumed hemodialysis and underwent aortic valve replacement. After 6 months, findings of a new CT scan and repeat endoscopy with biopsy specimen were unremarkable. After 13 months of antibiotherapy, the patient died from recurrent aortic valve stenosis. M. genavense is a fastidious nontuberculous mycobacterium (NTM) identified in 1991 (1). It is presumed to be ubiquitous in environmental reservoirs, including tap water, and the gastrointestinal tract of birds and mammals (2). Our patient’s household included four cats potentially contaminated with M. genavense. Disease is uncommon and has been described in immunocompromised patients (3, 4), but rarely in transplant recipients, with just 10 reported cases after heart (2 cases), liver (2 cases), and kidney (6 cases) transplantations (2–8). In our patient, chronic renal failure and the prolonged immunosuppressive therapy for multivisceral transplantation may have contributed to severe immune dysfunction. Including the present report, the age range of M. genavense infections in kidney transplant recipients was 41 to 73 years and infection was diagnosed at 7 months to 18 years after transplantation. The dominant presentation of M. genavense infection is gastrointestinal with diarrhea, abdominal pain (often associated with weight loss, fever, and/or night sweats), hepatosplenomegaly, abdominal lymphadenitis, and pancytopenia (2–12). Extra-abdominal presentations with pulmonary (2, 7) or subcutaneous (13) lesions are less frequent. Diagnosis in transplant recipients can be challenging because of the low index of suspicion, misdiagnosis as MMF toxicity, and the unique growth requirements of the pathogen (1). However, M. genavense infection should be rapidly identified and treated due to potentially lethal dissemination (4). A diagnosis of gastrointestinal involvement relies on endoscopic biopsies with histopathologic and specific microbiological analyses. The main endoscopic findings include thickening of the gastric and duodenal folds, villous flattening, nodular lesions with a velvety appearance, and superficial ulcers (8–12). The endoscopic aspect can be misdiagnosed as Whipple disease (14) or neoplasms. The main histopathologic feature of duodenal involvement is massive foamy histiocytic reaction in the submucosal connective tissue, which contains acid-fast bacilli (8–12). These clinical and histopathologic features are similar to those reported for infection with other NTM including MAC. However, with M. genavense, stools specimens are more often acid-fast bacillus smear positive although culture negative (1, 4). Moreover, in contrast with MAC, M. genavense grows poorly on the solid culture media routinely used for mycobacteria (1). It is essential to use broth media for primary culture and a prolonged incubation time (minimum of 8–12 weeks). Amplification by polymerase chain reaction or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue specimens. It is important to identify the specific species of NTM because of differences in susceptibility profiles between species. M. genavense shows a high level of resistance to standard antituberculosis drugs. Although optimal therapy for M. genavense infection has not been established, treatment usually includes clarithromycin (4) with rifabutin and either fluoroquinolone, ethambutol, or amikacin according to recommended dosages (15). When possible, immunosuppressive drugs are tapered, with close monitoring of anticalcineurin: rifabutin (less than rifampin) may accelerate, as clarithromycin may decelerate the metabolism of this immunosuppressant. Duration of treatment is not well defined and depends on the therapeutic response and degree of immunosuppression. Disseminated disease should be treated for a minimum of 12 months. In conclusion, a kidney transplant recipient with chronic diarrhea should prompt further evaluation that includes careful histologic and microbiological assessment of endoscopic tissue samples to detect potential NTMs such as M. genavense. Edith Renoult 1 Claude Fortin2 Judy Dorais3 Rachid Hadjeres4 Michel Pâquet1 Marie-Chantal Fortin1 Catherine Girardin1 Gilles St-Louis1 Héloïse Cardinal1 Renée Lévesque1 Marie-Josée Hébert1 1 Département de Médecine Service de néphrologie Centre Hospitalier de l’Université de Montréal Montreal, Quebec, Canada 2 Département de Microbiologie et Infectiologie Centre Hospitalier de l’Université de Montréal Montreal, Quebec, Canada 3 Département de Médecine Service de gastro-entérologie Centre Hospitalier de l’Université de Montréal Montreal, Quebec, Canada 4 Département de Pathologie Centre Hospitalier de l’Université de Montréal Montreal, Quebec, Canada

