Atsuhiro Ichihara

We found that when a site-specific binding protein interacts with the "handle" region of the prorenin prosegment, the prorenin molecule undergoes a conformational change to its enzymatically active state. This nonproteolytic activation is completely blocked by a decoy peptide with the handle region structure, which competitively binds to such a binding protein. Given increased plasma prorenin in diabetes, we examined the hypothesis that the nonproteolytic activation of prorenin plays a significant role in diabetic organ damage. Streptozotocin-induced diabetic rats were treated with subcutaneous administration of handle region peptide. Metabolic and renal histological changes and the renin-Ang system components in the plasma and kidneys were determined at 8, 16, and 24 weeks following streptozotocin treatment. Kidneys of diabetic rats contained increased Ang I and II without any changes in renin, Ang-converting enzyme, or angiotensinogen synthesis. Treatment with the handle region peptide decreased the renal content of Ang I and II, however, and completely inhibited the development of diabetic nephropathy without affecting hyperglycemia. We propose that the nonproteolytic activation of prorenin may be a significant mechanism of diabetic nephropathy. The mechanism and substances causing nonproteolytic activation of prorenin may serve as important therapeutic targets for the prevention of diabetic organ damage.

Rationale : The (pro)renin receptor [(P)RR], encoded in ATP6AP2 , plays a key role in the activation of local renin-angiotensin system (RAS). A truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H + -ATPase (V-ATPase), implicating a non–RAS-related function of ATP6AP2. Objective : We investigated the role of (P)RR/ATP6AP2 in murine cardiomyocytes. Methods and Results : Cardiomyocyte-specific ablation of Atp6ap2 resulted in lethal heart failure; the cardiomyocytes contained RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles, especially in the perinuclear regions. The myofibrils and mitochondria remained at the cell periphery. Cardiomyocyte death was accompanied by numerous autophagic vacuoles that contained undigested cellular constituents, as a result of impaired autophagic degradation. Notably, ablation of Atp6ap2 selectively suppressed expression of the V O subunits of V-ATPase, resulting in deacidification of the intracellular vesicles. Furthermore, the inhibition of intracellular acidification by treatment with bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes. Conclusions : Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non–RAS-related function that is essential for cell survival.

To collect outcome data in a large cohort of patients with aggressive pituitary tumours (APT)/carcinomas (PC) and specifically report effects of temozolomide (TMZ) treatment.Electronic survey to ESE members Dec 2015-Nov 2016.Reports on 166 patients (40 PC, 125 APT, 1 unclassified) were obtained. Median age at diagnosis was 43 (range 4-79) years. 69% of the tumours were clinically functioning, and the most frequent immunohistochemical subtype were corticotroph tumours (45%). Ki-67 index did not distinguish APT from PC, median 7% and 10% respectively. TMZ was first-line chemotherapy in 157 patients. At the end of the treatment (median 9 cycles), radiological evaluation showed complete response (CR) in 6%, partial response (PR) in 31%, stable disease (SD) in 33% and progressive disease in 30%. Response was more frequent in patients receiving concomitant radiotherapy and TMZ. CR was seen only in patients with low MGMT expression. Clinically functioning tumours were more likely to respond than non-functioning tumours, independent of MGMT status. Of patients with CR, PR and SD, 25, 40 and 48% respectively progressed after a median of 12-month follow-up. Other oncological drugs given as primary treatment and to TMZ failures resulted in PR in 20%.This survey confirms that TMZ is established as first-line chemotherapeutic treatment of APT/PC. Clinically functioning tumours, low MGMT and concurrent radiotherapy were associated with a better response. The limited long-term effect of TMZ and the poor efficacy of other drugs highlight the need to identify additional effective therapies.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than seven million people worldwide, contributing to 0.4 million deaths as of June 2020. The fact that the virus uses angiotensin-converting enzyme (ACE)-2 as the cell entry receptor and that hypertension as well as cardiovascular disorders frequently coexist with COVID-19 have generated considerable discussion on the management of patients with hypertension. In addition, the COVID-19 pandemic necessitates the development of and adaptation to a "New Normal" lifestyle, which will have a profound impact not only on communicable diseases but also on noncommunicable diseases, including hypertension. Summarizing what is known and what requires further investigation in this field may help to address the challenges we face. In the present review, we critically evaluate the existing evidence for the epidemiological association between COVID-19 and hypertension. We also summarize the current knowledge regarding the pathophysiology of SARS-CoV-2 infection with an emphasis on ACE2, the cardiovascular system, and the kidney. Finally, we review evidence on the use of antihypertensive medication, namely, ACE inhibitors and angiotensin receptor blockers, in patients with COVID-19.

Blockade of the renin-angiotensin system slows the progression of diabetic nephropathy but fails to abolish the development of end-stage nephropathy of diabetes. The prorenin-to-active renin ratio significantly increases in diabetes, and prorenin binding to its receptor in diabetic animal kidney induces the nephropathy without its conventional proteolytic activation, suggesting that angiotensin II (AngII) may not be the decisive factor causing the nephropathy. For identification of an AngII-independent mechanism, diabetes was induced in wild-type mice and AngII type 1a receptor gene-deficient mice by streptozotocin treatment, and their development and progression of diabetic nephropathy were assessed. In addition, prolonged inhibition of angiotensin-converting enzyme and prolonged prorenin receptor blockade were compared for their efficacy in preventing the nephropathy that occurred in diabetic AngII type 1a receptor gene-deficient mice. Only the prorenin receptor blockade with a short peptide of prorenin practically abolished the increased mitogen-activated protein kinase (MAPK) activation and nephropathy despite unaltered increase in AngII in diabetic kidney. These results indicate that the MAPK activation signal leads to the diabetic nephropathy but not other renin-angiotensin system-activated mechanisms in the glomeruli. It is not only AngII but also intraglomerular activation of MAPK by the receptor-associated prorenin that plays a pivotal role in diabetic nephropathy.

In contrast to proteolytic activation of inactive prorenin by cleavage of the N-terminal 43 residue peptide, we found that prorenin is activated without proteolysis by binding of the prorenin receptor to the pentameric "handle region" I(11P)LLKK(15P). We hypothesized that such activation occurs in hypertensive rats and causes cardiac renin-angiotensin system (RAS) activation and end-organ damage. To test this hypothesis, we devised methods of specifically inhibiting nonproteolytic activation by decapeptide spanning the pentameric handle region peptide as a decoy. In stroke-prone spontaneously hypertensive rats (SHRsp) fed a high-salt diet, arterial pressure started to rise significantly with a marked increase in the cardiac prorenin receptor mRNA level at 8 weeks of age, and cardiac fibrosis had developed by 12 weeks of age. By immunohistochemistry using antibodies to the active site of the renin molecule, we demonstrated increased proteolytic or nonproteolytic activation of prorenin in the heart but not in plasma of SHRsp. Continuous subcutaneous administration of the handle region peptide completely inhibited the increased staining by antibodies to the active site of the renin molecule, indicating the increased nonproteolytic but not proteolytic activation of prorenin in the heart of SHRsp. Administration of the handle region peptide also inactivated tissue RAS without affecting circulating RAS or arterial pressure and significantly attenuated the development and progression of cardiac fibrosis. These results clearly demonstrate the significant role of nonproteolytically activated tissue prorenin in tissue RAS activation leading to cardiac fibrosis and significant inhibition of the cardiac damage produced by chronic infusion of the handle region peptide.

For defining the pathogenic effects of the (pro)renin receptor-transgenic rat, strains that overexpressed the human receptor were generated. Although transgenic rats were normotensive and euglycemic and had a renal angiotensin II (AngII) level that was comparable to that of wild-type rats, transgenic rats developed proteinuria with aging and significant glomerulosclerosis at 28 wk of age. In kidneys of 28-wk-old transgenic rats, mitogen-activated protein kinases (MAPK) were activated without recognizable tyrosine phosphorylation of the EGF receptor, and expression of TGF-beta1 was enhanced. In vivo infusion of the (pro)renin receptor blocker peptide (formerly handle region decoy peptide) significantly inhibited the development of glomerulosclerosis, proteinuria, MAPK activation, and TGF-beta1 expression in the kidneys, but the angiotensin-converting enzyme inhibitor did not attenuate these changes despite a significant decrease in the renal AngII level. In addition, recombinant rat prorenin stimulated MAPK activation in the human receptor-expressed cultured cells, but human receptor was unable to evoke the enzyme activity of rat prorenin. Thus, human (pro)renin receptor elicits slowly progressive nephropathy by AngII-independent MAPK activation in rats. This study clearly provided in vivo evidence for the AngII-independent MAPK activation by human (pro)renin receptor and induction of glomerulosclerosis with increased TGF-beta1 expression.

The prorenin receptor is an accessory subunit of the vacuolar H(+)-ATPase, suggesting that it has fundamental functions beyond activation of the local renin-angiotensin system. Podocytes express the prorenin receptor, but its function in these cells is unknown. Here, podocyte-specific, conditional, prorenin receptor-knockout mice died of kidney failure and severe proteinuria within 4 weeks of birth. The podocytes of these mice exhibited foot process effacement with reduced and altered localization of the slit-diaphragm proteins nephrin and podocin. Furthermore, the podocytes contained numerous autophagic vacuoles, confirmed by enhanced accumulation of microtubule-associated protein 1 light chain 3-positive intracellular vesicles. Ablation of the prorenin receptor selectively suppressed expression of the V(0) c-subunit of the vacuolar H(+)-ATPase in podocytes, resulting in deacidification of intracellular vesicles. In conclusion, the prorenin receptor is important for the maintenance of normal podocyte structure and function.