Acute allograft glomerulopathy (AAG) is a distinct form of allograft rejection characterized by cytotoxic T-cell-mediated injury to the renal glomerular and arteriolar endothelium. Acute allograft glomerulopathy is characterized by mononuclear cell infiltration of glomerular capillary tufts in association with endothelial cell hypertrophy and injury. Intra-glomerular thrombi have been described in AAG, suggesting that overlapping features of AAG and post-transplant thrombotic microangiopathy (TMA) may coexist. We present a case suggesting that complement factor H deficiency, a known hereditary risk factor for TMA, may also favor development of AAG. We discuss the potential implications of factor H deficiency in the pathophysiology of renal allograft microvascular injury, leukocyte infiltration and formation of intraglomerular platelet thrombi. We propose that unopposed complement activation is a risk factor for both immune and nonimmune forms of microvascular injuries in renal allografts.

To date direct evidence for the presence of a H-K-ATPase in the medulla comes from proton and potassium transport studies performed on K-restricted animals and K dependent ATP hydrolysis and Rb uptake in both normal and K-depleted animals. The present work examines K-dependent acidification in the medulla of rabbits on normal K diets. A membrane vesicle preparation was developed that was enriched for apical membranes derived from the renal medulla. Adenosine triphosphate (ATP)-dependent vesicular acidification was present and the extent of vesicular acidification was dependent on ambient K concentration. Moreover, ATP hydrolysis was dependent on ambient K concentration. K-dependent acidification was inhibited by the specific inhibitor of the gastric H-K-ATPase, SCH28080. However, significant acidification was observed in the absence of K that was not inhibited by SCH28080. The data suggest that an H-K-ATPase similar to the gastric H-K-ATPase is present in the renal medulla of rabbits on a normal K diet. The component of acidification and ATP hydrolysis that is independent of K concentration likely represents the previously characterized vacuolar H-ATPase.

Understanding the mechanisms of injury associated with cardiac arrest is essential for defining strategies aimed at improving preservation and function of kidneys harvested in non-heart-beating (NHB) donors.We standardized a model of NHB donors in rats and studied the kinetics and types (apoptosis vs. necrosis) of renal cell death developing during cold storage. Using quantitative polymerase chain reaction, immunoblotting, and caspase inhibition, we also studied the molecular pathways regulating renal cell death in this model.The kinetics and extent of cell death developing in cortical tubules during cold storage were found to be increased in non-heart-beating (NHB) kidneys. Apoptosis of cortical tubules predominated in NHB kidneys exposed to 10 hr of cold storage, whereas necrosis increased after longer periods of cold ischemia. Shortly after cardiac arrest, a rapid up-regulation of Bax and Hsp 70 was found at the protein level in NHB kidneys. After 24 hr of cold storage, induction of Bax was maintained, whereas protein levels of Hsp70 returned to levels comparable to heart-beating (HB) controls. Also, mRNA levels of Bax were found to increase during cold storage in NHB kidneys. Cortical cell death was found to be largely caspase-independent but responsive to hydroxyl-radical scavenging with dimethyl sulfoxide (DMSO).Cardiac arrest promotes activation of death-inducing molecules such as Bax and is associated with increased development of caspase-independent renal cell death during cold storage. Developing strategies, such as free radical scavenging, aimed at inhibiting cell death during cold storage, could prove useful for improving preservation of NHB kidneys.

Low levels of organic and inorganic mercury compounds have been reported previously to induce cell death by apoptosis in human peripheral blood mononuclear cells (MNC), but little is known about their potential effects on the viability and death of polymorphonuclear neutrophils (PMN). In contrast to MNC, PMN are known to undergo readily spontaneous apoptosis both in vivo and in vitro. Therefore, it was hypothesized that PMN may differ from MNC in their reactions to low mercury levels. The effects of methylmercuric chloride (MeHgCl) and mercuric chloride (HgCl 2 ) were evaluated in concentration- response and time-course studies on human PMN viability and on their modes of cell death after in vitro incubation at 37°C. Cell death by apoptosis or necrosis was assessed by annexin V-fluorescein isothiocyanate binding to externalized phosphatidylserine in conjunction with propidium iodide, and flow cytometry analysis. Morphologic counting of pyknotic nuclei and the fluorescence properties of the DNA-binding dye Hoechst 33342 in combination with propidium iodide were used to further confirm apoptotic cell death and to characterize the sequence of Hg-induced cell death. Results show that low concentrations of MeHgCl (1-7.5 µ M ) that were cytotoxic to MNC actually inhibited PMN spontaneous apoptosis. Low-level HgCl 2 reproduced the anti-apoptotic effects of MeHgCl on PMN, but to a lower extent. Higher concentrations of MeHgCl and HgCl 2 were necro-genic to PMN, but MeHgCl was about an order of magnitude more toxic, and discrete differences were observed in the modalities of cell death induced by both species. These data reveal for the first time that (1) low levels of organic and inorganic mercury species protect human PMN from cell death via inhibition of spontaneous apoptosis, and (2) PMN are more resistant than MNC to mercury-induced cytotoxicity. Since delayed apoptosis and increased resistance to toxicant-induced cell death may lead to excessive accumulation of senescent PMN, evidence indicates that findings of this study may have implications for mercury-induced autoimmunity and inflammation.

Replacing a calcineurin inhibitor (CNI) with sirolimus (SRL) may preserve kidney graft function. However, at the present time, only short follow-up after conversion is available. The aim of this study was to assess whether conversion from a CNI-based to an SRL-based maintenance regimen was safe and effective. We performed a retrospective cohort study among kidney graft patients whose CNI was withdrawn to be replaced by SRL. Two-tailed paired t tests were used to compare glomerular filtration rates (GFRs) and proteinuria levels before and up to 2 years after conversion. We used linear regression to determine the factors associated with changes in renal function after conversion. The 193 study subjects had a mean GFR at conversion of 41 ± 16 mL/min/1.73 m2 a median proteinuria level of 0 g/L (interquartile range = 0–0.15). After conversion, the GFR was stable: at 1 year, the change was −0.34 mL/min/1.73 m2 (95% confidence interval [CI] = −2.71, 2.03) and at 2 years, −0.96 mL/min/1.73 m2 (95% CI = 4.26, 2.34). There was a small but significant increase in dipstick proteinuria at 1 year of +0.5 g/L, (95% CI = 0.20, 0.75). On multivariate analysis, proteinuria ≥ 1 g/L at the time of conversion was the only predictor of deteriorating GFR at 1 year (β: −7.91 mL/min/1.73 m2; 95% CI = −14.10, −1.70). SRL had to be discontinued in 31% of patients. Conversion from CNI to SRL resulted in stable graft function at 2 years and in a slight increase in proteinuria. Despite the relatively high reconversion rate, this strategy offers a reasonable alternative to CNIs for most patients.

Background Patients, families, and caregivers have a unique understanding of the diseases they live with and provide care for every day. Their experience and expertise are important and should be taken into consideration when determining research priorities. The aim of this study was to gather the perspectives of Canadian patients, families, caregivers, researchers, and healthcare professionals on what research priorities were important to them in the field of organ and hematopoietic cell transplantation (HCT) and donation within the Canadian National Transplant Research Program (CNTRP). Methods The CNTRP developed a national consultation process, which included a Web-based survey and in-person workshop, to ascertain and validate the viewpoints of the Canadian donation and transplant community. The Web-based survey identified 3 principal research priorities (increasing donation, developing better antirejection drugs and developing tolerance), which were further refined and prioritized during the one-and-a-half day national workshop held in Toronto in November 2015. Results A total of 505 participants answered the Web-based survey, and 46 participants (28 patients, 12 researchers and 6 healthcare professionals) participated in the in-person workshop. Workshop participants ranked the following 2 priorities as the most important in the fields of donation, HCT, and solid organ transplantation: methods for developing a culture of donation (within healthcare organizations and throughout society); and methods for improving graft survival and antirejection therapy. Conclusion The CNTRP will use these results to prioritize future research projects and studies in donation, HCT, and solid organ transplantation in the years to come.