Increased macula densa cyclooxygenase-2 (COX-2) is observed in diabetic rats and may contribute to hyperfiltration states. However, the signals mediating increased COX-2 expression in diabetic rats remain undetermined. We recently found that non-proteolytic activation of prorenin by site-specific binding proteins, such as prorenin receptor, plays a pivotal role in the development of diabetic nephropathy. The present study was designed to determine the contribution of prorenin receptor to renal cortical COX-2 expression. The COX-2 mRNA and protein levels of six 4-week-old male wild-type rats and six human prorenin receptor gene-transgenic (hProRenRcTg) rats were measured by real-time polymerase chain reaction methods, Western blotting, and immunohistochemistry, and compared. There were no differences between the two groups in arterial pressure measured by telemetry, urinary sodium excretion, or renal levels of rat prorenin receptor mRNA. The renal cortical COX-2 mRNA levels of the hProRenRcTg rats were significantly higher than those of the wild-type rats, and the renal cortical COX-2 protein levels were also higher in hProRenRcTg rats than in the wild-type rats. Immunohistochemical analysis revealed that COX-2 immunostaining was predominantly present in the macula densa cells, and significantly more COX-2-positive cells were present in the hProRenRcTg rats than in the wild-type rats. In addition, COX-2 inhibition with NS398 significantly decreased renal cortical blood flow in the hProRenRcTg rats but not in the wild-type rats. These results strongly suggest that human prorenin receptor directly or indirectly contributes to the regulation of renal cortical COX-2 expression. Increased macula densa cyclooxygenase-2 (COX-2) is observed in diabetic rats and may contribute to hyperfiltration states. However, the signals mediating increased COX-2 expression in diabetic rats remain undetermined. We recently found that non-proteolytic activation of prorenin by site-specific binding proteins, such as prorenin receptor, plays a pivotal role in the development of diabetic nephropathy. The present study was designed to determine the contribution of prorenin receptor to renal cortical COX-2 expression. The COX-2 mRNA and protein levels of six 4-week-old male wild-type rats and six human prorenin receptor gene-transgenic (hProRenRcTg) rats were measured by real-time polymerase chain reaction methods, Western blotting, and immunohistochemistry, and compared. There were no differences between the two groups in arterial pressure measured by telemetry, urinary sodium excretion, or renal levels of rat prorenin receptor mRNA. The renal cortical COX-2 mRNA levels of the hProRenRcTg rats were significantly higher than those of the wild-type rats, and the renal cortical COX-2 protein levels were also higher in hProRenRcTg rats than in the wild-type rats. Immunohistochemical analysis revealed that COX-2 immunostaining was predominantly present in the macula densa cells, and significantly more COX-2-positive cells were present in the hProRenRcTg rats than in the wild-type rats. In addition, COX-2 inhibition with NS398 significantly decreased renal cortical blood flow in the hProRenRcTg rats but not in the wild-type rats. These results strongly suggest that human prorenin receptor directly or indirectly contributes to the regulation of renal cortical COX-2 expression. Cyclooxygenase-2 (COX-2) is constitutively present in the macula densa cells and in medullary interstitial cells of the kidney.1.Kujubu D.A. Fletcher B.S. Varnum B.C. et al.TIS10, a phorbol ester tumor promoter-inducible mRNA from swiss3T3 cells, encodes a novel prlstaglandin synthase/cyclooxygenase homologue.J Biol Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar,2.Xie W. Chipman J.G. Robertson D.L. et al.Expression of a mitogen-responsive gene encoding prostaglandin systhase is regulated by mRNA splicing.Proc Natl Aca Sci USA. 1991; 88: 2692-2696Crossref PubMed Scopus (1643) Google Scholar COX-2 activity in the macula densa is stimulated by high renal perfusion pressure,3.Ichihara A. Imig J.D. Navar L.G. Cyclooxygenase-2 modulates afferent arteriolar responses to increases in pressure.Hypertension. 1999; 34: 843-847Crossref PubMed Google Scholar and the stimulated macula densa COX-2 catalyzes the synthesis of vasodilator prostaglandins counteracting the tubuloglomerular-feedback-mediated afferent arteriolar constriction.4.Ichihara A. Imig J.D. Inscho E.W. Navar L.G. Cyclooxygenase-2 participates in tubular flow-dependent afferent arteriolar tone: interaction with neuronal NOS.Am J Physiol. 1998; 275: F605-F612PubMed Google Scholar Thus, increased activity of macula densa COX-2 can elicit a decrease in afferent arteriolar resistance, which leads to elevation of intraglomerular pressure.5.Peterdi J.P. Komlosi P. Fuson A.L. et al.Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.J Clin Invest. 2003; 112: 76-82Crossref PubMed Scopus (121) Google Scholar Recent studies have shown increased expression of macula densa COX-2 in the kidneys of diabetic humans6.Khan K.N.M. Burke A. Stanfield K.M. et al.Expression of cyclooxygenase-2 in the macula densa of human kidney in hypertension, congestive heart failure, and diabetic nephropathy.Renal Failure. 2001; 23: 321-330Crossref PubMed Scopus (58) Google Scholar and rats.7.Komers R. Lindsly J.N. Oyama T.T. et al.Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes.J Clin Invest. 2001; 107: 889-898Crossref PubMed Scopus (174) Google Scholar Because dilated afferent arterioles and enlarged glomeruli are observed in the early stage of diabetic nephropathy, the increased expression of macula densa COX-2 may account for the hyperfiltration state of the glomeruli owing to afferent arteriolar dilation in diabetic kidneys. As glomerular hyperfiltration is well known to play an important role in the development of diabetic nephropathy, macula densa COX-2 may contribute to the development of nephropathy in diabetic kidneys. However, the mechanism of the upregulation of macula densa COX-2 expression in diabetic kidneys remained unknown. We recently found that site-specific prorenin-binding proteins, such as prorenin receptor,8.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1136) Google Scholar contribute to the development of diabetic nephropathy,9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar and we therefore hypothesized that the activation of prorenin receptor also contributes to the mechanism of the increased expression of COX-2 in the macula densa in diabetic kidneys. To test this hypothesis, in the present study we assessed the expression and function of renal cortical COX-2 in rats expressing a high level of human prorenin receptor (hProRenRc). Figure 1 shows that hProRenRc transgene was distributed to various tissues including brain, heart, kidney, liver, aorta, femoral artery, testis, lung, spleen, pancreas, adipose tissue, adrenal gland, ovary, and uterus. In the kidneys of hProRenRc-transgenic (Tg) rats, the majority of hProRenRc transgene was present in tubular cells, and a few were observed in the glomerular epithelium. As shown in Table 1, there were no significant differences in mean atrial pressure (MAP), urinary sodium excretion, plasma renin activity, plasma angiotensin II level, kidney angiotensin II level, renal plasma flow, and rat prorenin receptor (rProRenRc) mRNA levels between the Tg and wild-type rats. The microhematocrit of blood samples from wild-type and Tg rats was also similar and remained constant throughout the experiment. Urinary prostaglandin E2 was significantly higher in Tg rats than in wild-type rats. The mRNA expression of hProRenRc was detected only in Tg rats.Table 1Characterization of hProRenRc gene-transgenic (Tg) ratsWildTgP-valueNumber66MAP (mm Hg)88±288±10.54UNaV (mEq/day)3.67±0.193.58±0.300.84Urinary PGE2 (ng/h)20.3±1.833.7±2.0<0.001Microhematocrit (%)54.3±1.752.3±0.90.30PRA (ng/ml/h)3.3±0.9 (n=4)2.5±0.6 (n=19)0.43Plasma Ang II (fmol/l)48.6±7.2 (n=14)37.3±6.5 (n=19)0.26Kidney Ang II (fmol/g)53.7±7.840.1±9.00.30Renal plasma flow (ml/min/g)5.7±0.65.4±0.50.81Kidney rProRenRc mRNA (ratio to rat GAPDH mRNA)6.9±0.56.8±0.40.88Data are shown as means±s.d.Ang II, angiotensin II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mean arterial pressure; PGE2, prostaglandin E2; PRA, plasma renin activity; rProRenRc, rat prorenin receptor; UNaV, urinary sodium excretion. Open table in a new tab Data are shown as means±s.d. Ang II, angiotensin II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mean arterial pressure; PGE2, prostaglandin E2; PRA, plasma renin activity; rProRenRc, rat prorenin receptor; UNaV, urinary sodium excretion. Figure 2a shows the COX-2 mRNA levels in the renal cortex of the Tg and wild-type rats determined quantitatively by a real-time polymerase chain reaction method. Renal cortical COX-2 mRNA level in the Tg rats was 5.33±0.70 (ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA) and was significantly higher than in the wild-type rats (1.28±0.66 (ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA)). Figure 2b shows the COX-2 protein levels in the renal cortex of the Tg and wild-type rats determined by Western blot analysis. Renal cortical COX-2 protein levels (1.06±0.26 (ratio to α-tubulin)) were also higher in Tg rats than in the wild-type rats (2.18±0.17 (ratio to α-tubulin)). Figure 3 shows the results of immunostaining with anti-rat COX-2 antibody in the renal cortex of the Tg and wild-type rats. Strong COX-2 immunostaining was observed in the macula densa cells of the hProRenRcT rats, but weak staining in the renal cortex of the wild-type rats. Both the Tg and wild-type rats had a similar value of renal blood flow because they had similar values of renal plasma flow, as shown in Table 1, and microhematocrit throughout the experiment. Figure 4 shows the effects of the COX-2 inhibitor NS398 on renal blood flow assessed by an electromagnetic flow probe and renal cortical blood flow in the Tg and wild-type rats. There were no significantly changes in MAP in either group during COX-2 inhibition with NS398. Under anesthesia, MAP at the beginning and end of the study period measured 122±3 and 117±6 mm Hg, respectively, in the wild-type rats, and 118±2 and 120±3 mm Hg, respectively, in the Tg rats. NS398 did not significantly alter renal blood flow or renal cortical blood flow in the wild-type rats; however, the 1 and 10 ng/g bw (gram body weight) doses of NS398 significantly reduced the renal blood flow of the Tg rats from 2.91±0.23 to 2.39±0.19 and 2.03±0.41 ml/min, respectively, and decreased the renal cortical blood flow of the Tg rats by 27.2±3.6 and 42.3±7.9%, respectively. The decreases in renal blood flow and renal cortical blood flow in the Tg rats compared to the wild-type rats were significant. Figure 5 shows the phosphorylated and total extracellular signal-regulated kinase (ERK) levels in the renal cortex of the Tg and wild-type rats determined by Western blot analysis. The ratio of phospho-ERK level to total ERK level in the renal cortex was significantly higher in Tg rats (4.7±0.5) than in the wild-type rats (1.8±0.4). Transgenic expression of hProRenRc in rats did not affect MAP, urinary sodium excretion, or rProRenRc levels. However, macula densa COX-2 expression was significantly higher in the Tg rats than in the wild-type rats, and the increased COX-2 expression significantly contributed to the regulation of renal cortical blood flow. These results suggest that hProRenRc expression causes an increase in expression and function of macula densa COX-2 independently of blood pressure or urinary sodium excretion. When prorenin binds to the prorenin receptor, two important signals are thought to occur. One signal is the non-proteolytic activation of prorenin: prorenin undergoes a conformational change that includes exposure of its enzymatically active site, and the activated prorenin stimulates the renin–angiotensin system (RAS).9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar The other signal is an increase in mitogen-activated protein kinase (MAPK)-dependent intracellular transduction that is independent of the RAS.8.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1136) Google Scholar,10.Schefe J.H. Kaiser H.F. Jost A. Unger T. Signal transduction of the renin/prorenin receptor.J Hypertens. 2005; 123: S354-S355Google Scholar Thus, enhancement of macula densa COX-2 expression by hProRenRc is thought to be mediated through both a RAS-dependent mechanism and a RAS-independent mechanism. A recent study showed that angiotensin II stimulates renal COX-2 mRNA expression in 5/6 renal-ablated rats.11.Hernandez J. Astudillo H. Escalante B. Angiotenin II stimulates cyclooxygenase-2 mRNA expression in renal tissue from rats with kidney failure.Am J Physiol. 2002; 282: F592-F598PubMed Google Scholar Human ProRenRc may stimulate renal COX-2 mRNA expression through an increase in angiotensin II levels owing to RAS activation. In the present study, however, plasma angiotensin II levels were similar in the Tg and wild-type rats. In addition, Cheng et al.12.Cheng H.F. Wang J.L. Zhang M.Z. et al.Angiotensin II attenuates renal cortical cyclooxygenase-2 expression.J Clin Invest. 1999; 103: 953-961Crossref PubMed Scopus (189) Google Scholar clearly demonstrated that angiotensin II attenuates renal COX-2 expression localized to the cortical thick ascending limb of Henle's loop cells in the region of the macula densa in the rats. It is therefore unlikely that the RAS activation contributes to the increase in macula densa COX-2 expression by hProRenRc. Low-chloride medium stimulates COX-2 expression in primary cultured cortical thick ascending limb of Henle's loop cells13.Cheng H.F. Wang J.L. Zhang M.Z. et al.Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride.J Clin Invest. 2000; 106: 681-688Crossref PubMed Scopus (111) Google Scholar and a macula densa cell line14.Yang T. Park J.M. Arend L. et al.Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line.J Biol Chem. 2000; 275: 37922-37929Crossref PubMed Scopus (127) Google Scholar through phosphorylation of the three major MAPK pathways, p38, Jun N-terminal kinase, and ERK, suggesting that MAPK pathways play an important role in the expression of COX-2 localized to the region of macula densa. In the present study, the ratio of phospho-ERK to total ERK was significantly increased in the renal cortex from Tg rats compared to wild-type rats, suggesting that hProRenRc may stimulate the ERK activation. Therefore, phosphorylation of the MAPK pathways by hProRenRc can mediate the upregulation of macula densa COX-2 expression observed in the present study. Further studies are needed to clarify the mechanism of the upregulation of macula densa COX-2 by hProRenRc. As COX-2 mediates the kidney prorenin synthesis induced by a low-salt diet,15.Harding P. Sigmon D.H. Alfie M.E. et al.Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet.Hypertension. 1997; 29: 297-302Crossref PubMed Google Scholar increased levels and/or activity of macula densa COX-2 can activate RAS and lead to blood pressure elevation and a decreased urinary sodium excretion. In the present study, however, both the blood pressure and urinary sodium excretion of the Tg rats were similar to the values in the wild-type rats, and the plasma renin activity, plasma angiotensin II levels, and kidney angiotensin II levels of 4-week-old Tg rats were similar to those of 4-week-old wild-type rats. Thus, it appeared that the plasma or kidney RAS of the Tg rats is not activated at 4 weeks of age. Nevertheless, renal cortical ERK was activated in the Tg rats compared to the wild-type rats. Because of the high homology between the amino-acid sequences of rProRenRc and hProRenRc, rat prorenin is able to bind to hProRenRc. Our preliminary study using COS-7 cells that expressed hProRenRc showed that rat prorenin is capable of binding to hProRenRc but does not enzymatically activate prorenin. The maximum binding of hProRenRc to rat prorenin was 70% of the maximum binding of hProRenRc to human prorenin, and the Kd value of rat prorenin binding to hProRenRc was 3 nmol/l and approximately two-fold higher than the Kd of human prorenin binding to hProRenRc. In the presence of rat prorenin, however, sheep angiotensinogen was not converted to angiotensin I in the medium of COS-7 cells that expressed hProRenRc. Therefore, transgenic expression of hProRenRc was capable of stimulating the RAS-independent intracellular signals, but could not activate the RAS, because of competitive inhibition of rProRenRc binding to rat prorenin by hProRenRc. This may account for no activation of the RAS despite increased direct actions of prorenin in the Tg rats. In the present study, the clearance method and electromagnetic flow probe measurements confirmed a similar baseline renal plasma flow in wild-type and Tg rats despite enhanced COX-2 expression in the renal cortex of Tg rats. These results suggest that some compensative mechanisms worked to keep the renal plasma flow constant against increased COX-2 expression. Therefore, COX-2 inhibition with NS398 caused a significant decrease in renal cortical blood flow only in Tg rats, which had an increased level of macula densa COX-2. In conclusion, transgenic expression of hProRenRc resulted in stimulation of the expression and activity of macula densa COX-2. Thus, hProRenRc may influence the macula densa-dependent renal microcirculation by regulating COX-2 expression. These experiments were performed in accordance with the guidelines and practices established by the Keio University Animal Care and Use Committee. hProRenRc (GenBank accession number AF291814; nt 73–1301) plasmid vector, designated pCAGGS-hProRenRc, was used. The details of its use, including its construction, have been described previously.16.Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4424) Google Scholar Briefly, we constructed an expression vector by introducing a CAG promoter that comprises an AG promoter with a cytomegalovirus immediate early gene enhancer and rabbit β-globin gene terminator sequences including a polyadenylation signal. This vector, designated pCAGGS-hProRenRc, ubiquitously exhibited high-level production of hProRenRc in various organs. Transgenic rats were generated by pronuclear microinjection of fertilized Crj:Wistar rat eggs and reimplantation into pseudopregnant Crj:Wistar foster mothers. Six foster mothers carried 31 pups to term, three of which carried the transgene, as demonstrated by PCR analysis of DNA obtained from tail blood. Two of these founders transmitted the transgene to their progeny, and the transgenic lines hProRenRc no. 21 line and no. 29 line were established. Transgene of hProRenRc was ubiquitously observed in the brain, heart, kidney, liver, aorta, arteries, testis, lung spleen, pancreas, adipose tissue, adrenal gland, ovary, and uterus, as shown in Figure 1. The no. 21 line was used for the present study. Arterial pressure was determined under the unrestricted and untethered condition in 4-week-old rats using a telemetry device inserted into the abdominal aorta, and the 24-h urine was collected in metabolic cage. To determine p-aminohippurate sodium clearance as a renal plasma flow, renal clearance study was performed under anesthesia in the rats continuously infused with isotonic saline solution containing 1% albumin and 1.5% p-aminohippurate sodium (Merck & Co., Inc., Whitehouse Station, NJ, USA) at a rate of 20 μl/min, as described previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Two groups of rats (n=6 each) were used in this series of experiments: a wild-type group born of the hProRenRc line but to which the hProRenRc gene had not been transmitted (wild), and a group of hProRenRc gene-transgenic (Tg) rats. We decapitated six rats of each group to obtain the blood and kidneys. Immediately after decapitation, a 3-ml blood specimen was collected into a tube containing 30 μl of ethylenediaminetetraacetic acid (500 mM), 15 μl of enalaprilat (1 mM), and 30 μl of o-phenanthroline (24.8 mg/ml) and pepstatin (0.2 mM), and plasma samples were obtained by centrifugation. Plasma renin activity was determined with a radioimmunoassay coated-bead kit (Dinabott Radioisotope Institute, Tokyo, Japan). For the determination of kidney angiotensin II content, the removed kidney was weighed, placed in ice-cold methanol (10% wt/vol), homogenized with a chilled glass homogenizer, and centrifuged. The supernatant was then dried and reconstituted in 4 ml of 50 mM sodium phosphate buffer containing 1% albumin. Plasma and reconstituted samples from the kidneys were extracted with a Bond-Elut column (Analytichem, Harbor City, CA, USA), and the eluents were evaporated to dryness and reconstituted in angiotensin peptide assay diluent. The angiotensin II content was quantitatively determined by radioimmunoassay using rabbit anti-angiotensin II antiserum (Arnel, New York, NY, USA). The urine samples were collected in a metabolic cage, and the urinary concentration of prostaglandin E2 was determined by radioimmunoassay using a [125I] radioimmunoassay kit (PerkinElmer Life Sciences Inc., Boston, MA, USA) after the extraction by conventional methods and subsequent purification on Bond-Elut Si columns (Analytichem International, Harbor City, CA, USA). As described previously,9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar total RNA from part of the renal cortex removed from each animal was extracted with an RNeasy Mini Kit (QIAGEN KK, Tokyo, Japan), and real-time quantitative reverse transcriptase-PCR (RT-PCR) was performed by using the TaqMan One-Step RT-PCR Master Mix Reagents Kit with an ABI Prism 7700 HT Detection System (Applied Biosystems Inc., Foster City, CA, USA) and probes and primers for rat genes encoding the following: COX-2 (forward, 5′-TGTTCGCATTCTTTGCCCA-3′; reverse, 5′-TAAGTCCACTCCATGGCCCA-3′; probe, 5′-FAM-TCAGAAGCGAGGACCTGGGTTCACC-TAMRA-3′); rProRenRc (forward, 5′-CATTCGACACATCCCTGGTG-3′; reverse, 5′-AAGGTTGTAGGGACTTTGGGTG-3′; probe, 5′-FAM-AAGTCAAGGACCATCCTTGAGACGAAACAA-TAMRA-3′); hProRenRc (forward, 5′-AGATGACATGTACAGTCTTTATGGTGG-3′; reverse, 5′-TGCTGGGTTCTTCGCTTGT-3′; probe, 5′-FAM-TTTGACACCTCCCTCATTAGGAAGACAAGGACT-TAMRA-3′); and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-TGACAACTCCCTCAAGATTGTCA-3′; reverse, 5′-GGCATGGACTGTGGTCATGA-3′; probe, 5′-FAM-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3′). Renal localization of the transgene was assessed by in situ hybridization, as described previously.18.Burckle C.A. Jan Danser A.H. Muller D.N. et al.Elevated blood pressure and heart rate in human renin receptor Transgenic rats.Hypertension. 2006; 47: 1-5Crossref PubMed Scopus (186) Google Scholar Briefly, digoxigenin-labeled cRNA antisense and sense probes were synthesized using a 600-bp fragment of the AF291814 as template. Western blot analyses were performed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, the renal cortex was lysed in Tris (25 mmol/l) containing NaCl (150 mmol/l), glycerol (5%), and protease inhibitor (1 tablet/25 ml buffer), and after centrifugation at 10 000 g for 5 min followed by centrifugation at 24 000 g for 30 min, the supernatant was collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes, and after blocking the blots overnight with phosphate-buffered saline containing 5% bovine albumin and 0.5% Tween 20, they were incubated for 16 h with mouse monoclonal anti-rat COX-2 antibody (1:1000 dilution; Exalpha Biologicals, Watertown, MA, USA), mouse monoclonal anti-phospho-44/42 MAPK (E10) antibody that crossreacts with rat phosphorylated ERK1 and 2 (1:800 dilution; Cell Signaling Technology, Beverly, MA, USA), or mouse monoclonal anti-α-tubulin antibody (1:500 dilution; EMD Biosciences, La Jolla, CA, USA). Immunoreactivity was determined by horseradish peroxidase-conjugated donkey anti-mouse antibody and an enhanced chemiluminescence reaction, and the quantitative analyses were performed with Image 1D (Pharmacia, Peapack, NJ, USA). Immunohistochemical staining for COX-2 was performed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, part of the kidney was fixed in 4% paraformaldehyde phosphate buffer solution before paraffin embedding, and deparaffinized sections were pretreated with proteinase K. The sections were then boiled in citrate buffer with microwaves to unmask antigenic sites, and endogenous biotin was blocked with a biotin-blocking system (X0590; DAKO Corp., Kyoto, Japan). Next, the sections were immersed in 0.3% H2O2 in methanol to inhibit endogenous peroxidase and then precoated with 1% nonfat milk in phosphate-buffered saline to block nonspecific binding. Polyclonal rabbit anti-COX-2 antibody (160116, Cayman Chemical, Ann Arbor, MI, USA) (1:200–500 dilution) was used as the primary antibodies, and a biotinylated polyclonal goat anti-rabbit antibody (1:500 dilution) was applied as the second antibody. Immunoreaction was performed with a Vectastain ABC-Elite kit (Vector Laboratories, Burlingame, CA, USA) and visualized with 0.02% 3′,3′-diaminobenzidine tetrahydrochloride as a substrate, and then lightly counterstaining with hematoxylin. The effect of the COX-2 inhibitor NS398 (Cayman Chemical, Ann Arbor, MI, USA) on renal cortical blood flow was assessed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). The right femoral artery was cannulated, and the cannula was connected to a computer system (MacLab/8s, AD Instruments, Nagoya, Japan) via a pressure transducer to monitor arterial pressure. The right carotid artery was also cannulated to provide separate access for intra-arterial bolus doses of NS398. The left kidney was exposed by a flank incision and placed in a Lucite cup to stabilize it. A needle flow probe (500-μm diameter) connected to a laser-Doppler flowmeter (FloC1-Twin; Omegawave, Tokyo, Japan) was inserted into the kidney mass to a depth of 1 mm to position its tip in the superficial cortex, and used to measure relative changes in renal cortical blood flow. In addition, a small-diameter, noncannulating, and factory-precalibrated electromagnetic flow probe (1RB2949; Transonic Systems Inc., Ithaca, NY, USA), connected to a transit time flowmeter module (D-79232; Transonic Systems Inc.), was vertically fitted around the left renal artery to measure renal blood flow continuously. The rats were given intra-arterial bolus injections (100 μl) of increasing doses of NS398 (0, 1, and 10 ng/g) at 10-min intervals. Within-group statistical comparisons were made by one-way analysis of variance for repeated measures followed by the Neuman–Keuls post hoc test. Differences between two groups were evaluated by two-way analysis of variance for repeated measures combined with the Newman–Keuls post hoc test. A P-value of <0.05 was considered significant. Data are reported as means±s.e.m. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (16613002, 16790474, 17390249, and 41503340) and research Grant HL58205 from the United States National Institutes of Health.

The term "receptor-associated prorenin system" (RAPS) refers to the pathogenic mechanisms whereby prorenin binding to its receptor dually activates the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling via the receptor. The aim of the present study was to define the association of the RAPS with diabetes-induced retinal inflammation.Long-Evans rats, C57BL/6 mice, and angiotensin II type 1 receptor (AT1-R)-deficient mice with streptozotocin-induced diabetes were treated with (pro)renin receptor blocker (PRRB). Retinal mRNA expression of prorenin and the (pro)renin receptor was examined by quantitative RT-PCR. Leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal protein levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 were examined by ELISA. Retinal extracellular signal-regulated kinase (ERK) activation was analyzed by Western blotting.Induction of diabetes led to significant increase in retinal expression of prorenin but not the (pro)renin receptor. Retinal adherent leukocytes were significantly suppressed with PRRB. Administration of PRRB inhibited diabetes-induced retinal expression of VEGF and ICAM-1. To clarify the role of signal transduction via the (pro)renin receptor in the diabetic retina, we used AT1-R-deficient mice in which the RAS was deactivated. Retinal adherent leukocytes in AT1-R-deficient diabetic mice were significantly suppressed with PRRB. PRRB suppressed the activation of ERK and the production of VEGF, but not ICAM-1, in AT1-R-deficient diabetic mice.These results indicate a significant contribution of the RAPS to the pathogenesis of diabetes-induced retinal inflammation, suggesting the possibility of the (pro)renin receptor as a novel molecular target for the treatment of diabetic retinopathy.

Background Cushing's disease is a rare debilitating endocrine disorder for which few prospective interventional studies have been done. We report results of the first phase 3 trial assessing long-acting intramuscular pasireotide in patients with Cushing's disease. Methods In this phase 3 clinical trial we recruited patients aged 18 years or older with persistent, recurrent, or de-novo (non-surgical candidates) Cushing's disease who had a mean urinary free cortisol (mUFC) concentration (from three 24 h samples) of 1·5–5·0 times the upper limit of normal (ULN), a normal or greater than normal morning plasma adrenocorticotropic hormone concentration, and a pituitary source of Cushing's syndrome, from 57 sites across 19 countries. Exclusion criteria included previous pasireotide treatment, mitotane therapy within 6 months, and pituitary irradiation within 10 years. We randomly allocated patients 1:1 (block size of four) using an interactive-response-technology system to intramuscular pasireotide 10 mg or 30 mg every 4 weeks for 12 months (in the core phase). We stratified randomisation by screening mUFC concentration (1·5 to <2·0 × ULN and 2·0–5·0 × ULN). The dose could be uptitrated (from 10 mg to 30 mg or from 30 mg to 40 mg) at month 4 if the mUFC concentration was greater than 1·5 × ULN, and at month 7, month 9, or month 12 if the mUFC concentration was greater than 1·0 × ULN. Investigators, patients, site personnel, and those assessing outcomes were masked to dose group allocation. The primary endpoint was the proportion of patients in each group with an mUFC concentration of less than or equal to the ULN at month 7. Efficacy analyses were based on intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01374906. Findings Between Dec 28, 2011, and Dec 9, 2014, we randomly allocated 150 patients to receive pasireotide 10 mg (74 [49%] patients) or 30 mg (76 [51%] patients). The primary efficacy endpoint was met by 31 (41·9% [95% CI 30·5–53·9]) of 74 patients in the 10 mg group and 31 (40·8% [29·7–52·7]) of 76 in the 30 mg group. The most common adverse events were hyperglycaemia (36 [49%] in the 10 mg group and 36 [47%] in the 30 mg group), diarrhoea (26 [35%] and 33 [43%]), cholelithiasis (15 [20%] and 34 [45%]), diabetes mellitus (14 [19%] and 18 [24%]), and nausea (15 [20%] and 16 [21%]). Serious adverse events suspected to be study drug related were reported in eight (11%) patients in the 10 mg group and four (5%) in the 30 mg group. Two (3%) patients in the 30 mg group died during the study (pulmonary artery thrombosis and cardiorespiratory failure); neither death was judged to be related to the study drug. Interpretation Long-acting pasireotide normalised mUFC concentration in about 40% of patients with Cushing's disease at month 7 and had a similar safety profile to that of twice-daily subcutaneous pasireotide. Long-acting pasireotide is an efficacious treatment option for some patients with Cushing's disease who have persistent or recurrent disease after initial surgery or are not surgical candidates, and provides a convenient monthly administration schedule. Funding Novartis Pharma AG.

The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

Arterial stiffness assessed by pulse wave velocity (PWV) predicts all-cause and cardiovascular mortality in diabetic patients with end-stage renal disease. We studied the preventive effects of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin, on arterial PWV values in this population.Twenty-two patients with normal serum lipid levels received fluvastatin (20 mg/day p.o.) or a placebo for 6 months. Their serum lipid levels, serum levels of C-reactive protein (CRP), arterial PWV, and ankle brachial indexes (ABI) were determined before, and 3 and 6 months after taking the medication to evaluate arterial stiffness.At the beginning of the follow-up, there were no differences in age, blood pressure, body mass index, serum haemoglobin A1c level, serum CRP level, serum lipid levels, PWV or ABI between the placebo- (n=10) and the fluvastatin-treated patients (n=12). After 6 months, the PWV and the serum oxidized low-density lipoprotein cholesterol (LDL-C) level increased significantly (from 1969+/-140 to 2326+/-190 cm/s and 70.4+/-13.8 to 91.8+/-15.5 U/l, respectively) in the placebo-treated patients. However, the fluvastatin group had a significantly reduced PWV (from 1991+/-162 to 1709+/-134 cm/s), oxidized LDL-C serum levels (from 89.0+/-9.6 to 73.0+/-5.8 U/l) and CRP serum levels (from 0.97+/-0.32 to 0.26+/-0.16 mg/dl) compared with those in the placebo group.Long-term administration of fluvastatin prevents further worsening of arterial biomechanics in haemodialysis patients with type 2 diabetes mellitus, even in the presence of serum lipid levels in the normal range.

Prorenin is activated without proteolysis by binding of prorenin receptor to the pentameric "handle region" (HR) of prorenin prosegment. It was hypothesized that such activation occurs in the kidneys of hypertensive rats and causes tissue renin-angiotensin system (RAS) activation and end-organ damage. Because the HR's binding to its binding protein made the adjacent tetrameric "gate region" (GR) accessible to its specific antibody, immunohistochemistry of the GR was performed to test the hypothesis. Methods also were devised specifically to inhibit the nonproteolytic activation by the decapeptide corresponding to the HR as a decoy. Immunohistochemistry of the GR demonstrated that the majority of nonproteolytically activated prorenin is present in podocytes of the kidneys from stroke-prone spontaneously hypertensive rats, in which activation of renal tissue RAS, proteinuria, and glomerulosclerosis occurred. Continuous subcutaneous administration of the HR decoy peptide completely inhibited both nonproteolytic activation of tissue prorenin and activation of tissue RAS without affecting circulating RAS or arterial pressure and significantly attenuated the development and progression of proteinuria and glomerulosclerosis. These studies clearly demonstrated that nonproteolytic activation of prorenin in glomeruli is critically involved in renal tissue RAS activation, leading to renal damage in hypertensive animals.