To the Editor: Havasi and Borkan1.Havasi A. Borkan S.C. Apoptosis and acute kidney injury.Kidney Int. 2011; 80: 29-40Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar discussed recent observations that link apoptosis to cell deletion and loss of organ function after acute kidney injury. Although the major consequence of apoptosis is cell death, emerging evidence suggests that apoptosis is involved in tissue remodeling and fibrogenesis. The molecular machinery regulating the effector phase of apoptosis favors the extracellular translocation of a highly regulated set of mediators of importance in leukocyte trafficking and tissue remodeling. This molecular legacy mounts a communication network with surrounding cells to tissue repair in a paracrine manner, which is of importance in the setting of kidney injury.2.Cailhier J.F. Laplante P. Hebert M.J. Endothelial apoptosis and chronic transplant vasculopathy: recent results, novel mechanisms.Am J Transplant. 2006; 6: 247-253Crossref PubMed Scopus (72) Google Scholar Indeed, apoptotic cells release chemotactic factors, including fractalkine, monocyte chemoattractant protein 1, and lysophosphatidylcholine, to promote the recruitment of macrophages and monocytes at the site of apoptosis.2.Cailhier J.F. Laplante P. Hebert M.J. Endothelial apoptosis and chronic transplant vasculopathy: recent results, novel mechanisms.Am J Transplant. 2006; 6: 247-253Crossref PubMed Scopus (72) Google Scholar Apoptotic cells produce profibrogenic cytokines, including epidermal growth factor, and their engulfment by phagocytes promotes transforming growth factor-β secretion. Caspase-3 activation in apoptotic endothelial cells triggers the export of connective tissue growth factor, which in turn functions as a necessary co-factor of myofibroblast differentiation.3.Laplante P. Sirois I. Raymond M.A. et al.Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis.Cell Death Differ. 2010; 17: 291-303Crossref PubMed Scopus (60) Google Scholar Caspase-3 activation also leads to the externalization of cathepsin L, which in turn cleaves the extracellular matrix component perlecan generating a truncated C terminal fragment (LG3) that activates prosurvival pathways in fibroblasts and vascular smooth muscle cells.4.Laplante P. Raymond M.A. Labelle A. et al.Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation.J Biol Chem. 2006; 281: 30383-30392Crossref PubMed Scopus (64) Google Scholar In conclusion, apoptosis should be viewed not only as a cell-deletion pathway but also as a biological process that engages intercellular crosstalks of central importance in tissue remodeling and repair.

Vasculopathy caused by varicella zoster virus (VZV) reactivation is a rare but clinically relevant complication in kidney transplant recipients,1,2 as demonstrated by the following cases. CASE 1 A 64-year-old woman developed left lower limb paresis with sciatica 3 weeks after kidney transplantation, complicated by confusion, fever, and generalized zoster rash 2 weeks later. Her immunosuppression consisted of tacrolimus, mycophenolate mofetil (MMF), and prednisone without any antiviral prophylaxis (pretransplant serostatus positive for cytomegalovirus IgG, herpes simplex virus 1 IgG, Epstein-Barr virus nuclear antigen IgG, and VZV IgG). She had received an induction with basilixumab, and she never experienced acute rejection. On admission, cerebrospinal fluid (CSF) analysis showed mild pleocytosis with positive VZV using polymerase chain reaction. Brain magnetic resonance imaging (MRI) was normal. The MRI of the lumbar spine showed changes consistent with arachnoiditis. The MMF was discontinued and intravenous acyclovir (10 mg/kg 3 times daily) was started. Twelve days later, confusion worsened. Brain CT demonstrated subarachnoid hemorrhage within bilateral Sylvian fissures (Figure 1A), owing to presumed vessel damage by VZV. However, digital subtraction angiography revealed no vascular lesions. Brain MRI performed 17 days later revealed no infarct, hydrocephalus, or evidence of encephalitis. Acyclovir was discontinued after 21 days. The neurological status improved over the next 2 months but monoparesis remained.FIGURE 1: Axial unenhanced CTs revealing (A) bilateral Sylvian fissure subarachnoid hemorrhage (arrows) in case 1 and (B) right frontal and basal ganglia infarcts (arrows) in case 2.CASE 2 A 32-year-old man returned to dialysis after transplant failure, and some days later, he presented with right-sided zoster ophthalmicus with keratouveitis. Shingles responded to tacrolimus and MMF dose reduction and intravenous acyclovir (10 mg/kg every 8 hours adjusted to creatinine clearance) for 7 days followed by valacyclovir for 10 days. Five weeks later, he developed right third and sixth cranial nerve palsy. Brain MRI revealed inflammation of the right orbital apex and a right lenticulostriate infarct. Intravenous acyclovir was resumed. The MMF was withdrawn and tacrolimus through level was decreased to 3 ng/mL. One week later, the patient developed an acute left-sided sensorimotor stroke. Brain imaging indicated additional acute infarcts (Figure 1B). Digital subtraction angiography revealed multifocal arterial stenosis consistent with arteritis. The CSF analysis identified pleocytosis, increased protein levels, but negative VZV PCR (intrathecal anti-VZV antibodies were not checked). Within the next 2 weeks, the vasculopathy continued to progress with occurrence of new asymptomatic brain infarcts and disseminated to arteries of different sizes bilaterally. The condition finally stabilized with the discontinuation of immunosuppression and the allograft nephrectomy, aimed at restoring VZV immunity without precipitating graft rejection. Acyclovir was discontinued after 1 month. Eight years later, the patient remains with a right-eye keratitis and left-sided deficit. These 2 cases emphasize the protean CNS manifestations of VZV vasculopathy.3,4 Intracranial arteries of any size can be affected by this condition, with progressive multifocal stenoses, ectasia, and pseudoaneurysm, causing combinations of craniofacial pain, intracerebral or subarachnoid hemorrhage, and brain or cranial nerve ischemia. The spinal cord and nerve roots can also be affected. Neurological manifestations and CNS imaging findings are therefore variable and relatively nonspecific.3,4 Angiographic studies may even be normal, especially in cases only involving small vessels.3 Rapid viral diagnosis is important: mortality is high without treatment, and sequelae are frequent, especially in immunocompromised individuals.1,3,4 A temporal association of shingles with neurological symptoms suggests VZV-induced vasculopathy, which may be overlooked due to prolonged prodrome or absence of rash.1-4 Documentation of anti-VZV antibodies or VZV DNA in the CSF confirms the diagnosis. Both tests are recommended: VZV PCR may be positive during a short period.3,4 The VZV vasculitis is unlikely if both results are negative. Early virologic confirmation and prompt initiation of intravenous acyclovir (10–15 mg/kg every 8 hours for a minimum of 14 days) combined with tapering of immunosuppression are essential.2,3 Monitoring of VZV DNA in the CSF and of intrathecal antibody production was suggested to guide the duration of antiviral treatment.5 Regarding prophylaxis, there are no definitive recommendations in kidney transplantation.2