Activation of prorenin by (pro)renin receptor stimulates the tissue renin-angiotensin system and plays a significant role in the development of nephropathy in diabetic animals. This study examined whether (pro)renin receptor blockade inhibits the progression of nephropathy that has already developed in diabetic rats. Seventeen-week-old heminephrectomized streptozotocin-induced diabetic rats with an increased urinary protein excretion and a significant glomerulosclerosis had been treated for 12 wk with the (pro)renin receptor blocker (PRRB), angiotensin-converting enzyme inhibitor (ACEi), or vehicle peptide by using subcutaneously implanted osmotic minipumps. At the end of observation, in diabetic rats that were treated with vehicle, urinary protein excretion was progressively increased and a significant progression of glomerulosclerosis was observed. In diabetic rats that were treated with PRRB, however, no further increase in urinary protein excretion or glomerulosclerosis was observed, but 12-wk treatment with ACEi only attenuated further increases in urinary protein excretion and glomerulosclerosis. The enhanced expression of activated prorenin was observed in the kidneys of diabetic rats that were treated with vehicle, whereas it was markedly suppressed in the kidneys of diabetic rats that were treated with PRRB but not ACEi. These results suggest that (pro)renin receptor blockade does not only inhibit the progression of nephropathy but also reverses the glomerulosclerosis that has already developed in diabetic rats.

Despite the development of non-ionic radiographic contrast media (CM), CM-induced nephropathy is a clinically important problem in patients with pre-existing renal insufficiency. We examined the effects of non-ionic CM (iohexol) on renal function in conscious dogs with and without renal insufficiency, and evaluated the effects of a non-selective (theophylline), an A1 selective (KW-3902), and an A2 selective adenosine antagonist (KF17837) on the renal responses to CM. In sham-operated group, iohexol (2 ml/kg/min for 3 min) increased effective renal plasma flow (ERPF) and glomerular filtration rate (GFR), whereas in renal insufficiency group (with subtotal nephrectomy), following transient increases in ERPF and GFR, CM markedly decreased ERPF (-46.5 +/- 6.7%) and GFR (-51.2 +/- 7.1%). In sham-operated group, theophylline and KF17837 markedly attenuated CM-induced increases in ERPF and GFR, while KW-3902 had no effects on CM-induced increases in ERPF or GFR. In renal insufficiency group, initial increases in ERPF and GFR were blunted by theophylline and KF17837. In contrast, the subsequent decreases in ERPF and GFR were attenuated by theophylline (% delta ERPF, -12.2 +/- 3.2% vs. -46.6 +/- 6.7%, P < 0.01; % delta GFR, 4.3 +/- 2.5% vs. -51.0 +/- 7.1%, P < 0.01), and were completely prevented by KW-3902 (% delta ERPF, 10.8 +/- 2.9%; % delta GFR, 23.8 +/- 4.4%), whereas KF17837 aggravated ERPF (-73.3 +/- 5.3%) and GFR (-78.4 +/- 5.3%). These data indicate that in normal renal function, iohexol elicits renal vasodilation by activating mainly the adenosine A2 receptors. In contrast, in impaired renal function, CM induces both A2 and A1 activation; the former is associated with the initial renal vasodilation, while the latter is responsible for the sustained aggravation of renal hemodynamics.

The renin-angiotensin system is believed to influence blood pressure (BP) during pregnancy, but the associations between BP during pregnancy and the soluble form of the (pro)renin receptor (s[P]RR), a new component of the tissue renin-angiotensin system, remain undetermined. In this prospective cohort study of 437 pregnant women with normal BP (systolic <140 mm Hg and diastolic <90 mm Hg) during early pregnancy (<16 weeks of gestation) regression analysis was performed to examine the associations between plasma s(P)RR concentrations and BP in 3 gestational stages (20-24, 28-32, and 36-40 weeks of gestation) and logistic regression analysis to evaluate the incidence of preeclampsia. Plasma s(P)RR concentrations at early, middle (16-28 weeks), and late pregnancy (>28 weeks) and at delivery averaged 29.7 ± 10.0, 31.3 ± 12.0, 39.2 ± 8.9, and 40.4 ± 10.2 ng/mL (mean ± SD), respectively. A 1-ng/mL increase in plasma s(P)RR concentration in early pregnancy predicted systolic/diastolic BP elevation in the later 3 gestational stages: 0.11 (95% CI, 0.014-0.20)/0.093 (0.027-0.16) mm Hg for 20 to 24 weeks, 0.11 (0.029-0.19)/0.088 (0.027-0.15) mm Hg for 28 to 32 weeks, and 0.16 (0.058-0.26)/0.12 (0.043-0.19]) mm Hg for 36 to 40 weeks, respectively. Plasma s(P)RR concentrations in middle and late pregnancy were not associated with BP. Adjusted models revealed that women with plasma s(P)RR concentrations above the 75th percentile at delivery had a significantly increased risk of preeclampsia (odds ratio, 22.5 [95% CI, 1.8-279.9]). In conclusion, high circulating levels of s(P)RR at early pregnancy predicted a subsequent elevation in BP, and high concentrations at delivery were significantly associated with preeclampsia.

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.Background: The vacuolar H+-ATPase complex is thought to contribute to membrane fusion.Results: v-ATPase complex knock-out experiments in mice revealed that its absence does not affect phagosome-lysosome fusion.Conclusion: Participation of v-ATPase in phagosome-lysosome fusion is unlikely.Significance: Fusion between lysosomes/late endosomes and phagosomes is not controlled by the v-ATPase. The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. Background: The vacuolar H+-ATPase complex is thought to contribute to membrane fusion. Results: v-ATPase complex knock-out experiments in mice revealed that its absence does not affect phagosome-lysosome fusion. Conclusion: Participation of v-ATPase in phagosome-lysosome fusion is unlikely. Significance: Fusion between lysosomes/late endosomes and phagosomes is not controlled by the v-ATPase.

Within the kidney, the (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD), particularly in intercalated cells, and it is regulated by the PGE 2 receptor EP 4 . Notably, EP 4 also controls urinary concentration through regulation of aquaporin 2 (AQP2). Here, we tested the hypothesis that sequential activation of EP 4 and PRR determines AQP2 expression in the CD, thus mediating the antidiuretic action of vasopressin (AVP). Water deprivation (WD) elevated renal PRR expression and urinary soluble PRR excretion in rats. Intrarenal infusion of a PRR decoy peptide, PRO20, or an EP 4 antagonist partially prevented the decrease in urine volume and the increase in urine osmolality and AQP2 expression induced by 48-hour WD. In primary cultures of rat inner medullary CD cells, AQP2 expression induced by AVP treatment for 24 hours depended on sequential activation of the EP 4 receptor and PRR. Additionally, mice lacking PRR in the CD exhibited increased urine volume and decreased urine osmolality under basal conditions and impaired urine concentrating capability accompanied by severe volume loss and a dangerous level of plasma hyperosmolality after WD. Together, these results suggest a previously undescribed linear AVP/PGE 2 /EP 4 /PRR pathway in the CD for regulation of AQP2 expression and urine concentrating capability.

There are currently no factors that have been shown to predict gestational diabetes mellitus (GDM) during early pregnancy. The soluble (pro)renin receptor [s(P)RR] may contribute to the development of GDM. The objective of the study was to determine whether plasma s(P)RR concentrations during early pregnancy are associated with the development of GDM later in pregnancy. This prospective cohort study was conducted at a referral birth center. Pregnant women who first visited our hospital during the first trimester (<14 weeks of gestation) between 2010 and 2011 were enrolled. Inclusion criteria included singleton pregnancy and the absence of preexisting diabetes mellitus. A total of 716 women participated in this study. The association of plasma s(P)RR concentrations with the onset of GDM later in pregnancy was measured. Among 716 participants, 44 (6.1%) had GDM and 672 (93.9%) did not. There were 176 participants in the first plasma s(P)RR concentration quartile (Q1: < 25.8 ng/mL), 179 in the second (Q2: 25.8–30.2 ng/mL), 181 in the third (Q3: 30.2–34.2 ng/mL), and 180 in the fourth (Q4: > 34.2 ng/mL). GDM distribution was 7 (4.0%) in Q1, 5 (2.8%) in Q2, 13 (7.2%) in Q3, and 19 (10.6%) in Q4. A multivariate model adjusted for baseline characteristics, medical complications, and gestational characteristics revealed that the risk of developing GDM among women in Q4 compared with Q1 was 2.90 (95% confidence interval 1.11–7.49). Increased s(P)RR concentrations during the first trimester may predict the development of GDM later in pregnancy.

Choosing effective therapy for patients with malignant pheochromocytoma or paraganglioma (PPGL) is problematic and none of the options are curative. Although combination chemotherapy with cyclophosphamide, vincristine, and dacarbazine (CVD) is an established treatment option, only a limited number of case series have been reported in the literature. To determine the efficacy of CVD in patients treated at Tokyo Women's Medical University. Retrospective review of patients treated with CVD between 1989 and 2012 was conducted. Demographics, clinical presentation, imaging, and laboratory reports were reviewed and analyzed. Efficacy of CVD was ascertained from the biochemical and tumor responses. Twenty-three patients fulfilled study criteria and 6 of these were excluded due to inadequate follow-up or discontinuance by poor general condition or adverse effects. Thus, 17 cases were included in the study. The age and duration of the disease before initiation of CVD were 54.7 ± 12.0 years and 9.1 ± 8.1 years, respectively. The follow-up period after initiation of CVD ranged from 12 to 192 months (median, 60 months). Complete or partial biochemical and/or partial tumor response was achieved in 47.1 % (responders). No significant biochemical or tumor response was seen in 23.5 % and deterioration in biochemical and tumor outcomes was seen in 29.4 % (non-responders). No patient showed complete biochemical and tumor responses. In responders, these effects were documented within 4 months after initiation of CVD with a progression-free survival of 31 to 60 months (median, 40 months). Age at the first diagnosis with PPGL was younger (P < 0.05) and the lag time to eventual diagnosis of malignant disease was longer (P < 0.05) in responders than those in non-responders. The responders had improvements in hypertension and impaired glucose tolerance. Although CVD chemotherapy is not curative for patients with malignant PPGL, it does provide approximately half of the patients with biochemical, tumor, and hypertension benefits.

The (pro)renin receptor ((P)RR) is expressed in several tissues including kidney, heart and brain, and is thought to regulate the tissue renin-angiotensin system (RAS) through the non-proteolytic activation of prorenin. (P)RR is cleaved by furin to generate soluble (P)RR (s(P)RR), which is secreted into the extracellular space. s(P)RR is a candidate biomarker reflecting the status of the tissue RAS. Here, we investigated the relationship between background factors and serum s(P)RR levels. We measured s(P)RR levels in 122 patients with essential hypertension (EH) and assessed the relationships between background factors and s(P)RR levels. Serum s(P)RR levels were 19.0±4.9 ng ml(-1). Single regression analyses showed that age (r=0.251, P<0.01), serum creatinine levels (r=0.229, P<0.05) and urinary angiotensinogen excretion (r=0.196, P<0.05) were positively correlated with s(P)RR levels, whereas estimated glomerular filtration rate (eGFR; r=-0.337, P<0.001) were negatively correlated. Multiple regression analyses of age, blood pressure (BP), hemoglobin A1c (HbA1c) and s(P)RR levels revealed that age and s(P)RR levels were negatively correlated with the eGFR (P<0.05). In patients with EH, serum s(P)RR levels correlated positively with renal function independent of age, BP and HbA1c. These findings support s(P)RR as a useful biomarker that reflects the status of the tissue RAS.

The prorenin receptor (PRR), a recently discovered component of the renin-angiotensin system, is expressed in the nephron in general and the collecting duct in particular. However, the physiological significance of nephron PRR remains unclear, partly due to developmental abnormalities associated with global or renal-specific PRR gene knockout (KO). Therefore, we developed mice with inducible nephron-wide PRR deletion using Pax8-reverse tetracycline transactivator and LC-1 transgenes and loxP flanked PRR alleles such that ablation of PRR occurs in adulthood, after induction with doxycycline. Nephron-specific PRR KO mice have normal survival to ∼1 yr of age and no renal histological defects. Compared with control mice, PRR KO mice had 65% lower medullary PRR mRNA and protein levels and markedly diminished renal PRR immunofluorescence. During both normal water intake and mild water restriction, PRR KO mice had significantly lower urine osmolality, higher water intake, and higher urine volume compared with control mice. No differences were seen in urine vasopressin excretion, urine Na(+) and K(+) excretion, plasma Na(+), or plasma osmolality between the two groups. However, PRR KO mice had reduced medullary aquaporin-2 levels and arginine vasopressin-stimulated cAMP accumulation in the isolated renal medulla compared with control mice. Taken together, these results suggest nephron PRR can potentially modulate renal water excretion.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na(+) excretion and investigated the signaling mechanisms by which PRR regulates Na(+) balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na(+) diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na(+) balance with normal- and low-Na(+) intake. PRR KO mice had an attenuated hypertensive response and reduced Na(+) retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na(+) channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na(+) wasting and reduces the hypertensive response to ANG II.

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg-1·min-1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N ω -nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.

Although lowering blood pressure (BP) reduces aortic stiffness, achieving the recommended BP goal can be difficult. Recent studies have shown that short-term use of statins can reduce BP significantly. To determine the long-term effects of statins on BP and aortic stiffness, a single-blind randomized prospective study was performed on 85 hyperlipidaemic hypertensive patients whose BP was insufficiently controlled by antihypertensive therapy. Every 3 months, aortic stiffness was assessed by measuring pulse wave velocity (PWV). Patients were randomly allocated to groups treated with pravastatin, simvastatin, fluvastatin, or a nonstatin antihyperlipidaemic drug. No significant differences in patient characteristics, kinds of antihypertensive drugs, BP, ankle brachial index, PWV, or serum lipid, creatinine, or C-reactive protein levels were found between the four groups at the start of the study. During the 12-month treatment period, PWV did not change in the pravastatin group or nonstatin group, but it was transiently reduced in the simvastatin group and significantly decreased in the fluvastatin group, even though the doses of the statins used in this study were lower than the usually prescribed dose. All four antihyperlipidaemic drugs significantly decreased serum cholesterol levels without affecting BP, ankle brachial index, or serum triglyceride levels. The C-reactive protein serum levels decreased significantly in the three statin groups but not in the nonstatin group. These results suggest that long-term use of fluvastatin by hyperlipidaemic hypertensive patients is associated with a significant reduction in aortic stiffness without any effect on BP.

purpose. A recent study revealed that angiotensin receptor signaling mediates ocular inflammation and neovascularization. It was also found that prorenin undergoes nonproteolytic activation leading to upregulation of the renin-angiotensin system (RAS) when prorenin receptor interacts specifically with the handle region of prorenin. The purpose of the present study was to elucidate the role of the receptor-dependent nonproteolytic activation of prorenin in ocular inflammation in endotoxin-induced uveitis (EIU). methods. EIU was induced in Long-Evans rats by a single intraperitoneal injection of 100 μg lipopolysaccharide (LPS). Tissue localization of total prorenin, prorenin receptor, and activated prorenin in the EIU retina was examined by immunohistochemistry. To inhibit the prorenin receptor-mediated upregulation of the RAS, a decoy handle-region peptide (HRP) was intraperitoneally administered 24 hours before and immediately after the injection of LPS. Twenty-four hours after LPS injection, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. In addition, leukocyte infiltration into the vitreous cavity and protein concentration in the anterior chamber were also measured. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1, interleukin (IL)-6, and C-C chemokine ligand (CCL) 2/monocyte chemotactic protein (MCP)-1 were examined by RT-PCR and ELISA. results. Retinal vessels in rats with EIU were strongly positive for total prorenin, prorenin receptor, and activated prorenin. Systemic treatment with HRP resulted in dose- and time-dependent inhibition of the leukocyte adhesion and infiltration and the protein leakage, all of which were increased by the induction of EIU. Retinal mRNA expression and protein levels of ICAM-1, CCL2/MCP-1 and IL-6, induced in rats with EIU, were also significantly suppressed with application of HRP. conclusions. These findings demonstrate for the first time that nonproteolytically activated prorenin plays a significant role in the development of ocular inflammation in the EIU model. The present study suggests the potential use of HRP, a decoy peptide binding to the prorenin receptor, as a therapeutic agent to reduce ocular inflammation.

Hemodialysis patients have uremic dyslipidemia, represented by elevated serum intermediate-density lipoprotein cholesterol (IDL-C) levels, and an increased cardiovascular mortality rate. This study was performed to determine the low-dose effects of the angiotensin II receptor blocker losartan and the angiotensin-converting enzyme inhibitor trandolapril on pulse wave velocity (PWV), which predicts cardiovascular morbidity and mortality in hemodialysis patients.Serum lipid levels and PWV were monitored for 12 months in 64 hemodialysis patients who were administered low doses of losartan or trandolapril or a placebo.At the start of the study, there were no differences in patient characteristics among the 3 groups. PWV tended to increase in the placebo group during the 12-month study period, but decreased significantly in the losartan and trandolapril groups, and decreases in PWV were similar in the losartan and trandolapril groups. There were no changes in blood pressure, hematocrit, erythropoietin dose, ankle-brachial index, serum lipid levels, serum 8-isoprostane levels, or serum C-reactive protein levels during the 12-month study period, but there was an increase in serum triglyceride levels in the losartan group and a decrease in serum IDL-C levels in the losartan and trandolapril groups.In hemodialysis patients, trandolapril is as effective as losartan in decreasing PWV independent of its depressor effect and in suppressing elevated IDL-C levels. Long-term blockade of the renin-angiotensin system may have a beneficial effect on the acceleration of atherosclerosis and uremic dyslipidemia.

A direct renin inhibitor (DRI) had a benefit in decreasing albuminuria in type 2 diabetic patients having already been treated with angiotensin (Ang) II type 1 receptor blocker (ARB), suggesting that aliskiren may have another effect other than blockade of the traditional renin–angiotensin system (RAS). Recently, prorenin bound to (pro)renin receptor ((P)RR) was found and shown to evoke two pathways; the generation of Ang peptides and the receptor-dependent activation of extracellular signal–related protein kinase (ERK). Because (P)RR is present in the podocytes, a central component of the glomerular filtration barrier, we hypothesized that aliskiren influences the (P)RR-induced two pathways in human podocytes. Human podocytes were treated with 2nmol/l prorenin in the presence and absence of an angiotensin-converting enzyme inhibitor (ACEi) imidaprilat, an ARB candesartan, a DRI aliskiren, or the siRNA knocking down the (P)RR mRNA and the intracellular AngII levels and the phosphorylation of ERK were determined. The expression of (P)RR mRNA of human podocytes was unaffected by the treatment with RAS inhibitors, but decreased by 69% with the siRNA treatment. The basal levels of intracellular AngII and the prorenin-induced increase in intracellular AngII were significantly reduced by aliskiren and siRNA treatment, compared with imidaprilat and candesartan. The prorenin-induced ERK activation was reduced to control level by the siRNA treatment, but it was unaffected by imidaprilat, candesartan, or aliskiren. Aliskiren is the most potent inhibitor of intracellular AngII levels of human podocytes among RAS inhibitors, although it is incapable of inhibiting the (P)RR-dependent ERK phosphorylation.

Although Wnt/β-catenin signaling is known to be aberrantly activated in PDAC, mutations of CTNNB1, APC or other pathway components are rare in this tumor type, suggesting alternative mechanisms for Wnt/β-catenin activation. Recent studies have implicated the (pro)renin receptor ((P)RR) is related to the Wnt/β-catenin signaling pathway. We therefore investigated the possible role of (P)RR in pancreatic carcinogenesis. Plasma s(P)RR levels were significantly (P < 0.0001) higher in patients with PDAC than in healthy matched controls. We also identified aberrant expression of (P)RR in premalignant PanIN and PDAC lesions and all the PDAC cell lines examined. Inhibiting (P)RR with an siRNA attenuated activation of Wnt/β-catenin signaling pathway and reduced the proliferative ability of PDAC cells in vitro and the growth of engrafted tumors in vivo. Loss of (P)RR induced apoptosis of human PDAC cells. This is the first demonstration that (P)RR may be profoundly involved in ductal tumorigenesis in the pancreas.

The prorenin receptor ((P)RR) contributes to the regulation of the tissue renin-angiotensin system (RAS) and the function of V-ATPase, which are essential for Wnt signaling. Thus, (P)RRs may be involved in the control both of feto-placental and maternal circulation during pregnancy. This study was conducted to clarify how placental (P)RR expression and plasma soluble (P)RR [s(P)RR] levels are associated with blood pressure elevations and renal function during pregnancy.We conducted a cross-sectional study, conducted at Saitama medical center in 2010-2013. Preeclamptic women (n = 16) diagnosed according to the criteria of Japan Society of Obstetrics and Gynecology and normotensive pregnant women (n = 15) participated in the study. We measured the expression of (P)RR in the placenta, plasma s(P)RR levels, systolic blood pressure (SBP), and estimated glomerular filtration rate (eGFR).Placental expression of (P)RR was significantly higher in preeclamptic women than in normotensive pregnant women. The plasma s(P)RR levels were significantly higher in preeclamptic women than in normotensive pregnant women. Systolic blood pressure (SBP) was positively correlated with placental (P)RR levels (P = 0.0001) and plasma s(P)RR levels (P = 0.005) in all pregnant women. In preeclamptic women, SBP was positively correlated with placental (P)RR levels (P = 0.004), but not with plasma s(P)RR levels (P = 0.15). The eGFR was negatively correlated with placental (P)RR levels (P = 0.02) and plasma s(P)RR levels (P = 0.0002) in all pregnant women. In preeclamptic women, eGFR was negatively correlated with plasma s(P)RR levels (P = 0.006), but not with placental (P)RR levels (P = 0.93).Placental (P)RR can be involved in blood pressure regulation via the tissue RAS. On the other hand, plasma s(P)RR may be involved in the pathogenesis of decreased renal function in preeclampsia.

Mineralocorticoid receptor (MR) blockers are very beneficial for patients with hypertension and primary aldosteronism (PA). We investigated the efficacy and safety of a newly available nonsteroidal MR blocker, esaxerenone, in Japanese patients with hypertension and PA. A multicenter, open-label study was conducted in Japan between October 2016 and July 2017. Patients with hypertension and PA received 12 weeks of treatment with esaxerenone, initiated at 2.5 mg/day and escalated to 5 mg/day during week 2 or 4 of treatment, based on individual response. The only other permitted antihypertensive therapies were stable dosages of a Ca2+ channel blocker or α-blocker. The primary efficacy outcome was a change in sitting systolic and diastolic blood pressure (SBP/DBP) from baseline to the end of treatment. Forty-four patients were included; dose escalation to 5 mg/day was implemented for 41 of these patients. Significant decreases in SBP and DBP were observed (point estimates [95% confidence interval] -17.7 [-20.6, -14.7] and -9.5 [-11.7, -7.3] mmHg, respectively; both p < 0.0001 at the end of treatment). Significant BP reductions were evident from week 2 and continued through to week 8; BP remained stable until week 12. The antihypertensive effect of esaxerenone on SBP was significantly greater in females and in patients receiving monotherapy. The major drug-related adverse events were serum K+ increase and estimated glomerular filtration rate decrease (both 4.5%, n = 2); no gynecomastia or breast pain was observed. We conclude that esaxerenone is a potent MR blocker with favorable efficacy and safety profiles in patients with hypertension and PA.

Preeclampsia is a hypertensive disorder in pregnancy characterized by placental malperfusion and subsequent multi-organ injury. It accounts for approximately 14% of maternal deaths and 10-25% of perinatal deaths globally. In addition, preeclampsia has been attracting attentions for its association with risks for developing chronic diseases in later life for both mother and child. This mini-review discusses on latest knowledge on prediction, prevention, management, and long-term outcomes of preeclampsia and also touches on association between COVID-19 and preeclampsia. HTN hypertension, HDP hypertensive disorders of pregnancy, PE preeclampsia, BP blood pressure, cfDNA cell-free DNA, ST2 human suppression of tumorigenesis 2, sFlt-1 soluble fms-like tyrosine kinase-1, PIGF placental growth factor, VEGF vascular endothelial growth factor, VEGFR VEGF receptor, TGFβ transforming growth factor β, ENG endoglin, sENG soluble ENG, PRES posterior reversible encephalopathy syndrome, AKI acute kidney injury, CVD cardiovascular disease, ESKD end-stage kidney disease, ACE angiotensinogen converting enzyme, Ang angiotensin.

Rapid right ventricular pacing could induce congestive heart failure in conscious dogs with significant increase in plasma concentration of arginine vasopressin (AVP) (from 1.2 +/- 0.2 to 3.4 +/- 0.6 pg/ml). In this experimental model of heart failure, oral administration of the selective AVP V1 receptor antagonist OPC-21268 significantly increased cardiac output and improved renal function without significant changes in serum electrolytes and hormones. Oral administration of the selective AVP V2 receptor antagonist OPC-31260 induced marked water diuresis, which resulted in significant increases in serum sodium concentration, plasma renin activity, and plasma concentration of AVP, although it did not produce hemodynamic improvement. Combined administration of OPC-21268 and OPC-31260 showed supra-additive hemodynamic responses as well as additive renal and metabolic responses, i.e., it showed prolonged decrease in mean arterial pressure and profound increase in cardiac output. These results suggest that AVP plays a significant role in elevation of vascular tone through V1 receptors and plays a major role in retaining free water through V2 receptors in this model of heart failure. Furthermore, combined administration of V1 and V2 receptor antagonists could induce not only metabolic and hormonal responses but also more beneficial hemodynamic responses than those observed following treatment with V1 receptor antagonist alone.

Recently, it was revealed that the inhibition of nonproteolytic activation of prorenin led to significant suppression of ocular inflammation in endotoxin-induced uveitis. The purpose of the present study was to investigate whether nonproteolytically activated prorenin plays a role in ischemia-induced retinal neovascularization.C57BL/6 neonatal mice were reared in an 80% concentration of oxygen from postnatal (P) day 7 to P12, followed by room-air breathing to P17 to induce ischemia-initiated retinal neovascularization. Tissue localization of activated prorenin and prorenin receptor was examined by immunohistochemistry. Animals received intraperitoneal injections of handle-region peptide (HRP), a decoy peptide corresponding to the handle region of prorenin, which inhibits prorenin receptor-mediated upregulation of the renin-angiotensin system (RAS). A concanavalin A lectin perfusion-labeling technique was used to evaluate the areas of physiologic and pathologic retinal new vessels and the number of leukocytes adhering to the vasculature. Retinal mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by RT-PCR and ELISA.Retinal vessels in ischemic retinopathy eyes were positive for activated prorenin and prorenin receptor. Pathologic, but not physiologic, retinal neovascularization was significantly attenuated in HRP-treated mice compared with vehicle- or control peptide-treated animals. The number of adherent leukocytes was also significantly reduced. Retinal mRNA expression and protein levels of ICAM-1, VEGF, VEGFR-1, and VEGFR-2 in ischemic retinopathy were also significantly suppressed by the application of HRP.The present findings suggest that nonproteolytic activation of prorenin selectively promotes pathologic, but not physiologic, retinal neovascularization through the inflammatory processes related to pathologic neovascularization.

In order to determine whether the ovarian follicles of the domestic cat which normally ovulates following copulation, were similar to those of the rabbit steroidogenically, the following experiments were carried out. Follicles were dissected out of ovaries from nine estrous cats throughout the year and incubated in medium alone or with luteinizing hormone (LH) or human chorionic gonadotropin (HCG). Media were removed every 15 min and stored frozen until analyzed for testosterone using established radioimmunoassay procedures. Although LH and HCG caused slight increases in testosterone production the amounts produced were much less than that produced by rabbit follicles under identical conditions. However, when follicles were incubated for a total of 8 h without removal of media, marked changes in testosterone and estradiol production occurred. These data suggest that the ovarian follicles of the estrous cat are steroidogenically active.