The most common complications of irradiated implant-based mammary reconstruction are fibrosis and capsular contracture. The indications for postmastectomy adjuvant radiotherapy have considerably broadened. Facing an increased number of patients who will require radiotherapy, most guidelines recommend delaying reconstruction after radiotherapy to prevent long-term fibrotic complications. Does radiotherapy permanently alter cellular properties which will adversely affect implant-based reconstruction? If so, is there a benefit in delaying reconstruction after radiotherapy? Our in vitro model simulates two implant-based mammary reconstruction approaches: the irradiated implant and the delayed implant reconstructions by using an implant inset beneath healthy non-irradiated tissue post radiotherapy. We performed cell culture of fibroblasts and endothelial cells to simulate these two surgical conditions. Irradiated fibroblasts simulate the capsular tissue seen around the breast implant. The delayed reconstruction approach is simulated by non-irradiated fibroblasts conditioned with supernatant culture media obtained from irradiated endothelial cells. Irradiation induced fibrosis through fibroblast differentiation into myofibroblasts, as demonstrated by increased α-smooth-muscle actin levels in fibroblasts. This constitutes the basis for scar tissue contraction observed in irradiated implant-based breast reconstruction. Irradiation of endothelial cells induced irreversible cell cycle arrest known as senescence and secretion of the profibrotic connective tissue growth factor. Non-irradiated fibroblasts conditioned with culture media obtained from irradiated endothelial cells exhibited myofibroblast differentiation and the expression of fibrotic phenotype akin to capsular contracture. Our results demonstrate that radiotherapy causes irreversible cellular changes, which permanently alter the microenvironment in favor of fibrosis. Given that these changes are permanent, delaying reconstruction does not present an advantage in preventing capsular contracture.