The receptor-associated prorenin system (RAPS) refers to pathogenic mechanisms whereby prorenin binding to its receptor activates both the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling pathways. Although we found significant involvement of angiotensin II type 1 receptor (AT1-R)-mediated inflammation in choroidal neovascularization (CNV), a central abnormality of vision-threatening age-related macular degeneration, the association of receptor-associated prorenin system with CNV has not been defined. Here, (pro)renin receptor blockade in a murine model of laser-induced CNV led to the significant suppression of CNV together with macrophage infiltration and the up-regulation of intercellular adhesion molecule-1, (ICAM-1) monocyte chemotactic protein-1, (MCP-1) vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2. To clarify the role of signal transduction via the (pro)renin receptor in CNV, we used mice in which renin-angiotensin system was deactivated by either the pharmacological blockade of AT1-R with losartan or the genetic ablation of AT1-R or angiotensinogen. Compared with wild-type controls, these mice exhibited significant reduction of CNV and macrophage infiltration, both of which were further suppressed by (pro)renin receptor blockade. The (pro)renin receptor and phosphorylated extracellular signal-regulated kinases (ERK) were co-localized in vascular endothelial cells and macrophages in CNV. (Pro)renin receptor blockade suppressed ERK activation and the production of MCP-1 and VEGF, but not ICAM-1, VEGFR-1, or VEGFR-2, in AT1-R-deficient mice with CNV and in losartan-treated microvascular endothelial cells and macrophages. These results indicate the significant contribution of RAPS to CNV pathogenesis. The receptor-associated prorenin system (RAPS) refers to pathogenic mechanisms whereby prorenin binding to its receptor activates both the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling pathways. Although we found significant involvement of angiotensin II type 1 receptor (AT1-R)-mediated inflammation in choroidal neovascularization (CNV), a central abnormality of vision-threatening age-related macular degeneration, the association of receptor-associated prorenin system with CNV has not been defined. Here, (pro)renin receptor blockade in a murine model of laser-induced CNV led to the significant suppression of CNV together with macrophage infiltration and the up-regulation of intercellular adhesion molecule-1, (ICAM-1) monocyte chemotactic protein-1, (MCP-1) vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2. To clarify the role of signal transduction via the (pro)renin receptor in CNV, we used mice in which renin-angiotensin system was deactivated by either the pharmacological blockade of AT1-R with losartan or the genetic ablation of AT1-R or angiotensinogen. Compared with wild-type controls, these mice exhibited significant reduction of CNV and macrophage infiltration, both of which were further suppressed by (pro)renin receptor blockade. The (pro)renin receptor and phosphorylated extracellular signal-regulated kinases (ERK) were co-localized in vascular endothelial cells and macrophages in CNV. (Pro)renin receptor blockade suppressed ERK activation and the production of MCP-1 and VEGF, but not ICAM-1, VEGFR-1, or VEGFR-2, in AT1-R-deficient mice with CNV and in losartan-treated microvascular endothelial cells and macrophages. These results indicate the significant contribution of RAPS to CNV pathogenesis. Although several types of organ damage are known to result from the activation of tissue renin-angiotensin system (RAS), the precise mechanism for activating tissue RAS is not fully understood. (Pro)renin receptor, a recently identified transmembrane protein consisting of 350 amino acids, interacts with prorenin to exert renin activity through the conformational change of the prorenin molecule instead of the conventional proteolysis of the prorenin prosegment achieved by processing enzymes such as cathepsin B. Since the membrane-bound (pro)renin receptor is reported to exist in the major organs but not in the circulation,1Nguyen G Delarue F Burckle C Bouzhir L Giller T Sraer JD Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1228) Google Scholar the nonproteolytic activation of prorenin is hypothesized to play a critical role in the activation of tissue, but not circulatory, RAS. 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AMD is complicated by choroidal neovascularization (CNV), leading to severe vision loss due to hemorrhage and exudation from the immature new vessels.8Ferris 3rd, FL Fine SL Hyman L Age-related macular degeneration and blindness due to neovascular maculopathy.Arch Ophthalmol. 1984; 102: 1640-1642Crossref PubMed Scopus (925) Google Scholar, 9Klein R Wang Q Klein BE Moss SE Meuer SM The relationship of age-related maculopathy, cataract, and glaucoma to visual acuity.Invest Ophthalmol Vis Sci. 1995; 36: 182-191PubMed Google Scholar Epidemiological risk factors for AMD were reported to include hypertension,10Klein R Klein BE Tomany SC Cruickshanks KJ The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam eye study.Ophthalmology. 2003; 110: 1273-1280Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar dyslipidemia,10Klein R Klein BE Tomany SC Cruickshanks KJ The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam eye study.Ophthalmology. 2003; 110: 1273-1280Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar and atherosclerosis,11van Leeuwen R Ikram MK Vingerling JR Witteman JC Hofman A de Jong PT Blood pressure, atherosclerosis, and the incidence of age-related maculopathy: the Rotterdam Study.Invest Ophthalmol Vis Sci. 2003; 44: 3771-3777Crossref PubMed Scopus (220) Google Scholar all of which are related to the metabolic syndrome. Recently, angiotensin II type 1 receptor (AT1-R) signaling has been shown to play a significant role in various pathological processes complicating the metabolic syndrome such as angiogenesis and inflammation.12Sola S Mir MQ Cheema FA Khan-Merchant N Menon RG Parthasarathy S Khan BV Irbesartan and lipoic acid improve endothelial function and reduce markers of inflammation in the metabolic syndrome: results of the irbesartan and lipoic acid in endothelial dysfunction (ISLAND) study.Circulation. 2005; 111: 343-348Crossref PubMed Scopus (261) Google Scholar, 13Ferder L Inserra F Martinez-Maldonado M Inflammation and the metabolic syndrome: role of angiotensin II and oxidative stress.Curr Hypertens Rep. 2006; 8: 191-198Crossref PubMed Scopus (91) Google Scholar, 14Vaziri ND Xu ZG Shahkarami A Huang KT Rodriguez-Iturbe B Natarajan R Role of AT-1 receptor in regulation of vascular MCP-1. IL-6, PAI-1, MAP kinase, and matrix expressions in obesity.Kidney Int. 2005; 68: 2787-2793Crossref PubMed Scopus (46) Google Scholar, 15Brasier AR Recinos 3rd, A Eledrisi MS Vascular inflammation and the renin-angiotensin system.Arterioscler Thromb Vasc Biol. 2002; 22: 1257-1266Crossref PubMed Scopus (536) Google Scholar CNV has proven to be an inflammatory disorder depending on intercellular adhesion molecule (ICAM)-1,16Sakurai E Taguchi H Anand A Ambati BK Gragoudas ES Miller JW Adamis AP Ambati J Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 2743-2749Crossref PubMed Scopus (119) Google Scholar monocyte chemotactic protein (MCP)-117Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (418) Google Scholar and vascular endothelial growth factor (VEGF).18Ishibashi T Hata Y Yoshikawa H Nakagawa K Sueishi K Inomata H Expression of vascular endothelial growth factor in experimental choroidal neovascularization.Graefes Arch Clin Exp Ophthalmol. 1997; 235: 159-167Crossref PubMed Scopus (234) Google Scholar We have recently shown that AT1-R-mediated up-regulation of these inflammatory and angiogenic molecules is required for the development of CNV19Nagai N Oike Y Izumi-Nagai K Urano T Kubota Y Noda K Ozawa Y Inoue M Tsubota K Suda T Ishida S Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.Arterioscler Thromb Vasc Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (118) Google Scholar; however, the role of (pro)renin receptor as a trigger to activate tissue RAS in CNV has not been defined. Although we have further shown that tissue RAS promoting retinal inflammation20Satofuka S Ichihara A Nagai N Yamashiro K Koto T Shinoda H Noda K Ozawa Y Inoue M Tsubota K Suzuki F Oike Y Ishida S Suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin.Invest Ophthalmol Vis Sci. 2006; 47: 2686-2692Crossref PubMed Scopus (89) Google Scholar and neovascularization21Satofuka S Ichihara A Nagai N Koto T Shinoda H Noda K Ozawa Y Inoue M Tsubota K Itoh H Oike Y Ishida S Role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization.Invest Ophthalmol Vis Sci. 2007; 48: 422-429Crossref PubMed Scopus (66) Google Scholar is activated by nonproteolytic activation of prorenin, it has not been determined whether (pro)renin receptor-mediated intracellular signaling, the other pathway of RAPS, is pathogenic in the eye. We therefore hypothesize that prorenin binding to its receptor promotes CNV by dually activating tissue RAS and RAS-independent ERK pathway via the receptor. In the present paper, we report the first evidence of significant relationship between RAPS and CNV together with underlying molecular and cellular mechanisms related to inflammation. Male C57BL/6J mice (CLEA, Tokyo, Japan) at the age of 6 to 9 weeks, age- and sex-matched AT1-R-deficient mice22Sugaya T Nishimatsu S Tanimoto K Takimoto E Yamagishi T Imamura K Goto S Imaizumi K Hisada Y Otsuka A Uchida H Sugiura M Fukuta K Fukamizu A Murakami K Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia.J Biol Chem. 1995; 270: 18719-18722Crossref PubMed Scopus (358) Google Scholar (based on the C57BL/6J strain and donated by Tanabe Seiyaku Co., Ltd., Osaka, Japan), angiotensinogen (AGT)-deficient mice23Tanimoto K Sugiyama F Goto Y Ishida J Takimoto E Yagami K Fukamizu A Murakami K Angiotensinogen-deficient mice with hypotension.J Biol Chem. 1994; 269: 31334-31337Abstract Full Text PDF PubMed Google Scholar (based on the C57BL/6J strain and purchased from YS Institute Inc, Tochigi, Japan) and Long-Evans rats (SLC, Shizuoka, Japan) were used. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. To cover the handle region (positions 11–15) of the prorenin molecule, which is the binding site of (pro)renin receptor,24Suzuki F Hayakawa M Nakagawa T Nasir UM Ebihara A Iwasawa A Ishida Y Nakamura Y Murakami K Human prorenin has “gate and handle” regions for its non-proteolytic activation.J Biol Chem. 2003; 278: 22217-22222Crossref PubMed Scopus (170) Google Scholar we designed a decoy peptide, NH2-IPLKKMPS-COOH (positions 11 to 18), as murine (pro)renin receptor blocker (PRRB) and purified it by high performance liquid chromatography, as described previously.3Ichihara A Suzuki F Nakagawa T Kaneshiro Y Takemitsu T Sakoda M Nabi AH Nishiyama A Sugaya T Hayashi M Inagami T Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice.J Am Soc Nephrol. 2006; 17: 1950-1961Crossref PubMed Scopus (250) Google Scholar The specific inhibitory action of PRRB against RAPS was confirmed in our recent in vivo data.3Ichihara A Suzuki F Nakagawa T Kaneshiro Y Takemitsu T Sakoda M Nabi AH Nishiyama A Sugaya T Hayashi M Inagami T Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice.J Am Soc Nephrol. 2006; 17: 1950-1961Crossref PubMed Scopus (250) Google Scholar, 5Ichihara A Hayashi M Kaneshiro Y Suzuki F Nakagawa T Tada Y Koura Y Nishiyama A Okada H Uddin MN Nabi AH Ishida Y Inagami T Saruta T Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (402) Google Scholar As a negative control for PRRB, we also prepared a control peptide (CP), NH2-MTRLSAE-COOH (positions 30 to 36) with an amino acid sequence outside the handle region. Laser-induced CNV is widely used as an animal model for neovascular AMD and reflects the pathogenesis of CNV-related inflammation seen in AMD. In this model, new vessels from the choroid invade the subretinal space after photocoagulation. Laser photocoagulation was performed around the optic nerve with the wavelength of 532 nm, the power of 200 mW, the duration of 100 ms and the spot size of 75 μm for mice or 100 μm for rats using a slit lamp delivery system (Novus Spectra; Lumenis, Tokyo, Japan), as described previously.17Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (418) Google Scholar Mice received intraperitoneal injections of vehicle (PBS), CP (1.0 mg/kg), PRRB (1.0 mg/kg), or losartan (2, 20, or 50 mg/kg; Cayman Chemical, Ann Arbor, MI) 1 day before photocoagulation and the treatments were continued daily until the end of the study. The present dose of PRRB is equivalent to that applied to significantly reduce retinal neovascularization in mice.21Satofuka S Ichihara A Nagai N Koto T Shinoda H Noda K Ozawa Y Inoue M Tsubota K Itoh H Oike Y Ishida S Role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization.Invest Ophthalmol Vis Sci. 2007; 48: 422-429Crossref PubMed Scopus (66) Google Scholar As for losartan, the dose of 20 mg/kg was the most potent in inhibiting CNV (data not shown) and used as the maximal-effect dose in the present data. One week after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde. Eye cups obtained by removing anterior segments were incubated with 0.5% fluorescein-isothiocyanate-isolectin B4 (Vector Laboratories, Burlingame, CA). CNV was visualized with blue argon laser on a confocal microscope (FV1000; Olympus, Tokyo, Japan). Horizontal optical sections of CNV were obtained at every 1-μm step from the surface to the deepest focal plane. The area of CNV-related fluorescence was measured by National Institutes of Health ImageJ (Bethesda, MD). The summation of the whole fluorescent area was used as the volume of CNV, as described previously.17Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (418) Google Scholar Three days after laser injury, whole-mount retinal pigment epithelium (RPE)-choroid complex was incubated with a goat polyclonal antibody against platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and a rat polyclonal antibody against F4/80 (Serotec, Oxford, UK). Alexa 488- and Alexa 546-tagged secondary antibodies (Molecular Probes, Eugene, OR) were then applied. PECAM-1-stained area of CNV and F4/80-positive macrophages were evaluated, and the volume-adjusted number of macrophages was calculated. We isolated total RNA from the RPE-choroid and performed quantitative reverse transcription (RT)-PCR with an ABI Prism 7700 HT Detection System (Applied Biosystems, Foster City, CA); and probes and primers for the rat genes that encode prorenin, (pro)renin receptor, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as described previously.5Ichihara A Hayashi M Kaneshiro Y Suzuki F Nakagawa T Tada Y Koura Y Nishiyama A Okada H Uddin MN Nabi AH Ishida Y Inagami T Saruta T Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (402) Google Scholar, 7Takahashi H Ichihara A Kaneshiro Y Inomata K Sakoda M Takemitsu T Nishiyama A Itoh H Regression of nephropathy developed in diabetes by (pro)renin receptor blockade.J Am Soc Nephrol. 2007; 18: 2054-2061Crossref PubMed Scopus (125) Google Scholar, 25Ichihara A Kaneshiro Y Takemitsu T Sakoda M Suzuki F Nakagawa T Nishiyama A Inagami T Hayashi M Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension.Hypertension. 2006; 47: 894-900Crossref PubMed Scopus (230) Google Scholar Three days after laser injury, the RPE-choroid complex was carefully isolated and placed into the lysis buffer. After blocking nonspecific binding with 5% skim milk, polyvinylidene fluoride membranes were incubated with a goat polyclonal antibody against angiotensin II (Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal antibody against phosphorylated ERK1/2 (Cell Signaling Technology, Beverly, MA), total ERK1/2 (Cell Signaling Technology) or α-tubulin (Sigma, St. Louis, MO). Membranes were then incubated with biotin-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA) followed by avidin-biotin complex (Vectastain ABC Elite Kit; Vector Laboratories). Finally the signals were detected through enhanced chemiluminescence (ECL Blotting Analysis System; GE Health Care). Protein extracts were obtained from the RPE-choroid complex 3 days after photocoagulation. The protein levels of ICAM-1, MCP-1, VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 were determined with the enzyme-linked immunosorbent assay (ELISA; R&D Systems). Three days after photocoagulation, rat eye cups were incubated with a goat anti-rat (pro)renin receptor antibody together with fluorescein-isothiocyanate-isolectin B4 (Vector Laboratories) or a rabbit anti-EMR 1 (corresponding to murine F4/80) antibody (Santa Cruz Biotechnology). The anti-(pro)renin receptor antibody was raised by using the previously established COS-7 cells producing rat (pro)renin receptor protein.5Ichihara A Hayashi M Kaneshiro Y Suzuki F Nakagawa T Tada Y Koura Y Nishiyama A Okada H Uddin MN Nabi AH Ishida Y Inagami T Saruta T Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (402) Google Scholar Alexa 488- and Alexa 546-tagged secondary antibodies (Molecular Probes) were then applied. For immunohistochemical staining of phosphorylated ERK1/2, a goat polyclonal antibody against rat phosphorylated ERK1/2 (Santa Cruz Biotechnology) was applied as the primary antibody. Murine brain-derived capillary endothelial cells (b-End3) were cultured with Dulbecco's modified Eagle's medium (Sigma). After 6-hour incubation with tumor necrosis factor(TNF)-α (Sigma, 1 ng/ml) plus losartan (10 μmol/L), or TNF-α plus PRRB (100 μmol/L) and losartan (10 μmol/L), the supernatant and cell lysate were collected for protein analyses, and then the concentration of MCP-1 in the supernatant and ICAM-1, VEGFR-1 and VEGFR-2 in the cell lysate were measured by the ELISA kits (R&D Systems). Murine macrophages (RAW264.7) were treated with Dulbecco's modified Eagle's medium containing lipopolysaccharide (100 ng/ml) plus losartan (10 μmol/L) or lipopolysaccharide plus PRRB (100 μmol/L) and losartan (10 μmol/L). After 6-hour incubation, supernatant was processed for ELISA analyses for VEGF (R&D Systems). All results were expressed as mean ± SEM. The values were processed for statistical analyses (Mann-Whitney test). Differences were considered statistically significant when the P values were <0.05. To elucidate the involvement of prorenin and (pro)renin receptor in the pathogenesis of CNV, we first performed quantitative RT-PCR analyses for prorenin and (pro)renin receptor in the RPE-choroid complex. Prorenin mRNA levels (ratio to GAPDH mRNA) were up-regulated (P < 0.05) in the RPE-choroid of laser-treated rats, compared with age-matched normal controls (Figure 1A). In contrast, mRNA levels of (pro)renin receptor showed no significant difference (P > 0.05) between laser-treated rats and normal controls (Figure 1B). The CNV volume was measured to evaluate the effects of PRRB on the development of CNV. CP treatment did not significantly (P > 0.05) change the CNV volume (501,810 ± 66,820 μm3), compared with vehicle-treated animals (504,411 ± 49,791 μm3) (Figure 1, C and D). However, PRRB-treated mice showed a significant (P < 0.01) decrease in the CNV volume (208,355 ± 29,388 μm3), compared with vehicle-treated mice (504,411 ± 49,791 μm3) or CP-treated mice (501,810 ± 66,820 μm3) (Figure 1, C and D). As the cellular mechanism in the pathogenesis of CNV, infiltration of inflammatory cells including macrophages plays a critical role. We compared the number of macrophages, which was adjusted by the area of CNV, between mice treated with PRRB versus vehicle. PRRB-treated mice showed a significant decrease in the number of F4/80-positive macrophages, compared with vehicle-treated animals (P < 0.01, 2.84 ± 0.31/10,000 μm3 versus 4.39 ± 0.41/10,000 μm3) (Figure 2, A and B). To investigate the effect of PRRB on angiotensin II generation during CNV, we analyzed angiotensin II levels in the RPE-choroid complex. RPE-choroidal levels of angiotensin II were higher (P < 0.01) in animals with CNV than in age-matched normal controls (Figure 2, C and D). Application of PRRB significantly suppressed protein levels of angiotensin II in the RPE-choroid (P < 0.05) (Figure 2, C and D). To determine whether PRRB affects angiogenic and inflammatory molecules related to the pathogenesis of CNV, protein levels of ICAM-1, MCP-1, VEGF, VEGFR-1, and VEGFR-2 in the RPE-choroid complex were analyzed by ELISA. RPE-choroidal protein levels of ICAM-1, MCP-1, VEGF, VEGFR-1, and VEGFR-2 were up-regulated by inducing CNV. Systemic administration of PRRB significantly suppressed protein levels of ICAM-1 (P < 0.01), MCP-1 (P < 0.05), VEGF (P < 0.05), VEGFR-1 (P < 0.05), and VEGFR-2 (P < 0.01) (Figure 2, E–I). To clarify the role of RAS-independent intracellular signaling via (pro)renin receptor, we used mice in which RAS was deactivated by pharmacological blockade of AT1-R with losartan or genetic ablation of AT1-R or AGT. Compared with vehicle-treated wild-type animals (504,411 ± 49,791 μm3), these mice exhibited a significant (P < 0.01) reduction of CNV (305,244 ± 37,883 μm3 for losartan treatment, 314,120 ± 34,023 μm3 for AT1-R deficiency, 278,811 ± 30,462 μm3 for AGT deficiency), which was further suppressed by additional PRRB application (212,643 ± 38,779 μm3 for losartan treatment [P < 0.01], 198,206 ± 15,536 μm3 for AT1-R deficiency [P < 0.05], 163,457 ± 23,767 μm3 for AGT deficiency [P < 0.01]) (Figure 3, A and B). We further examined the role of RAS-independent intracellular signaling via (pro)renin receptor in macrophage infiltration into CNV. Compared with wild-type animals (4.39 ± 0.41/10,000 μm3), AGT-deficient mice exhibited a significant (P < 0.05) decrease in the number of F4/80-positive macrophages (2.85 ± 0.38/10,000 μm3), which was further attenuated by PRRB treatment (1.89 ± 0.31/10,000 μm3, P < 0.05) (Figure 3, C and D). To examine the expression and tissue localization of (pro)renin receptor and phosphorylated ERK1/2, a known downstream pathway via (pro)renin receptor, rat CNV tissues were immunostained with antibodies against (pro)renin receptor and phosphorylated ERK1/2 together with isolectin B4 or an anti-EMR 1 antibody, markers for vascular endothelial cells or macrophages, respectively. The immunohistochemical analyses of rat CNV tissues showed (pro)renin receptor immunoreactivity in isolectin B4-positive endothelial cells (Figure 4A) and EMR1-positive macrophages (Figure 4B), both of which were positive for phosphorylated ERK1/2. We examined the effect of PRRB on the activation of ERK1/2 in the RPE-choroid excised from mice with CNV. The relative ratio of phosphorylated to total ERK1/2 in the RPE-choroid, increased by inducing CNV, was significantly (P < 0.01) suppressed by PRRB treatment (Figure 4, C and D), while no significant (P > 0.05) difference was detected in total ERK1/2 protein levels. To further determine whether RAS-independent intracellular signaling via (pro)renin receptor contributes to the activation of ERK1/2 in CNV, phosphorylated ERK1/2 was examined in the RPE-choroid from AT1-R-deficient mice with CNV. PRRB treatment significantly (P < 0.05) suppressed phosphorylated but not total ERK1/2 (Figure 5A). Relative phosphorylation of ERK1/2 was significantly (P < 0.05) suppressed by PRRB application, compared with vehicle-treated AT1-R-deficient mice (Figure 5B). To examine whether RAS-independent intracellular signaling via (pro)renin receptor contributes to the up-regulation of the inflammatory and angiogenic molecules responsible for CNV (Figure 2, E–I), CNV was induced in AT1-R-deficient mice to measure protein levels of ICAM-1, MCP-1, VEGF, VEGFR-1, and VEGFR-2 in the RPE-choroid. PRRB application to AT1-R-deficient mice with CNV led to significant suppression of MCP-1 (P < 0.01) (Figure 5C) and VEGF (P < 0.05) (Figure 5D), but not ICAM-1, VEGFR-1, or VEGFR-2 (P > 0.05, data not shown), compared with vehicle treatment to AT1-R-deficient mice with CNV. To confirm the in vivo molecular mechanisms mediated by RAS-independent pathway via (pro)renin receptor (Figure 5, C and D), we further performed in vitro analyses, using murine cell lines including b-End3 microvascular endothelial cells (Figure 5E) and RAW264.7 macrophages (Figure 5F), both of which were treated by losartan. We analyzed protein levels of ICAM-1, MCP-1, VEGFR-1, and VEGFR-2 inTNF-α-stimulated endothelial cells and of VEGF in lipopolysaccharide-stimulated macrophages. In losartan-treated endothelial cells, protein levels of MCP-1 were significantly (P < 0.01) suppressed by the treatment with PRRB (Figure 5E). In contrast, no significant difference was detected in protein levels of ICAM-1, VEGFR-1, or VEGFR-2 following PRRB treatment (data not shown). In losartan-treated macrophages, VEGF protein levels were significantly (P < 0.05) suppressed by the treatment with PRRB (Figure 5F). The present study reveals, for the first time to our knowledge, several important findings concerning the role of (pro)renin receptor in CNV generation. First, CNV development was associated with up-regulation of prorenin expression in the RPE-choroid complex and PRRB treatment showed a significant decrease in the CNV volume, indicating that prorenin binding with its receptor contributes to CNV (Figure 1). Second, the cellular and molecular mechanisms in the PRRB-induced suppression of CNV included the inhibitory effects on macrophage infiltration into CNV, angiotensin II generation and the up-regulated expression of inflammatory and angiogenic molecules such as ICAM-1, MCP-1, VEGF, VEGFR-1, and VEGFR-2, all of which were downstream molecules of angiotensin II19Nagai N Oike Y Izumi-Nagai K Urano T Kubota Y Noda K Ozawa Y Inoue M Tsubota K Suda T Ishida S Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.Arterioscler Thromb Vasc Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (118) Google Scholar (Figure 2). Although the detailed molecular and cellular mechanisms underlying CNV are not fully clarified, ICAM-1 expression16Sakurai E Taguchi H Anand A Ambati BK Gragoudas ES Miller JW Adamis AP Ambati J Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 2743-2749Crossref PubMed Scopus (119) Google Scholar, 26Yeh DC Bula DV Miller JW Gragoudas ES Arroyo JG Expression of leukocyte adhesion molecules in human subfoveal choroidal neovascular membranes treated with and without photodynamic therapy.Invest Ophthalmol Vis Sci. 2004; 45: 2368-2373Crossref PubMed Scopus (43) Google Scholar and macrophage infiltration18Ishibashi T Hata Y Yoshikawa H Nakagawa K Sueishi K Inomata H Expression of vascular endothelial growth factor in experimental choroidal neovascularization.Graefes Arch Clin Exp Ophthalmol. 1997; 235: 159-167Crossref PubMed Scopus (234) Google Scholar, 19Nagai N Oike Y Izumi-Nagai K Urano T Kubota Y Noda K Ozawa Y Inoue M Tsubota K Suda T Ishida S Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.Arterioscler Thromb Vasc Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (118) Google Scholar were observed in CNV tissues from human eyes with AMD and the laser-induced murine model, suggesting the close association of inflammation with the progression of CNV. Pharmacological depletion of macrophages17Sakurai E Anand A Ambati BK van Rooijen N Ambati J Macrophage depletion inhibits experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3578-3585Crossref PubMed Scopus (418) Google Scholar, 27Espinosa-Heidmann DG Suner IJ Hernandez EP Monroy D Csaky KG Cousins SW Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization.Invest Ophthalmol Vis Sci. 2003; 44: 3586-3592Crossref PubMed Scopus (338) Google Scholar or genetic ablation of CCR2,28Tsutsumi C Sonoda KH Egashira K Qiao H Hisatomi T Nakao S Ishibashi M Charo IF Sakamoto T Murata T Ishibashi T The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization.J Leukoc Biol. 2003; 74: 25-32Crossref PubMed Scopus (239) Google Scholar a receptor for MCP-1, was shown to result in the reduction of CNV, suggesting that macrophages, recruited by MCP-1 released from RPE or vascular endothelial cells, facilitate the development of CNV by producing VEGF. In concert with our previous data,19Nagai N Oike Y Izumi-Nagai K Urano T Kubota Y Noda K Ozawa Y Inoue M Tsubota K Suda T Ishida S Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.Arterioscler Thromb Vasc Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (118) Google Scholar the currently observed PRRB-induced suppression of CNV indicates that tissue RAS is activated during CNV by (pro)renin receptor-mediated nonproteolytic activation of prorenin, leading to AT1-R signaling-mediated up-regulation of CNV-related inflammatory molecules. The present study further revealed the role of RAS-independent (pro)renin receptor signaling in CNV generation (Figure 3). This study is the first to show the involvement of RAPS, (pro)renin receptor-mediated signal transduction and tissue RAS activation, in in vivo angiogenesis as well as in ocular pathogenesis. We recently showed the contribution of (pro)renin receptor signaling to diabetic nephropathy using AT1-R-deficient mice.3Ichihara A Suzuki F Nakagawa T Kaneshiro Y Takemitsu T Sakoda M Nabi AH Nishiyama A Sugaya T Hayashi M Inagami T Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice.J Am Soc Nephrol. 2006; 17: 1950-1961Crossref PubMed Scopus (250) Google Scholar AT1-R-deficient mice with streptozotocin-induced diabetes exhibited reduced proteinuria and glomerulosclerosis in the early phase as compared to wild-type diabetes, indicating a significant role of tissue RAS in diabetic nephropathy. Surprisingly, these renal events in AT1-R-deficient diabetes later progressed to the equivalent levels seen in wild-type diabetic mice. The glomerulosclerosis observed in AT1-R-deficient diabetic mice was associated with ERK activation, which was completely blocked together with the phenotype by sustained application of PRRB, suggesting that the redundant pathways of RAPS were involved in the pathogenesis of diabetic nephropathy. In the present study, we administered PRRB to CNV mice receiving the AT1-R blocker losartan or genetically deficient in AT1-R or AGT, and these three different methods for deactivating RAS confirmed the significant role of intracellular signaling via (pro)renin receptor in the development of CNV (Figure 3, A and B). The data are compatible with the result of parallel experiments showing that macrophage infiltration into CNV was also suppressed by PRRB in AGT-deficient mice (Figure 3, C and D). Importantly, (pro)renin receptor was present in macrophages and vascular endothelial cells, the major cellular components in CNV tissues, together with the activation of ERK 1/2, a known intracellular signaling via (pro)renin receptor (Figure 4, A and B). Indeed, in vivo quantitative analyses for ERK 1/2 revealed PRRB-induced suppression of the phosphorylation of ERK1/2, which was enhanced following CNV induction (Figure 4, C and D). However, because AT1-R has been shown to mediate CNV generation,19Nagai N Oike Y Izumi-Nagai K Urano T Kubota Y Noda K Ozawa Y Inoue M Tsubota K Suda T Ishida S Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.Arterioscler Thromb Vasc Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (118) Google Scholar the data (Figure 4) could not exclude the possibility of ERK activation via AT1-R as well as (pro)renin receptor, leading us to further perform the following in vivo and in vitro experiments (Figure 5) to confirm that (pro)renin receptor signaling per se caused the activation of ERK and up-regulation of inflammatory molecules responsible for CNV formation. Importantly, PRRB application to AT1-R-deficient mice with CNV led to significant suppression of ERK activation (Figure 5, A and B). Out of the CNV-related molecules, the expression of which was inhibited by PRRB (Figure 2, E–I), our in vivo (Figure 5, C and D) and in vitro (Figure 5, E and F) data showed that MCP-1 and VEGF were also regulated by (pro)renin receptor signaling per se. These new findings (Figure 3, Figure 4, Figure 5) clarified molecular and cellular mechanisms mediated by RAS-independent intracellular signaling via (pro)renin receptor in CNV generation. In addition to our recent reports3Ichihara A Suzuki F Nakagawa T Kaneshiro Y Takemitsu T Sakoda M Nabi AH Nishiyama A Sugaya T Hayashi M Inagami T Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice.J Am Soc Nephrol. 2006; 17: 1950-1961Crossref PubMed Scopus (250) Google Scholar, 5Ichihara A Hayashi M Kaneshiro Y Suzuki F Nakagawa T Tada Y Koura Y Nishiyama A Okada H Uddin MN Nabi AH Ishida Y Inagami T Saruta T Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (402) Google Scholar, 6Ichihara A Kaneshiro Y Takemitsu T Sakoda M Nakagawa T Nishiyama A Kawachi H Shimizu F Inagami T Contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage.J Am Soc Nephrol. 2006; 17: 2495-2503Crossref PubMed Scopus (129) Google Scholar, 25Ichihara A Kaneshiro Y Takemitsu T Sakoda M Suzuki F Nakagawa T Nishiyama A Inagami T Hayashi M Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension.Hypertension. 2006; 47: 894-900Crossref PubMed Scopus (230) Google Scholar showing that RAPS contributes to glomerulosclerosis in the kidney and fibrosis in the heart, the present data are the first to show the association of RAPS with inflammation and angiogenesis in the eye. Although hypertension is a known risk factor predisposing to AMD, there are indeed a large number of normotensive patients with CNV who have the potential risk of hypotension caused by the use of antihypertensive agents including AT1-R blockers and angiotensin-converting enzyme inhibitors. In contrast, since (pro)renin receptor is present in the major organs but not in the circulation, PRRB does not affect circulatory RAS or systemic blood pressure.5Ichihara A Hayashi M Kaneshiro Y Suzuki F Nakagawa T Tada Y Koura Y Nishiyama A Okada H Uddin MN Nabi AH Ishida Y Inagami T Saruta T Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (402) Google Scholar, 7Takahashi H Ichihara A Kaneshiro Y Inomata K Sakoda M Takemitsu T Nishiyama A Itoh H Regression of nephropathy developed in diabetes by (pro)renin receptor blockade.J Am Soc Nephrol. 2007; 18: 2054-2061Crossref PubMed Scopus (125) Google Scholar Interestingly, PRRB treatment to CNV was shown to cause not only tissue RAS deactivation but also additional suppression of (pro)renin receptor signaling-mediated expression of MCP-1 and VEGF, the major pathogenic factors responsible for CNV formation. Collectively, inhibition of RAPS with PRRB may prove more useful as a novel therapeutic strategy for CNV than RAS suppression with conventional AT1-R blockers or angiotensin-converting enzyme inhibitors. Prorenin and the (Pro)renin Receptor in Ocular PathologyThe American Journal of PathologyVol. 173Issue 6PreviewThis commentary reports on the protective effects of a (pro)renin receptor inhibitor in an experimental model of age-related macular degeneration. Full-Text PDF

The role of the prorenin receptor (PRR) in the regulation of ureteric bud (UB) branching morphogenesis is unknown. Here, we investigated whether PRR acts specifically in the UB to regulate UB branching, kidney development and function. We demonstrate that embryonic (E) day E13.5 mouse metanephroi, isolated intact E11.5 UBs and cultured UB cells express PRR mRNA. To study its role in UB development, we conditionally ablated PRR in the developing UB (PRR (UB-/-)) using Hoxb7 (Cre) mice. On E12.5, PRR (UB-/-) mice had decreased UB branching and increased UB cell apoptosis. These defects were associated with decreased expression of Ret, Wnt11, Etv4/Etv5, and reduced phosphorylation of Erk1/2 in the UB. On E18.5, mutants had marked kidney hypoplasia, widespread apoptosis of medullary collecting duct cells and decreased expression of Foxi1, AE1 and H(+)-ATPase α4 mRNA. Ultimately, they developed occasional small cysts in medullary collecting ducts and had decreased nephron number. To test the functional consequences of these alterations, we determined the ability of PRR (UB-/-) mice to acidify and concentrate the urine on postnatal (P) day P30. PRR (UB-/-) mice were polyuric, had lower urine osmolality and a higher urine pH following 48 hours of acidic loading with NH4Cl. Taken together, these data show that PRR present in the UB epithelia performs essential functions during UB branching morphogenesis and collecting duct development via control of Ret/Wnt11 pathway gene expression, UB cell survival, activation of Erk1/2, terminal differentiation and function of collecting duct cells needed for maintaining adequate water and acid-base homeostasis. We propose that mutations in PRR could possibly cause renal hypodysplasia and renal tubular acidosis in humans.