Transplantation is invariably associated with organ injury following donor organ procurement. Death of cells can negatively impact organ function if sufficient numbers of parenchymal cells are eliminated and not replaced as part of remodelling. Additionally by the release of contents, the death of cells can alter immune responses that are related to ischemia-reperfusion injury and alloimmune rejection. There is increasing awareness of the link between innate and adaptive immunity, and the profound influence inflammation has on organ function, tolerance induction, rejection responses and perhaps survival. Unfortunately, long-term survival of transplants has not been greatly impacted by advances in transplant strategies that solely target recipient immune responses. Therefore, a focus on better understanding tissue injury, and characterizing how newly described forms of cell death influence inflammation in transplanted organs, is timely and likely to yield more effective strategies. Until recently, programmed cell death was relegated entirely to apoptosis, a form of death that is largely, but not exclusively, silent immunologically. In contrast, necrosis is highly pro-inflammatory, but has not been considered to be regulatable, and is thus beyond therapeutic targeting. This partitioning of cell death has become blurred as regulated forms of necrosis such as necroptosis have been identified and immune consequences of apoptosis are characterized. While these share in a common capacity to promote inflammation, they vary considerably in specific pathways, susceptibility to therapeutic intervention, and indeed, in specific organ relevance. This review will discuss current understanding of apoptosis and regulated forms of necrosis in organ injury with specific reference to ischemia-reperfusion injury and allogeneic solid organ transplantation.

Nanoparticles made of the coat protein of papaya mosaic virus (PapMV) and a single-strand RNA were previously shown to be an efficient antigen presentation system for the trigger of cellular immunity. Engineering of PapMV nano with a cytotoxic T lymphocyte epitope was previously shown activating specific T lymphocytes through a proteasome-independent major histocompatibility complex class I (MHC-I) cross-presentation. In this study, we provide new insights into the mechanism of the MHC-I cross-presentation mediated by PapMV nanoparticles. We demonstrate that PapMV nanoparticles do not require the transporter associated with antigen presentation (TAP), but rather depend on lysosome acidification and cathepsin S protease activity for presentation of the T cell epitope. We have also linked the induction of autophagy with this vacuolar MHC-I cross-presentation process. Interestingly, autophagy is induced in antigen-presenting cells after PapMV nanoparticles exposure and inhibition of autophagy reduce MHC-I cross-presentation. This study demonstrates that autophagy is associated with TAP- and proteasome-independent MHC-I cross-presentation. A deeper understanding of the autophagy-dependent MHC-I cross-presentation will be useful in designing vaccination platforms that aim to trigger an efficient cytotoxic T lymphocyte response.

To explore the views of physicians on the use of personalized medicine tools to develop a new method for selecting potential recipients of a renal allograft.A total of 22 semidirected interviews, using clinical case studies.According to the participants, this method has several possible applications within renal transplantation (individualizing immunosuppressive therapy, help with decision making, and possibly with the selection of patients). It could be more effective than the method presently used. The method must be validated scientifically, and must also involve clinical judgment.The use of personalized medicine within transplantation must be in the best interests of the patient. An ethical reflection is necessary in order to focus on the possibility of patients being excluded, as well as on the resolution of the equity/efficacy dilemma. Empirical research has shown itself to be essential for ascertaining the views of the clinicians who will be working with the tools provided by personalized medicine.

SUMMARY While SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTR) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCW), the second dose induced weak humoral responses in SOTR, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, while the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.

Herpes zoster (HZ) usually begins with radicular pain in 1 to 3 dermatomes followed by the appearance of a vesicular eruption over the affected area. In transplant recipients who are at high risk of life-threatening visceral dissemination of varicella zoster virus (VZV), neuropathic pain (the prodrome) may precede the appearance of a rash by weeks and shingles may be absent.1 We aimed to investigate whether VZV DNA detection in plasma could have value for an early diagnosis of HZ during the prodromal phase. We evaluated 11 kidney transplant recipients who developed HZ in the first year after transplantation and from whom 2 to 7 blood samples had been systematically collected in the first 6 months and stored in our transplant biobank, approved by the local Institutional Review Board (BH 07-002). Plasma VZV DNA was retrospectively analyzed by a qualitative real-time PCR analysis2 of these stored samples. The 11 subjects (Table 1) underwent transplantation between 2009 and 2013. The mean time from transplantation to the onset of shingles was 129 days (range, 12-307 days). Prodromal symptoms occurred at 5.5 days on average (range, 0-19 days) before skin eruptions. A total of 41 blood samples from our study cohort were available for analysis, at a median of 4 specimens per patient. Plasma VZV DNA was not detected in 7 individuals. It was detected during HZ infection, at 2 to 53 days after rash onset, in 3 patients. Varicella zoster virus replication was also noted during the prodromal phase, in 1 of these 3 subjects (patient 10, who was diagnosed with HZ ophthalmicus 6 days later) and in another recipient (patient 8, who developed VZV vasculopathy 12 days later).TABLE 1: VZV DNA detected by PCR in plasma in 11 kidney transplant recipients with herpes zosterOur retrospective analysis of available archived blood specimens in a small number of patients had obvious limitations but our findings provide interesting PCR evidence of VZV reactivation during HZ in transplant recipients. These data document the presence of VZV in the plasma of 4 patients diagnosed with HZ. In 3 of these cases, VZV DNA was detectable in blood taken up to 53 days after the onset of shingles. Blood VZV DNA has previously been detected during acute HZ,3,4 in 16% to 100% of the patient populations analyzed by molecular assays. Some previous studies have revealed a higher VZV load in immunocompromised patients. In addition, VZV DNemia can be documented up to 6 months after skin eruptions. An even more interesting observation from our results was the detection of VZV DNA before rash onset in 2 individuals. In both of these patients, the prodromal phase was particularly painful and prolonged, and the clinical features of HZ were quite severe, particularly in 1 patient who developed a VZV vasculopathy as described elsewhere.5 Analysis of VZV DNA in plasma could therefore be used for the early diagnosis of atypical HZ and avoid delayed initiation of treatment. However, the precise diagnostic value of this marker for these purposes requires additional research.3

The aim of this study was to assess the relationship between cyclosporine (CyA) trough level (C0) and 2-hour postdose (C2) and total cholesterol (TC) in kidney transplant (KT) recipients on Neoral maintenance immunosuppression. In KT recipients who had more than 5 years of follow-up, stable graft function, and stable Neoral dose, we measured C2 and C0 blood levels, serum creatinine, mean total cholesterol (TC) over the last 5 years, prednisone dose, use of beta-blockers and thiazides. Correlations between C0 and C2 levels and TC were performed with the Pearson coefficient. Receiver operating characteristics (ROCs) were used to define the threshold with greater accuracy for significant variables at the correlation test. Statistical tests were performed with SPSS 9.5 The C2 correlated with TC (0.31; P=.008) whereas C0 did not. The C2 level was an independent predictor for TC after adjusting for recipient age, gender, dose of prednisone, creatinine clearance, and use of beta-blockers and thiazides (B coefficient=1.124(E-3); P=.009). A threshold C2 value of 700 microg/L yielded to a TC level of 5.2 mmol/L. This is the first study to report a correlation between C2 levels and TC. Although C2 explained a small fraction of TC variability, it is an independent predictor of TC in KT recipients on Neoral maintenance immunosuppression. A long-term C2 value under 700 microg correlates with better control of hypercholesterolemia.

Background. Both angiotensin II receptor autoantibodies (ATRabs) and autoantibodies to LG3 have been linked to kidney graft rejection with alloimmune vascular injury (AVI). We aimed to examine whether positivity for both anti-LG3 and ATRabs is associated with rejection with AVI in kidney transplant recipients. Methods. We performed a retrospective cohort study including consecutive kidney transplant recipients between 2013 and 2017 at a single center. The primary outcome was acute rejection with AVI (Banff grade 2 or 3 T-cell-mediated rejection and/or antibody-mediated rejection) in the first 3 mo posttransplant. The secondary outcome was death-censored allograft loss. The independent variables, anti-LG3 and ATRab, were measured pretransplant. Results. Among the 328 study participants, 68 experienced acute rejection with AVI and 23 experienced graft loss over a median follow-up of 4.5 y. In a multivariable model, double pretransplant positivity for anti-LG3/ATRab was associated with acute rejection with AVI (odds ratio: 2.73, 95% confidence interval: 1.06-7.05). We did not observe an association between double positivity for anti-LG3/ATRab and death-censored graft loss. Conclusions. Double positivity for anti-LG3/ATRabs pretransplant is associated with a higher risk of acute rejection with AVI. Whether therapies that remove antibodies could decrease that risk remains to be studied. Supplemental Visual Abtract: http://links.lww.com/TXD/A494.