Background It is widely acknowledged that the (pro)renin receptor mediates angiotensin (Ang) II-dependent and Ang II-independent effects of prorenin. Method We examined the effect of prorenin on vascular smooth muscle cell (VSMC) signal transduction, proliferation, and hypertrophy. Results Recombinant rat prorenin dose-dependently increased extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylation in rat VSMCs. Prorenin also significantly increased cell number, and [3H]-thymidine and [3H]-leucine incorporation, which were attenuated by pretreatment with inhibitors for ERK kinase and phosphatidylinositol 3 kinase. Prorenin was also found to stimulate epidermal growth factor (EGF) receptor and Src phosphorylation. Pretreatment of VSMCs with an EGF receptor tyrosine kinase inhibitor and a Src inhibitor significantly attenuated the prorenin-induced increase in ERK 1/2 and Akt phosphorylation, as well as DNA and protein synthesis. Prorenin-induced phosphorylation of the EGF receptor, ERK 1/2, and Akt, as well as DNA and protein synthesis were all blocked by (pro)renin receptor siRNA, but not by an Ang II type 1 receptor blocker, candesartan, nor an Ang-converting enzyme inhibitor, captopril. Conclusion These results reveal that prorenin directly stimulates VSMC proliferative and hypertrophic changes, dependent on the (pro)renin receptor, independent of Ang II. Furthermore, EGF receptor-mediated ERK 1/2 and Akt activation contributes to prorenin-dependent proliferative and hypertrophic effects in VSMCs.

To assess the efficacy, safety, and pharmacokinetics of metyrosine (an inhibitor of catecholamine synthesis) in patients with pheochromocytoma/paraganglioma (PPGL), we conducted a prospective, multi-center, open-label study at 11 sites in Japan. We recruited PPGL patients aged ≥12 years requiring preoperative or chronic treatment, receiving α-blocker treatment, having baseline urinary metanephrine (uMN) or normetanephrine (uNMN) levels ≥3 times the upper limit of normal values, and having symptoms associated with excess catecholamine. Metyrosine treatment was started at 500 mg/day and modified according to dose-adjustment criteria up to 4,000 mg/day. The main outcome measure was the proportion of patients who achieved at least 50% reduction in uMN or uNMN levels from baseline. Sixteen patients (11 males/5 females) aged 12-86 years participated. After 12 weeks of treatment and at the last evaluation of efficacy, the primary endpoint was achieved in 31.3% of all patients, including 66.7% of those under preoperative treatment and 23.1% of those under chronic treatment. Sedation, anemia, and death were reported in 1 patient each as serious adverse drug reactions during the 24-week treatment. Metyrosine was shown to be tolerated and to relieve symptoms by reducing excess catecholamine in PPGL patients under both preoperative and chronic treatment.

In 2021, 217 excellent manuscripts were published in Hypertension Research. Editorial teams greatly appreciate the authors' contribution to hypertension research progress. Here, our editorial members have summarized twelve topics from published work and discussed current topics in depth. We hope you enjoy our special feature, "Update on Hypertension Research in 2021".

To delineate the microvascular role of cyclooxygenase-2 (Cox-2) in modulating tubuloglomerular feedback (TGF) signals and to determine its relationship to neuronal nitric oxide synthase (nNOS), afferent (AA) and efferent (EA) arteriolar diameters of rat kidneys were assessed using the blood-perfused juxtamedullary nephron technique. The Cox-2 inhibitor NS-398 (10 μM) did not alter AA diameters in untreated kidneys but significantly constricted AAs by 17.0 ± 2.2% in kidneys treated with 10 mM acetazolamide, which enhances TGF-mediated AA constriction by increasing distal volume delivery. The NS-398-induced AA constriction was prevented after interruption of distal delivery by transection of the loops of Henle. The effect was selective for AAs since NS-398 did not influence EAs of untreated or acetazolamide-treated kidneys. Pretreatment with the nNOS inhibitor S-methyl-l-thiocitrulline (10 μM) prevented the NS-398-induced AA constriction observed during acetazolamide treatment. Although we previously demonstrated that acetazolamide treatment enhanced AA constrictor response to S-methyl-l-thiocitrulline, the enhancement by acetazolamide was inhibited by pretreatment with 10 μM NS-398 (16.4 ± 1.9 and 15.0 ± 0.5% with and without acetazolamide, respectively, P &gt; 0.05). These results indicate that, during increased activation of TGF-dependent vasoconstrictor signals, Cox-2 generates vasodilatory metabolites in response to increased nNOS activity and thus participates in the counteracting modulation of TGF-mediated AA constriction.

Prorenin binding to the (pro)renin receptor not only causes a nonproteolytic activation of prorenin leading to the activation of the renin-angiotensin system (RAS), but also stimulates the receptor's own intracellular signaling pathways independent of the RAS. Within the kidneys, the (pro)renin receptor is present in the glomerular mesangium and podocytes, which play an important role in the maintenance of the glomerular filtration barrier. Therefore, prorenin-receptor blockers, which competitively bind to the receptor as a decoy peptide, have superior benefits with regard to proteinuria and glomerulosclerosis in experimental animal models with elevated plasma prorenin levels such as diabetes and hypertension compared with conventional RAS inhibitors, possibly by inhibiting both the nonproteolytic activation of prorenin and RAS-independent intracellular signals.

For determining the effects of anandamide (ANA) on renal hemodynamics and microcirculation, a clearance study was performed in Sprague-Dawley rats that received injections of ANA in doses of 15, 150, and 1500 pmol/kg. At doses up to 150 pmol/g, ANA significantly decreased GFR and increased renal blood flow (RBF) without affecting mean arterial pressure (MAP). In the presence of the cannabinoid type 1 (CB1) receptor antagonist AM251, only the 15-pmol/kg dose significantly increased GFR and RBF without altering MAP, with higher doses having no effect on GFR, RBF, or MAP. By contrast, AM281, which antagonizes cannabinoid receptors nonselectively, inhibited the GFR, RBF, and MAP responses to ANA. The arteriolar responses to ANA were also assessed in vitro by the blood-perfused juxtamedullary nephron technique. Higher doses of ANA significantly increased the diameter of both afferent and efferent arterioles, whereas lower doses elicited predominant efferent arteriolar dilation. AM251 attenuated the afferent arteriolar response to ANA and inhibited the efferent arteriolar response to ANA, whereas AM281 inhibited the responses in both arterioles. The CB1 receptor mRNA was expressed in afferent arterioles, and immunohistochemical staining demonstrated the presence of CB1 receptors in both afferent and efferent arterioles. These results suggest that ANA causes afferent arteriolar dilation via both CB1 and non-CB1 receptors and greater efferent arteriolar dilation via CB1 receptors, resulting in a decreased GFR and an increased RBF without affecting MAP.

Recent studies have shown that blocking non‐proteolytically activated prorenin with a decoy peptide for the handle region of the prorenin prosegment (HRP) inhibits the development of microvascular complications in diabetic animals. In the present study, we investigated whether non‐proteolytic activation of prorenin contributes to the development of fructose‐induced insulin resistance. Rats were fed a standard diet ( n = 10), a 60% high fructose diet ( n = 16), or a high fructose diet + HRP (0.1 mg kg −1 day −1 , n = 16) for 10 weeks. Fructose‐fed rats showed higher systolic blood pressure (SBP), fasting plasma triglycerides, total cholesterol and insulin levels; which, except for SBP, were suppressed by HRP. The responses of plasma glucose and insulin levels to oral glucose loading were significantly greater in fructose‐fed rats than in standard diet‐fed rats. The HRP normalized the enhanced responses of plasma glucose and insulin levels that were observed in fructose‐fed rats. Moreover, HRP suppressed the enhanced prorenin activation and angiotensin II formation in the soleus muscle of fructose‐fed rats. These data suggest that local angiotensin II generation in skeletal muscle, induced by non‐proteolytic activation of prorenin, contributes to the development of insulin resistance induced by a high fructose diet.

An important role of the decoy peptide sequence has recently been suggested in vitro for the binding of prorenin to the (pro)renin receptor [(P)RR]. In this study, other prospective crucial regions in renin and prorenin responsible for their interaction with (P)RR were investigated using various kinds of peptides, e.g., the “hinge” S149QGVLKEDVF158 designed from the structure of renin also common to prorenin, L1PPTDTTTF8P, L1PPTDTTTFKRIFLKR15P and the decoy (R10PIFLKRMPSI19P) designed from the predicted structure of prorenin. For the kinetic analysis, the recombinant h(P)RR was immobilized on the biosensor surface through a specific anti-(P)RR antibody. In case of the equilibrium state analysis, the (P)RR was directly adsorbed on plastic wells for observing the bindings of renin/prorenin. The dissociation constants (KD) for the bindings of renin and prorenin to the pre-adsorbed receptors were 4.5 and 1.0 nM, respectively, similar to those stated in the kinetic study by BIAcore assay. The “hinge” region peptide bound to (P)RR in a dose-dependent manner with a KD estimated 17.0 nM which was five times higher than that of the decoy. The KD values for L1PPTDTTTF8P and L1PPTDTTTFKRIFLKR15P were 52 and 7.6 nM, respectively. The “hinge” peptide, as the decoy, inhibited the bindings of renin and prorenin to (P)RR. The inhibition constant (Ki) for the binding of renin and prorenin by the decoy and “hinge” were 16.7 and 15.1, and 37.1 and 30.7 nM, respectively. These in vitro studies suggest that renin has a single and prorenin has at least two high affinity binding sites for the (P)RR.

&lt;i&gt;Background/Aims:&lt;/i&gt; This study was conducted to determine the effect of telmisartan on the cardio-ankle vascular index (CAVI), a novel blood pressure (BP)-independent marker for arterial stiffness in hypertensive patients. &lt;i&gt;Methods:&lt;/i&gt; One hundred consecutive hypertensive patients were randomly assigned either to a group treated with calcium channel blocker (CCB)-based therapy or a group treated with telmisartan-based therapy. Clinical and biological parameters were then measured before and 12 months after the start of this study. &lt;i&gt;Results:&lt;/i&gt; CAVI, the logarithm of urinary albumin excretion, and BP were reduced significantly after telmisartan-based therapy. The decreases in 24-hour diastolic BP and daytime systolic BP associated with telmisartan-based therapy were significantly greater than those associated with CCB-based therapy. Both therapies significantly and similarly decreased the clinical BP, 24-hour systolic BP, daytime diastolic BP and serum levels of low-density lipoprotein cholesterol. No significant differences in the metabolic parameters were observed between the two therapies. &lt;i&gt;Conclusion:&lt;/i&gt; Telmisartan-based therapy had beneficial effects on arterial stiffness assessed by CAVI, albuminuria, 24-hour BP and metabolism compared with CCB-based therapy. Since these markers are known to influence the future risk of cardiovascular events, telmisartan could be a useful drug for hypertensive patients.

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H+-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

We have reported previously that thyroid hormone activates the circulating and tissue renin-angiotensin systems without involving the sympathetic nervous system, which contributes to cardiac hypertrophy in hyperthyroidism. This study examined whether the circulating or tissue renin-angiotensin system plays the principal role in hyperthyroidism-induced cardiac hypertrophy. The circulating renin-angiotensin system in Sprague-Dawley rats was fixed by chronic angiotensin II infusion (40 ng/min, 28 days) via mini-osmotic pumps. Daily i.p. injection of thyroxine (0.1 mg/kg per day, 28 days) was used to mimic hyperthyroidism. Serum free tri-iodothyronine, plasma renin activity, plasma angiotensin II, cardiac renin and cardiac angiotensin II were measured with RIAs. The cardiac expression of renin mRNA was evaluated by semiquantitative reverse transcriptase-polymerase chain reaction. Plasma renin activity and plasma angiotensin II were kept constant in the angiotensin II and angiotensin II+thyroxine groups (0.12+/-0.03 and 0.15+/-0.03 microgram/h per liter, 126+/-5 and 130+/-5 ng/l respectively) (means+/-s.e.m.). Despite stabilization of the circulating renin-angiotensin system, thyroid hormone induced cardiac hypertrophy (5.0+/-0.5 vs 3.5+/-0.1 mg/g) in conjunction with the increases in cardiac expression of renin mRNA, cardiac renin and cardiac angiotensin II (74+/-2 vs 48+/-2%, 6.5+/-0.8 vs 3.8+/-0.4 ng/h per g, 231+/-30 vs 149+/-2 pg/g respectively). These results indicate that the local renin-angiotensin system plays the primary role in the development of hyperthyroidism-induced cardiac hypertrophy.

Aortic stiffness is assessed by pulse wave velocity (PWV) and predicts the cardiovascular morbidity and mortality of hypertensive patients. To determine the long-term effects of intensive blood pressure (BP) lowering by antihypertensive drug therapy on aortic stiffness assessed by PWV, a single-blind randomized prospective study was performed.One hundred forty nondiabetic hypertensive patients (67.6 +/- 0.9 years old; systolic/diastolic BP: 177 +/- 1/101 +/- 1 mm Hg) were assigned to an intensive control group (IC) with a target BP of <130/85 mm Hg (n = 71) or a moderate control group (MC) with a target BP of <140/90 mm Hg (n = 69), and aortic stiffness was assessed every 3 months by measuring aortic PWV with a pulse pressure analyzer.During the 12-month treatment period, BP significantly decreased to 129 +/- 1/78 +/- 1 mm Hg and 152 +/- 2/87 +/- 1 mm Hg in the IC and MC, respectively. At the beginning of the study, PWV in the IC and MC was similar, averaging 1779 +/- 41 and 1885 +/- 50 cm/sec, respectively. By the end of the treatment period, however, PWV had decreased to 1621 +/- 34 cm/sec in the IC, but had not changed significantly in the MC. In the IC, the ratio of the change in PWV to the change in BP increased with the duration of BP lowering. Clinical and biological parameters were similar in both groups, except that higher doses of amlodipine were used in the IC.Long-term intensive BP lowering in the hypertensive patients was associated with a significant reduction in aortic stiffness distinct from its acute depressor effect.

Background Patients with kidney failure treated with hemodialysis have a high incidence of cardiovascular diseases caused by accelerated arteriosclerosis. However, accurate evaluation of the extent of arteriosclerosis is difficult. This study sought to compare the strength of predictions of arterial fibrosis by using a new parameter, the cardio-ankle vascular index (CAVI), versus pulse wave velocity (PWV) in patients with kidney failure treated with hemodialysis. Study Design Diagnostic test study. Setting & Participants 103 patients with kidney failure undergoing surgical construction of an arteriovenous access for hemodialysis therapy. Index Test CAVI and PWV. Reference Test Arterial fibrosis, evaluated by using Masson trichrome stain on part of the brachial artery obtained during surgery, expressed as percentage of fibrosis of the layer of vascular smooth muscle cells. Results Median percentage of arterial stiffness was 52.85%. Mean PWV and CAVI were 18.3 ± 5.6 (SD) m/s and 9.9 ± 2.6, respectively. Multivariate regression analysis showed that arterial fibrosis was significantly associated with older age (0.247%/y; 95% confidence interval, 0.013 to 0.482) and CAVI (6.117%/unit; 95% confidence interval, 4.764 to 4.740), but not with systolic blood pressure (0.039%/mm Hg; 95% confidence interval, −0.076 to 0.153) or PWV (−0.044%/m/s; 95% confidence interval, −0.646 to 0.558). The area under the receiver operating characteristic curve to predict greater than median percentage of arterial stiffness was 0.892 for CAVI and 0.779 for PWV (P = 0.006). Limitation It is unclear whether arterial fibrosis of the brachial artery evaluated by using CAVI is applicable for arteriosclerosis of other arterial districts, and clinical outcomes were not evaluated in this study. Conclusion CAVI reflects the histological arterial fibrosis of hemodialysis patients and is a useful clinical marker for evaluating arterial stiffness in these patients. Patients with kidney failure treated with hemodialysis have a high incidence of cardiovascular diseases caused by accelerated arteriosclerosis. However, accurate evaluation of the extent of arteriosclerosis is difficult. This study sought to compare the strength of predictions of arterial fibrosis by using a new parameter, the cardio-ankle vascular index (CAVI), versus pulse wave velocity (PWV) in patients with kidney failure treated with hemodialysis. Diagnostic test study. 103 patients with kidney failure undergoing surgical construction of an arteriovenous access for hemodialysis therapy. CAVI and PWV. Arterial fibrosis, evaluated by using Masson trichrome stain on part of the brachial artery obtained during surgery, expressed as percentage of fibrosis of the layer of vascular smooth muscle cells. Median percentage of arterial stiffness was 52.85%. Mean PWV and CAVI were 18.3 ± 5.6 (SD) m/s and 9.9 ± 2.6, respectively. Multivariate regression analysis showed that arterial fibrosis was significantly associated with older age (0.247%/y; 95% confidence interval, 0.013 to 0.482) and CAVI (6.117%/unit; 95% confidence interval, 4.764 to 4.740), but not with systolic blood pressure (0.039%/mm Hg; 95% confidence interval, −0.076 to 0.153) or PWV (−0.044%/m/s; 95% confidence interval, −0.646 to 0.558). The area under the receiver operating characteristic curve to predict greater than median percentage of arterial stiffness was 0.892 for CAVI and 0.779 for PWV (P = 0.006). It is unclear whether arterial fibrosis of the brachial artery evaluated by using CAVI is applicable for arteriosclerosis of other arterial districts, and clinical outcomes were not evaluated in this study. CAVI reflects the histological arterial fibrosis of hemodialysis patients and is a useful clinical marker for evaluating arterial stiffness in these patients.

Human (pro)renin receptor ((P)RR) has been implicated in the augmentation of many biological and cellular processes through bindings to its ligands, renin and prorenin. In this study, we investigated the effects of aliskiren, a direct oral renin inhibitor, on the activities of free and (P)RR-bound forms of human mature renin. We also elucidated the effect of aliskiren on the 'renin activity' of the receptor-bound form of prorenin. Aliskiren had an IC(50) of 0.72 nmol l(-1) against renin. The compound competitively inhibited renin activity with an inhibitory constant (K(i)) of 0.18 nmol l(-1). Furthermore, the dissociation constants (K(D)) for aliskiren from renin and prorenin bound to (P)RR were determined using surface plasmon resonance in a BIAcore assay system (Uppsala, Sweden). These values were estimated to be 0.46 ± 0.03 and 0.25 ± 0.01 nmol l(-1), respectively. The compound competitively inhibited the renin activities of (P)RR-bound forms of both renin and prorenin with a K(i) of 0.14 and 0.15 nmol l(-1), respectively. These results indicate that aliskiren could be a potent inhibitor of the free forms of mature renin and of the receptor-bound forms of renin and prorenin.

(Pro)renin receptor [(P)RR], also known as Atp6ap2, has attracted growing attention as a key molecule for tissue renin-angiotensin system (RAS). In addition to its role in tissue RAS activation, Atp6ap2/(P)RR was originally identified as an accessory subunit for vacuolar H(+)-ATPase (v-ATPase), which is a multisubunit proton pump involved in diverse and fundamental cellular physiology. In this study, to elucidate the physiological function of Atp6ap2/(P)RR during retinal development in mammals, we used Cre-LoxP system to generate photoreceptor-specific conditional knock-out (CKO) mice, and revealed a critical role of Atp6ap2/(P)RR in photoreceptor development. Deletion of photoreceptor Atp6ap2/(P)RR did not affect retinal cell differentiation, but led to laminar disorganization around the outer nuclear layer together with severe dysfunction of photoreceptor cells. In the CKO mice, cell adhesion and polarity molecules, some of which were colocalized with Atp6ap2/(P)RR at the apical edge of the wild-type developing retina, were substantially dispersed together with mislocalization of retinal progenitor cells apart from the apical surface. Among theses molecules, coimmunoprecipitation using retinal homogenates and ATP6AP2/(P)RR-transfected cells showed that Atp6ap2/(P)RR interacted with partitioning defective 3 homolog (PAR3) protein, which is known to function in the Par-atypical protein kinase C (aPKC) system. Furthermore, yeast two-hybrid assays demonstrated direct molecular interaction between ATP6AP2/(P)RR and PAR3. Our present data revealed the novel function of Atp6ap2/(P)RR required for laminar formation during retinal development. We propose that this cellular activity associated with the Par-aPKC system, in addition to the v-ATPase function and tissue RAS activation, is the third biological role of Atp6ap2/(P)RR.

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; Obesity is an independent risk factor for morbidity and mortality in cardiovascular diseases not only in the general population, but also in hemodialysis (HD) patients. We previously reported that an increased visceral fat area (VFA) determined using computed tomography (CT) scans was associated with atherosclerosis in HD patients. However, whether a high VFA is associated with increased cardiovascular mortality in HD patients remains unknown. Therefore, we investigated the relationship between VFA and prognosis in HD patients. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; VFA was estimated in 126 patients on maintenance HD using CT scans. These patients were followed for 60 months. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; Kaplan-Meier analysis revealed that the cardiovascular survival rate was significantly lower in the high-VFA group, with a VFA of 71.5 cm&lt;sup&gt;2&lt;/sup&gt; or greater, than in the low-VFA group, with a VFA of less than 71.5cm&lt;sup&gt;2&lt;/sup&gt;. In univariate Cox proportional hazards analyses, age, albumin, low-density lipoprotein cholesterol, cardio-thoracic ratio and VFA above 71.5 cm&lt;sup&gt;2&lt;/sup&gt; were significantly correlated with cardiovascular deaths. In multivariate analyses testing these factors as dependent variables, VFA above 71.5 cm&lt;sup&gt;2&lt;/sup&gt; was estimated to be an independent predictor of cardiovascular deaths. &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; These results suggest that an increased VFA is a stronger risk factor for cardiovascular deaths in HD patients. Measuring VFA may be recommended for predicting the risk of cardiovascular diseases in HD patients.

Abstract To extend life expectancy and ensure healthy aging, it is crucial to prevent and minimize age‐induced skeletal muscle atrophy, also known as sarcopenia. However, the disease's molecular mechanism remains unclear. The age‐related Wnt/β‐catenin signaling pathway has been recently shown to be activated by the (pro)renin receptor ((P)RR). We report here that (P)RR expression was increased in the atrophied skeletal muscles of aged mice and humans. Therefore, we developed a gain‐of‐function model of age‐related sarcopenia via transgenic expression of (P)RR under control of the CAG promoter. Consistent with our hypothesis, (P)RR‐Tg mice died early and exhibited muscle atrophy with histological features of sarcopenia. Moreover, Wnt/β‐catenin signaling was activated and the regenerative capacity of muscle progenitor cells after cardiotoxin injury was impaired due to cell fusion failure in (P)RR‐Tg mice. In vitro forced expression of (P)RR protein in C2C12 myoblast cells suppressed myotube formation by activating Wnt/β‐catenin signaling. Administration of Dickkopf‐related protein 1, an inhibitor of Wnt/β‐catenin signaling, and anti‐(P)RR neutralizing antibody, which inhibits binding of (P)RR to the Wnt receptor, significantly improved sarcopenia in (P)RR‐Tg mice. Furthermore, the use of anti‐(P)RR neutralizing antibodies significantly improved the regenerative ability of skeletal muscle in aged mice. Finally, we show that Yes‐associated protein (YAP) signaling, which is coordinately regulated by Wnt/β‐catenin, contributed to the development of (P)RR‐induced sarcopenia. The present study demonstrates the use of (P)RR‐Tg mice as a novel sarcopenia model, and shows that (P)RR‐Wnt‐YAP signaling plays a pivotal role in the pathogenesis of this disease.

Internet-based information and communication technology is altering our lives. Although medicine is traditionally conservative, it can benefit in many ways from adopting new technology and styles of care. Hypertension is a prime condition for the practical application of digital health management because it is prevalent and undercontrolled, and its primary index, home blood pressure, can be effectively telemonitored. Compared to other conditions that require laboratory measures or the use of drugs with frequent side effects, hypertension can be managed without actual office visits with sufficiently low risk. In this review of hypertension in Japan, we discuss the current and somewhat fragmented state of internet technology and the components and processes necessary for smooth, integrated, and multidisciplinary care in the future. Although further clinical trials are required to show the safety and efficacy of information and communication technology-based care for hypertension, the deployment of telemonitoring and telemedicine in daily practice should be expedited to solve the hypertension paradox. Challenges remain relating to cost, data integration, the redesigning of team-based care, and the improvement of user experience, but information and communication technology-based hypertension management is sure to become pivotal in improving public health.

This study was designed to examine the contribution of cyclooxygenase-2 (COX-2) in the afferent arteriolar autoregulatory responses to increases in perfusion pressure and its relationship with neuronal nitric oxide synthase (nNOS). In rat kidneys, afferent arteriolar diameter responses to increases in perfusion pressure were assessed in vitro with the blood-perfused juxtamedullary nephron technique. Basal afferent arteriolar diameter at 100 mm Hg averaged 21.0+/-1.2 microm (n=7), and the vasoconstrictor response to increasing perfusion pressure to 160 mm Hg averaged 18.4+/-1.2%. Superfusion with the COX-2 inhibitor NS398 (10 micromol/L) did not influence basal diameters, but it did significantly enhance the vasoconstrictor response to the increase in perfusion pressure (32.9+/-4.0%). In contrast to previous findings that the nNOS inhibitor S-methyl-L-thiocitrulline (10 micromol/L) enhanced afferent arteriolar autoregulatory responses in normal rat kidneys, in this study, administration of 10 micromol/L S-methyl-L-thiocitrulline did not further modulate the vasoconstrictor response to increases in perfusion pressure in the NS398-treated kidneys of normal rats (31.8+/-4.7%). When tubuloglomerular feedback activity was interrupted by papillectomy and the addition of 50 micromol/L furosemide to the blood perfusate (n=5 for each), the afferent arteriolar constrictor responses to increasing perfusion pressure to 160 mm Hg averaged 7.9+/-0.9% and 10.7+/-0.7%, respectively, and they were significantly attenuated compared with the responses observed in control kidneys. NS398 treatment did not modulate the afferent arteriolar autoregulatory responses in papillectomized or furosemide-treated kidneys. These results indicate that COX-2-derived metabolites contribute to the nNOS modulation of pressure-mediated afferent arteriolar autoregulatory responses.

It is well known that renal hypertrophy is induced by hyperthyroidism; however, the mechanism is not fully understood. We recently reported that cardiac hypertrophy in hyperthyroidism is mediated by enhanced cardiac expression of renin mRNA. The present study addresses the hypothesis that renal hypertrophy in hyperthyroidism is mediated by amplification of renal expression of renin mRNA. Twenty Sprague-Dawley rats were divided into control (n=5) and hyperthyroid groups by daily intraperitoneal injections of saline vehicle or thyroxine. The hyperthyroid group was subdivided further into hyperthyroid-vehicle (n=5), hyperthyroid-losartan (n=5), and hyperthyroid-nicardipine (n=5) groups by daily intraperitoneal injections of saline vehicle, losartan, or nicardipine. All rats were killed at 4 weeks, and the blood and kidneys were collected. The kidney-to-body weight ratio increased in the hyperthyroid groups (+34%). Radioimmunoassays and reverse transcriptase-polymerase chain reaction revealed increased renal renin (+91%) and angiotensin II (+65%) levels and enhanced renal renin mRNA expression (+113%) in the hyperthyroid groups. Losartan and nicardipine decreased systolic blood pressure to the same extent, but only losartan caused regression of thyroxine-induced renal hypertrophy. These results suggest that thyroid hormone activates the intrarenal renin-angiotensin system via enhancement of renal renin mRNA expression, which then leads to renal hypertrophy.

When the 'handle region' of the prorenin prosegment interacts with the (pro)renin receptor, the prorenin molecule partially changes the conformation to an enzymatically active state. On the other hand, the receptor triggers its own intracellular signalling pathways independent of the renin-angiotensin system (RAS). The 'handle region' peptide competitively binds to the receptor as a decoy peptide and inhibits both the non-proteolytic activation of prorenin and the RAS-independent intracellular signals. Therefore, prorenin receptor blockers including the decoy peptide may have superior benefits on end-organ damage in diabetes and hypertension compared with conventional RAS inhibitors.

The present study was designed to determine whether adding amlodipine further improved functional and structural cardiovascular damage in hypertensive patients whose blood pressure was already well controlled with an angiotensin II type 1 receptor blocker (ARB). The cardiothoracic ratio on chest radiographs, level of urinary albumin excretion, pulse wave velocity (PWV), intima-media thickness (IMT) of the carotid arteries, and 24 hour ambulatory blood pressure (BP) were evaluated before and 12 months after the start of add-on of amlodipine or placebo in 50 hypertensive patients being treated with an ARB. The add-on amlodipine therapy significantly improved the PWV from 1689 +/- 61 to 1447 +/- 47 cm/s and the IMT from 0.88 +/- 0.08 to 0.75 +/- 0.06 mm in the hypertensive patients treated with an ARB without altering their mean 24 hour ambulatory BP values, but did not alter the cardiothoracic ratio or urinary albumin excretion. Amlodipine also significantly decreased the variability of ambulatory BP, but the decrease did not significantly contribute to the changes in PWV or IMT. Thus, the add-on low-dose amlodipine therapy had benefits in terms of the vascular function and vascular structure of hypertensive patients treated with an ARB that were independent of its depressor effects. The antiatherogenic pleiotropic properties of amlodipine have a preventive effect on the progression of arterial stiffness in hypertensive patients treated with an ARB.

(Pro)renin receptor-bound prorenin not only causes the generation of angiotensin II via the nonproteolytic activation of prorenin, it also activates the receptor's own intracellular signaling pathways independent of the generated angiotensin II. Within the kidneys, the (pro)renin receptor is not only present in the glomerular mesangium, it is also abundant in podocytes, which play an important role in the maintenance of the glomerular filtration barrier. Recent in vivo studies have demonstrated that the overexpression of the (pro)renin receptor to a degree similar to that observed in hypertensive rat kidneys leads to slowly progressive nephropathy with proteinuria. In addition, the handle region peptide, which acts as a decoy peptide and competitively inhibits the binding of prorenin to the receptor, is more beneficial than an angiotensin-converting enzyme inhibitor with regard to alleviating proteinuria and glomerulosclerosis in experimental animal models of diabetes and essential hypertension. Thus, the (pro)renin receptor may be upregulated in podocytes under hypertensive conditions and may contribute to the breakdown of the glomerular filtration barrier.

A 30-kDa protein in the medium of cultured human umbilical vein endothelial cells (HUVECs) was identified as (pro)renin receptor, (P)RR, by western blot analysis using anti-human (P)RR antibodies. The protein bound recombinant human prorenin with a K(D) of 4.0 nmol l(-1) and activated prorenin. These observations suggest the presence of soluble (P)RR, s(P)RR, in the medium of cultured HUVECs. For quantification of the s(P)RR in the medium, an enzyme-linked immunosorbent assay (ELISA) was established. The quantitative range of the ELISA was validated over a nominal range of 7.5-300 pmol l(-1) in the wells of a microtiter plate. The assay system showed good linearity (r(2)=0.99) with interassay (5.8-9.7%) and intraassay (2.1-7.0%) precision. Using this method, the concentration of s(P)RR in the culture medium of HUVECs was measured to be 32 pmol l(-1). Therefore, these results show qualitative and quantitative evidence that prorenin can be activated after binding to s(P)RR secreted from cultured HUVECs.

The clinical syndrome of adult growth hormone deficiency (AGHD) was widely recognized in the 1980s. In this review, we first describe the clinical features and diagnosis of AGHD and then state the effects of growth hormone (GH) therapy for these patients. The main characteristics of AGHD are abnormal body composition, dyslipidemia, insulin resistance, and an impaired quality of life (QoL) due to decreased psychological well-being. For diagnosing AGHD, the international consensus guidelines have suggested that an insulin tolerance test (ITT) is the gold standard, but in Japan, the growth hormone releasing peptide-2 (GHRP-2) test is available and is recommended as a convenient and safe GH stimulating test. The cut-off for diagnosing severe AGHD is a peak GH concentration of 9 g/L during the GHRP-2 test. Since 2006, GH therapy has been approved for Japanese patients with severe AGHD. For adults, GH replacement therapy should be initiated at a low dose (3 g/kg body weight/day), followed by individualized dose titration while monitoring patients’ clinical status and serum insulin-like growth factor-I (IGF-I) concentrations. A variety of favorable effects of GH replacement have been indicated; however, it has not yet been established fully whether there is a direct effect of GH treatment on reducing mortality.

The (pro)renin receptor ((P)RR) is known to play an important role in the pathogenesis of vascular complications in diabetes mellitus and hypertension through its function in activating the local renin-angiotensin system. Recent studies have shown that the (P)RR is an accessory protein of the vacuolar H(+)-ATPase, suggesting a more fundamental and developmental function. In this study, smooth muscle cell-specific (P)RR/Atp6ap2 conditional knockout mice were generated. Smooth muscle cell-specific ablation of the (P)RR resulted in nonatherogenic sclerosis in the abdominal aorta. The deletion of the (P)RR did not affect ambulatory blood pressure levels. In cultured murine vascular smooth muscle cells (VSMCs), ablation of the (P)RR suppressed the expression of the Vo subunit c of the vacuolar H(+)-ATPase and impaired the cell recycling system, leading to autophagic cell death. In addition, loss of the (P)RR in VSMCs induced the expression of monocyte chemotactic protein-1 and interleukin-6 mRNAs. These results suggest that the (P)RR is essential for cell survival and downregulation of vascular inflammation in murine VSMCs through maintaining normal function of the vacuolar H(+)-ATPase.

The renin-angiotensin system (RAS) is involved in inflammation. The signaling via the ANG II type 1 receptor in human lymphocytes and monocytes, which play key roles in pathophysiology of glomerulonephritis (GN), can enhance inflammation. However, the role of the (pro)renin receptor [(P)RR], a component of the RAS, in inflammatory reactions is unknown. We assessed whether (P)RR is expressed in human lymphocytes and monocytes by RT-PCR, Western blotting, flow cytometry, and immunohistochemistry, and whether (P)RR functions in inflammation. (P)RR mRNA and protein were expressed in human peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis revealed high expression of (P)RR on monocytes. (P)RR was present on PBMCs, infiltrating lymphocytes, and macrophages around glomeruli with a crescent in anti-neutrophil cytoplasmic antibody (ANCA)-associated GN. Renin stimulation of PBMCs from healthy subjects in the presence of the ANG II type 1 receptor and ANG II type 2 receptor blockers induced ERK1/2 phosphorylation and release of IL-6 and expression of cyclooxygenase-2 (COX-2). The increases in cytokine release and COX-2 expression were inhibited in the presence of an ERK1/2 inhibitor. (P)RR knockdown by small interfering RNA in U937 cells, a human leukemic monocyte lymphoma cell line, significantly decreased ERK1/2 phosphorylation after renin stimulation. Thus (P)RR expressed in human inflammatory cells might contribute to inflammation in ANCA-associated GN.

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.

The Na/K ratio is considered to be a useful index, the monitoring of which allows an effective Na reduction and K increase, because practical methods (self-monitoring devices and reliable individual estimates from spot urine) are available for assessing these levels in individuals. An intervention trial for lowering the Na/K ratio has demonstrated that a reduction of the Na/K ratio mainly involved Na reduction, with only a small change in K. The present study aimed to clarify the relationship between dietary Na intake and the urinary Na/K molar ratio, using standardized low- and high-salt diets, with an equal dietary K intake, to determine the corresponding Na/K ratio. Fourteen healthy young adult volunteers ingested low-salt (3 g salt per day) and high-salt (20 g salt per day) meals for seven days each. Using a portable urinary Na/K meter, participants measured their spot urine at each voiding, and 24-h urine was collected on the last day of each diet period. On the last day of the unrestricted, low-salt, and high-salt diet periods, the group averages of the 24-h urine Na/K ratio were 4.2, 1.0, and 6.9, while the group averages of the daily mean spot urine Na/K ratio were 4.2, 1.1, and 6.6, respectively. The urinary Na/K ratio tracked changes in dietary salt intake, and reached a plateau approximately three days after each change in diet. Frequent monitoring of the spot urine Na/K ratio may help individuals adhere to an appropriate dietary Na intake.

The (pro)renin receptor [(P)RR] is cleaved by furin to generate soluble (P)RR [s(P)RR], which reflects the status of the tissue renin-angiotensin system. Hemodialysis patients have advanced atherosclerosis. The aim of this study was to investigate the relationships between serum s(P)RR levels and background factors, including indices of atherosclerosis, in hemodialysis patients. Serum s(P)RR levels were measured in hemodialysis patients and clearance of s(P)RR through the membrane of the dialyzer was examined. Furthermore, relationships between serum s(P)RR levels and background factors were assessed. Serum s(P)RR levels were significantly higher in hemodialysis patients (30.4 ± 6.1 ng/ml, n = 258) than those in subjects with normal renal function (21.4 ± 6.2 ng/ml, n = 39, P < 0.0001). Clearance of s(P)RR and creatinine were 56.9 ± 33.5 and 147.6 ± 9.50 ml/min, respectively. Serum s(P)RR levels were significantly higher in those with ankle-brachial index (ABI) of < 0.9, an indicator of severe atherosclerosis, than those with ABI of ≥ 0.9 (32.2 ± 5.9 and 30.1 ± 6.2 ng/ml, respectively, P < 0.05). An association between low ABI and high serum s(P)RR levels was observed even after correction for age, history of smoking, HbA1c, and LDL-C. Serum s(P)RR levels were significantly higher in hemodialysis patients when compared with subjects with normal renal function, although s(P)RR is dialyzed to some extent, but to a lesser extent than creatinine. High serum s(P)RR levels may be associated with atherosclerosis independent of other risk factors, suggesting that serum s(P)RR could be used as a marker for atherosclerotic conditions in hemodialysis patients.

To assess the afferent arteriolar autoregulatory response during increased activity of the tubuloglomerular feedback (TGF) mechanism and to delineate the contribution of neuronal nitric oxide synthase (nNOS) to this response, afferent arteriolar diameter responses to changes in renal perfusion pressure (RPP) were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. At RPP of 100 mmHg, basal afferent arteriolar diameter averaged 21.1 +/- 1.4 micrometer (n = 9). The initial and sustained constrictor responses of afferent arterioles to a 60-mmHg increase in RPP averaged 14.8 +/- 1.4% and 13.3 +/- 1.3%, respectively. Acetazolamide treatment, which enhances TGF responsiveness by increasing distal nephron volume delivery, significantly decreased basal afferent arteriolar diameter by 8.2 +/- 0.5% and enhanced the initial response (25.5 +/- 2.3%) to a 60-mmHg increase in RPP but did not alter the sustained response (14.3 +/- 1.5%). In another series of experiments, nNOS inhibition with 10 microM S-methyl-L-thiocitrulline (L-SMTC) significantly decreased afferent arteriolar diameter from 20.3 +/- 1.3 to 18.3 +/- 1.1 micrometer (n = 7) and enhanced both the initial (34.4 +/- 3.5%) and sustained constrictor responses (27.6 +/- 2.9%) to a 60-mmHg increase in RPP. Treatment with acetazolamide further enhanced both initial (56.4 +/- 3.0%) and sustained responses (54.6 +/- 2.7%). Interruption of distal delivery by transection of the loops of Henle prevented the enhanced responses to increases in RPP elicited with either acetazolamide or L-SMTC. These results indicate that nNOS contributes to the counteracting resetting process of biphasic afferent arteriolar constrictor responses to increases in RPP through a TGF-dependent mechanism.

The objective of this study was to determine the contribution of renal nerves to the enhanced afferent arteriolar reactivity observed in angiotensin II (Ang II)-induced hypertension. Uninephrectomized Sprague-Dawley rats were divided into four groups: sham rats, renal-denervated rats, Ang II-infused (at 40 ng/min for 13 days) rats, and Ang II-infused+renal-denervated rats. With the use of an implanted arterial catheter, mean arterial pressure (MAP) was monitored in conscious rats. Ang II infusion resulted in a progressive increase in MAP from 98±1 (day 0) to 166±7 mm Hg (day 13). This increase in MAP was attenuated in denervated rats and averaged 136±3 mm Hg on day 13. Kidneys were harvested on day 13 for microcirculatory experiments or measurement of intrarenal Ang II levels. Basal afferent arteriolar diameter was similar in all groups, and group averages ranged from 19.6 to 20.7 μm. Chronic Ang II infusion increased intrarenal Ang II levels. Renal denervation did not alter this effect. Increasing perfusion pressure from 100 to 160 mm Hg reduced afferent arteriolar diameter significantly by 11.2±0.6% in the sham group and by a similar degree in the remaining three groups. Superfusion with Ang II (10 nmol/L) reduced afferent arteriolar diameter by 34.3±2.0% in the sham group. This response was enhanced in Ang II-infused (62.3±3.4%) but not in renal-denervated or Ang II-infused+renal-denervated rats. Additionally, the enhanced afferent arteriolar reactivity to Ang II was not influenced by adrenergic receptor blockade. The afferent arteriolar response to norepinephrine was enhanced in renal-denervated, Ang II-infused, and Ang II-infused+renal-denervated rats compared with sham controls. Administration of the calcium ionophore A23187 decreased afferent arteriolar diameter similarly in all four groups. These results indicate that renal nerves contribute to the development of hypertension and to the enhanced afferent arteriolar responsiveness to Ang II elicited by chronic Ang II infusion.

This study was designed to determine the influence of increased superoxide anion in neuronal nitric oxide synthase (nNOS)-dependent regulation of afferent arterioles in spontaneously hypertensive rats (SHR). Afferent arteriolar diameters of male Wistar-Kyoto rats (WKY) and SHR were assessed in vitro with the blood-perfused juxtamedullary nephron technique and averaged 21.6+/-1.6 (n=6) and 18.8+/-1.2 (n=7) micrometer, respectively. The superoxide dismutase mimetic Tempol (1, 10, and 100 micromol/L) did not influence afferent arterioles of WKY but significantly increased afferent arteriolar diameters of SHR by 20.6+/-5.5%, 25.2+/-5.4%, and 23.3+/-4.9%, respectively. In WKY (n=6), the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC; 10 micromol/L) and the NOS inhibitor N(omega)-nitro-L-arginine (L-NNA; 100 micromol/L) significantly decreased afferent arteriolar diameters (19.6+/-1.6 micrometer) by 11.9+/-3.1% and 21.0+/-3.9%, respectively. In SHR (n=7), L-SMTC did not influence afferent arteriolar diameters (21.0+/-1.5 micrometer), but L-NNA exerted an afferent arteriolar constriction (14.8+/-3.2%) that was similar to the response observed in WKY. Experiments were also performed in the presence of 100 micromol/L Tempol. In afferent arterioles of WKY (n=6), Tempol treatment did not modulate the basal diameters (21.5+/-1.2 micrometer) or the constrictor response to L-SMTC (10.6+/-2.1%) or L-NNA (19.3+/-3.3%). In SHR (n=8), Tempol significantly increased afferent arteriolar diameters by 22.5+/-4.3% and enhanced afferent arteriolar constrictor responses to L-SMTC (18.4+/-2.7%) and L-NNA (31.9+/-2.6%). However, the nitric oxide donor S-nitroso-N-acetylpenicillamine (10 micromol/L), which similarly increased afferent arteriolar diameters (17.2+/-2.3%, n=6), did not affect afferent arteriolar responses to L-SMTC (1.5+/-2.7%) or L-NNA (18.6+/-2.3%). These suggest that superoxide anion inhibits the control of afferent arteriolar diameters by nNOS in SHR.

The present study was conducted to determine the contribution of nitric oxide to angiotensin II (Ang II) reactivity of afferent and efferent arterioles from Ang II-infused hypertensive rats. Experiments were performed in vitro with the blood-perfused juxtamedullary nephron technique in kidneys harvested from hypertensive Sprague-Dawley rats (181+/-1 mm Hg) that had received 60 ng/min Ang II subcutaneously for 13 days. Superfusion with 0.1, 1, and 10 nmol/L Ang II reduced afferent arteriolar diameter (18.1+/-0.6 microm; n=12) by 10.0+/-0.7%, 28.1+/-1.7%, and 52.8+/-1.9%, respectively, and efferent arteriolar diameter (17.2+/-1.4 microm; n=8) decreased by 9.3+/-0.7%, 27.0+/-1.2%, and 50.4+/-1.6%, respectively. Nitric oxide synthase inhibition with 100 micromol/L N(omega)-nitro-L-arginine (NLA) reduced resting afferent and efferent arteriolar diameters to 14.7+/-0.4 and 14.3+/-1.2 microm, respectively, and enhanced afferent but not efferent arteriolar reactivity to Ang II. The enhanced afferent arteriolar reactivity to Ang II was eliminated by addition of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 10 micromol/L), which reversed the NLA-induced decrease in diameter. Addition of 10 micromol/L SNAP, without NLA, blunted efferent but not afferent arteriolar reactivity to Ang II. Afferent (n=7) and efferent arteriolar diameters (n=6) decreased by 48.5+/-2.2% and 41.0+/-1.9%, respectively, in response to 10 nmol/L Ang II. These results suggest that in this model of hypertension, maintained nitric oxide production in afferent arterioles counteracts the enhanced afferent arteriolar reactivity that occurs in Ang II-induced hypertension.

In conscious dogs, 36-h water deprivation induced a significant increase in renal blood flow (RBF) with elevation of the plasma arginine vasopressin (AVP) concentration to 9.6 +/- 1.8 pg/ml. To simulate such a condition, a mild elevation of plasma AVP was produced by infusing AVP intravenously at a dose of 0.1 ng.kg-1.min-1 for 20 min. The plasma AVP concentration then increased to 6.8 +/- 0.7 pg/ml. This dose of AVP increased the RBF by 21.7 +/- 2.6% and decreased the renal vascular resistance by 18.1 +/- 2.3% without significant changes in mean arterial pressure, cardiac output, or heart rate. The mechanism of this renal vasodilatory action was examined using newly developed, orally effective, selective AVP antagonists OPC-21268 (a V1-receptor antagonist) and OPC-31260 (a V2-receptor antagonist). In 36-h water-deprived dogs, V2-receptor blockade with OPC-31260 significantly decreased the RBF by 20.5 +/- 2.6% without significant changes in cardiac output. The exogenous AVP-induced renal vasodilatory response tended to be augmented when V1 receptors were blocked by pretreatment with OPC-21268, but the change did not achieve statistical significance. On the other hand, V2-receptor blockade by either pretreatment with OPC-31260 or simultaneous infusion of OPC-31260 inhibited this vasodilatory response. Furthermore, intravenous infusion of 1-desamino-8-D-arginine vasopressin (DDAVP) at a dose of 0.3 ng.kg-1 x min-1 for 20 min significantly increased the RBF by 36.5 +/- 1.7%, and this DDAVP-induced renal vasodilation was inhibited by simultaneous infusion of V2-receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Background. To identify differences between the effects of calcitriol and the calcitriol analogue, maxacalcitol, on parathyroid hormone (PTH) and bone metabolisms, we conducted a randomized prospective multicentre study on patients on chronic haemodialysis.

Blood pressure (BP) variability is estimated as the standard deviation of 24-h ambulatory BP. The present study was performed to determine the effect of the mean 24-h ambulatory BP values and standard deviations on arterial wall stiffness assessed by brachial-ankle pulse wave velocity (baPWV). Brachial-ankle pulse wave velocity, carotid intima-media thickness (IMT), urinary albumin excretion (UAE) and 24-h ambulatory BP were measured before the start of antihypertensive therapy in 203 newly diagnosed hypertensive patients (53.3+/-0.7 years old; clinic systolic/diastolic BP: 154+/-1/98+/-1 mm Hg), and univariate and multivariate regression analyses of these clinical and biological parameters were performed. Univariate regression analyses revealed a significant association between mean baPWV values and the standard deviations of ambulatory systolic/diastolic BP. Mean ambulatory systolic/diastolic BP values were also associated with UAE, and the standard deviations of ambulatory systolic BP were associated with maximum carotid IMT. Quintile analyses showed that patients with a mean 24-h ambulatory mean BP value and standard deviation below 110 and 20 mm Hg, respectively, had the lowest baPWV. Moreover, the multivariate regression analyses confirmed a significant correlation between baPWV and the standard deviation of 24-h ambulatory systolic BP. In conclusion, untreated hypertensive patients with a higher 24-h ambulatory systolic BP variability had stiffer arterial walls. Ambulatory systolic BP variability may be involved in stiffening of the arteries of hypertensive patients.

Previous studies have demonstrated that prorenin plays a significant role in the development and progression of nephropathy in streptozotocin-induced diabetic animals, a model for type 1 diabetes, through a (pro)renin receptor-dependent mechanism. However, whether this novel mechanism also contributes to the mechanism of diabetic nephropathy in type 2 diabetes has remained undetermined. In 16-week-old db/db mice, a model for type 2 diabetes, we found a significant degree of glomerulosclerosis, enhanced immunostaining for the active site of renin (representing non-proteolytically activated prorenin), and an increased immunoreactivity to activated extracellular-signal-related protein kinase 1/2 in the kidneys. These changes were blocked by the chronic subcutaneous administration (1 mg/kg/day) of a decoy peptide with the "handle region" structure, which competitively inhibits prorenin binding to a "handle region"-specific binding protein, such as the (pro)renin receptor. The kidneys of db/db mice also contained increased angiotensin (Ang) I and II levels, eliciting significant microalbuminuria. Treatment with the "handle region" peptide significantly decreased the renal content of Ang I and II and inhibited the development of microalbuminuria. Thus prorenin also contributes to the development of nephropathy in type II diabetes, probably through a (pro)renin receptor-dependent mechanism.

Numerous in vitro and in vivo animal studies using the (pro)renin receptor (P)RR blocker handle region peptide have suggested an important role of (P)RR in the pathogenesis of end-stage organ damage in patients with diabetes and hypertension. In addition, a limited number of clinical studies have suggested an association between (P)RR gene polymorphisms and blood pressure levels and between (P)RR mRNA levels and angiotensin-converting enzyme mRNA levels in human arteries. However, recent studies have shown that the (P)RR is divided into its soluble form and a residual hydrophobic part, which includes ATPase 6 associated protein 2, within cells. Therefore, the (P)RR may have a more complex function than previously thought. In addition, the physiological roles of the (P)RR remain undetermined, because the construction of (P)RR null mice has not been successful. As a next step for research in this area, a method for determining the soluble (P)RR levels in plasma and urine and the construction of tissue-specific (P)RR-knockout mice are needed to elucidate the roles of the (P)RR in physiology and pathophysiology.

Hypertension frequently requires combination therapy to attain efficient control to prevent cardiovascular diseases effectively. This study was conducted to determine which add-on treatment is better, namely calcium channel blockers or diuretics, in improving vascular damage. In 70 nondiabetic chronic kidney disease stage 1/2 patients who had been already treated with angiotensin II type 1 receptor blocker valsartan for at least 12 months, amlodipine or hydrochlorothiazide was added to their existing medication. Pulse wave velocity (PWV), intima-media thickness (IMT) of the carotid arteries, urinary albumin excretion (UAE), and 24-hour ambulatory blood pressure (BP) were determined before and 12 months after the start of add-on treatments. Add-on amlodipine and add-on hydrochlorothiazide significantly and similarly decreased 24-hour ambulatory BP by 18 and 19 mm Hg, respectively, PWV by 206 and 184 cm/s, respectively, and UAE, but did not change the IMT. The decreases in BP significantly contributed to the decreases in PWV and UAE and suggested that the decrease in serum cholesterol level brought about by add-on amlodipine also contributed to the decrease in UAE. These results suggest that 12 months of add-on treatment with either amlodipine or hydrochlorothiazide could have beneficial effects in nondiabetic chronic kidney disease stage 1/2 patients already being treated with valsartan.

Since renin inhibition interferes with the first and rate-limiting steps in the renin-angiotensin system, the renin step is a very attractive target for lowering blood pressure and minimizing target-organ damage. The newly developed direct renin inhibitor aliskiren has several attractive characteristics: it definitively reduces plasma renin activity among inhibitors of the renin-angiotensin system, is remarkably specific for human renin, exhibits a long half-life in plasma comparable to that of amlodipine, and has a high affinity for renal glomeruli and vasculature. Although these characteristics suggest the clinical usefulness and safety of aliskiren, several problems remain unsolved. Why does aliskiren have beneficial effects on the heart and kidneys of patients treated with angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II type 1-receptor blockers (ARBs)? Is the blood-pressure-lowering effect of aliskiren dependent on the plasma renin activity? Does aliskiren exert a possible adverse effect via (pro)renin receptor-dependent intracellular signals? Here, we review the characteristics and usefulness of aliskiren and discuss the current issues associated with this direct renin inhibitor.

Despite suppression of the circulating renin-angiotensin system (RAS), high salt intake (HSI) aggravates kidney injury in chronic kidney disease. To elucidate the effect of HSI on intrarenal RAS, we investigated the levels of intrarenal prorenin, renin, (pro)renin receptor (PRR), receptor-mediated prorenin activation, and ANG II in chronic anti-thymocyte serum (ATS) nephritic rats on HSI. Kidney fibrosis grew more severe in the nephritic rats on HSI than normal salt intake. Despite suppression of plasma renin and ANG II, marked increases in tubular prorenin and renin proteins without concomitant rises in renin mRNA, non-proteolytically activated prorenin, and ANG II were noted in the nephritic rats on HSI. Redistribution of PRR from the cytoplasm to the apical membrane, along with elevated non-proteolytically activated prorenin and ANG II, was observed in the collecting ducts and connecting tubules in the nephritic rats on HSI. Olmesartan decreased cortical prorenin, non-proteolytically activated prorenin and ANG II, and apical membranous PRR in the collecting ducts and connecting tubules, and attenuated the renal lesions. Cell surface trafficking of PRR was enhanced by ANG II and was suppressed by olmesartan in Madin-Darby canine kidney cells. These data suggest the involvement of the ANG II-dependent increase in apical membrane PRR in the augmentation of intrarenal binding of prorenin and renin, followed by nonproteolytic activation of prorenin, enhancement of renin catalytic activity, ANG II generation, and progression of kidney fibrosis in the nephritic rat kidneys on HSI. The origin of the increased tubular prorenin and renin remains to be clarified. Further studies measuring the urinary prorenin and renin are needed.

The (pro)renin receptor [(P)RR] is a molecule that binds prorenin and renin in tissues, leading not only to their activation, but also carrying out intracellular signaling. As a key player in the tissue renin-angiotensin system, (P)RR activation plays an important role in the development of end-organ damage in hypertension and diabetes. One fragment of (P)RR is also known as ATP6AP2 because it is associated with vacuolar H(+)-ATPase (V-ATPase). V-ATPase is a multi-subunit proton pump involved in diverse and fundamental aspects of cellular physiology, including receptor-mediated endocytosis and recycling, processing of proteins and signaling molecules, membrane sorting and trafficking, and activation of lysosomal/autophagosomal enzymes. The role of (P)RR in the function of V-ATPase has been investigated in recent studies using conditional knockout mice. Furthermore, the novel function of (P)RR as an adaptor protein between the Wnt receptor complex and V-ATPase has been demonstrated. Thus, (P)RR is a multi-functional molecule that has complex structure and functionality. This review focuses on current insights into the possibility of (P)RR acting as a modulator of V-ATPase and future perspectives in translational research.

Visit-to-visit blood pressure variability has been shown to be an independent risk factor for cardiovascular diseases. High visit-to-visit blood pressure variability and endothelial dysfunction are observed in patients with chronic kidney disease. It is therefore assumed that high variability in visit-to-visit blood pressure measurements may be associated with endothelial dysfunction in these patients. The present study investigated the associations between visit-to-visit blood pressure variability and renal and endothelial function in patients with chronic kidney disease. We analyzed 150 consecutive patients with predialysis chronic kidney disease who visited our outpatient clinic from January 2006 to December 2010. The study examined the relationships between variability in visit-to-visit systolic blood pressure levels or mean systolic blood pressure (M SBP) and estimated glomerular filtration rate (eGFR) and flow-mediated dilation, an index of endothelial function. Variability in visit-to-visit systolic blood pressure showed a significant negative association with eGFR, independent of age, hemoglobin A1c, low-density lipoprotein (LDL) cholesterol and uric acid, whereas M SBP did not. Similarly, variability in SBP showed a significant negative association with flow-mediated dilation, independent of age, eGFR, HbA1c, LDL cholesterol and M SBP. These data indicate that variability in visit-to-visit blood pressure measurements is associated with impaired renal and endothelial function in patients with chronic kidney disease. This finding suggests that reducing blood pressure fluctuations might have beneficial effects in patients with chronic kidney disease, although this point needs to be addressed by future studies.

The renin-angiotensin system (RAS) affects inflammatory responses during sepsis. Nonproteolytic activation of prorenin by the (pro)renin receptor has recently been shown to stimulate the tissue RAS. In the present study, the effect of (pro)renin receptor blocker (PRRB) pretreatment on sepsis in a rat cecal ligation and puncture (CLP) model was investigated.Male Sprague-Dawley rats underwent CLP and were randomly divided into two groups: PRRB-treated group and control peptide-treated group. Survival was analyzed for 7 d after CLP. The serum concentrations of cytokines and high-mobility group box chromosomal protein 1 (HMGB1) were measured at three time points (0, 3, and 6 h after CLP). Hematoxylin-eosin staining and immunohistochemical staining for nonproteolytically activated prorenin and HMGB1 were performed on the cecum to assess pathologic changes found 6 h after CLP.Treatment with PRRB improved the survival rate of the post-CLP septic rats (P = 0.023). PRRB also significantly reduced serum tumor necrosis factor-α, interleukin-1β, and HMGB1 levels 6 h after CLP. In CLP rats that were treated with control peptide, the expression of activated prorenin was elevated in peritoneal foam cells. Moreover, expression of HMGB1 was increased in peritoneal inflammatory cells. In contrast, both were markedly suppressed in CLP rats that were treated with PRRB.PRRB significantly improved the survival rate of rats with clinically relevant sepsis, possibly by attenuating a sepsis-induced systemic inflammatory response. We propose that overactivation of the RAS by activation of prorenin in foam cells may be a significant contributor to sepsis.

The receptor-associated prorenin system refers to the pathogenic mechanism whereby prorenin binding to (pro)renin receptor [(P)RR] dually activates the tissue renin-angiotensin system (RAS) and RAS-independent signaling, and its activation contributes to the molecular pathogenesis of various ocular diseases. We recently developed a new single-stranded RNAi agent targeting both human and mouse (P)RR ((P)RR-proline-modified short hairpin RNA [(P)RR-PshRNA]), and confirmed its therapeutic effect on murine models of ocular inflammation. Here, we investigated the efficacy of (P)RR-PshRNA against laser-induced choroidal neovascularization (CNV) and subretinal fibrosis, both of which are involved in the pathogenesis of age-related macular degeneration (AMD). Administration of (P)RR-PshRNA in mice significantly reduced CNV formation, together with the expression of inflammatory molecules, macrophage infiltration, and extracellular signal-regulated kinase (ERK) 1/2 activation. In addition, (P)RR-PshRNA attenuated subretinal fibrosis, together with epithelial-mesenchymal transition (EMT)-related markers including phosphorylated SMAD2. The suppressive effect of (P)RR-PshRNA is comparable with aflibercept, an anti-vascular endothelial growth factor drug widely used for AMD therapy. AMD patient specimens demonstrated (P)RR co-localization with phosphorylated ERK1/2 in neovascular endothelial cells and retinal pigment epithelial cells. These results indicate that (P)RR contributes to the ocular pathogenesis of both inflammation-related angiogenesis and EMT-driven fibrosis, and that (P)RR-PshRNA is a promising therapeutic agent for AMD.

Hyperuricaemia is recognized as an independent risk marker for cardiovascular and renal diseases. However, uric acid is a powerful free-radical scavenger, and the optimal level of serum uric acid (SUA) determining outcomes is unknown. This study explored whether interventional treatments for excessive SUA reduction were harmful and what constituted the optimal lowering of SUA levels for the prevention of events in patients with asymptomatic hyperuricaemia.This was a post hoc analysis of a randomized trial (Febuxostat for Cerebral and CaRdiorenovascular Events PrEvEntion StuDy [FREED]) in which 1070 older patients with asymptomatic hyperuricaemia were enrolled and allocated to febuxostat (n = 537) or non-febuxostat treatment group (n = 533). We assessed the relationship between the endpoint (withdrawal or study completion) SUA levels and clinical outcomes. Primary endpoint was defined as a composite of all-cause mortality, cerebral and cardiorenovascular events.In the febuxostat group, patients achieving SUA levels ≤4 mg/dl (hazard ratio: 2.01 [95% CI: 1.05, 3.87]), >4 to ≤5 mg/dl (2.12 [1.07, 4.20], >6 to ≤7 mg/dl (2.42 [1.05, 5.60]), and >7 mg/dl (4.73 [2.13, 10.5]) had significantly higher risks for a primary composite event than those achieving SUA levels >5 to ≤6 mg/dl (P = 0.003 [log-rank test]). This J-shaped relationship applied to patients with renal impairment (P = 0.007 [Gray's test]) and was not significant in the non-febuxostat treatment group (P = 0.212 [log-rank test]).Optimal SUA level by febuxostat treatment is 5-6 mg/dl for reducing all-cause mortality, cerebral, cardiovascular and renal events. Excessive SUA reduction may be harmful in older hyperuricaemic populations.ClinicalTrial.gov, https://clinicaltrials.gov, NCT01984749.

Introduction: This study aimed to explore the impact of comorbid hyperuricemia on disease severity in Japanese patients with coronavirus disease 2019 .This retrospective cohort study included patients with COVID-19 between July 2020 and February 2021. Methods:We divided patients into mild, moderate, and severe groups according to the degree of disease severity.Clinical and biochemical parameters on admission and comorbidities were compared between the mild and severe groups. Results:We enrolled 146 patients in this study: 36 patients were allocated to the mild group, 96 to the moderate group, and 14 to the severe group.The male sex, age, body mass index (BMI), systolic blood pressure, pulse rate, white blood cell counts, levels of serum urea nitrogen and uric acid were significantly higher in the severe group than in the mild group (p<0.05),while lymphocyte counts and estimated glomerular filtration rate were significantly lower (p<0.05).As for comorbidities, malignant tumor, diabetes mellitus, hypertension, chronic kidney disease (CKD), hyperlipidemia, and hyperuricemia were associated with COVID-19 severity.Logistic regression analysis indicated that hyperuricemia was significantly positively associated with the severity of COVID-19 independent of age, sex, BMI, comorbidities of diabetes mellitus, and malignant tumor.However, the association between hyperuricemia and COVID-19 severity was eliminated by correction with hypertension or CKD. Conclusion:These data suggested that comorbidities of hyperuricemia may indicate an increased risk of COVID-19 progression.Furthermore, patients with hyperuricemia comorbidities may require careful and intensive multidisciplinary treatment for hyperuricemia and hypertension and/or CKD to prevent progression of COVID-19.