Akira Nishiyama

The Intrarenal Renin-Angiotensin System: From Physiology to the Pathobiology of Hypertension and Kidney Disease

In recent years, the focus of interest on the role of the renin-angiotensin system (RAS) in the pathophysiology of hypertension and organ injury has changed to a major emphasis on the role of the local RAS in specific tissues. In the kidney, all of the RAS components are present and intrarenal angiotensin II (Ang II) is formed by independent multiple mechanisms. Proximal tubular angiotensinogen, collecting duct renin, and tubular angiotensin II type 1 (AT1) receptors are positively augmented by intrarenal Ang II. In addition to the classic RAS pathways, prorenin receptors and chymase are also involved in local Ang II formation in the kidney. Moreover, circulating Ang II is actively internalized into proximal tubular cells by AT1 receptor-dependent mechanisms. Consequently, Ang II is compartmentalized in the renal interstitial fluid and the proximal tubular compartments with much higher concentrations than those existing in the circulation. Recent evidence has also revealed that inappropriate activation of the intrarenal RAS is an important contributor to the pathogenesis of hypertension and renal injury. Thus, it is necessary to understand the mechanisms responsible for independent regulation of the intrarenal RAS. In this review, we will briefly summarize our current understanding of independent regulation of the intrarenal RAS and discuss how inappropriate activation of this system contributes to the development and maintenance of hypertension and renal injury. We will also discuss the impact of antihypertensive agents in preventing the progressive increases in the intrarenal RAS during the development of hypertension and renal injury.

Possible Contributions of Reactive Oxygen Species and Mitogen-Activated Protein Kinase to Renal Injury in Aldosterone/Salt-Induced Hypertensive Rats

Abstract—Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 &mgr;g/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165±5 mm Hg) and urinary excretion of protein (106±24 mg/d) than vehicle-infused rats (118±3 mm Hg and 10±3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23±0.02) than vehicle-infused rats (0.09±0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127±2 and 125±5 mm Hg), and the elevations of urinary excretion of protein (10±2 and 9±2 mg/day) or TBARS contents (0.08±0.01 and 0.11±0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.

Prorenin Receptor Blockade Inhibits Development of Glomerulosclerosis in Diabetic Angiotensin II Type 1a Receptor–Deficient Mice

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.

Nonproteolytic Activation of Prorenin Contributes to Development of Cardiac Fibrosis in Genetic Hypertension

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” I11PLLKK15P. 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.

Role of NAD(P)H Oxidase- and Mitochondria-Derived Reactive Oxygen Species in Cardioprotection of Ischemic Reperfusion Injury by Angiotensin II

Reactive oxygen species (ROS) participate in cardioprotection of ischemic reperfusion (I/R) injury via preconditioning mechanisms. Mitochondrial ROS have been shown to play a key role in this process. Angiotensin II (Ang II) exhibits pharmacological preconditioning; however, the involvement of NAD(P)H oxidase, known as an ROS-generating enzyme responsive to Ang II stimuli, in the preconditioning process remains unclear. We compared the effects of 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial ATP-sensitive potassium channels), apocynin (an NAD(P)H oxidase inhibitor), and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger) on pharmacological preconditioning by Ang II in rat cardiac I/R injury in vivo. Treatment with a pressor dose of Ang II before a 30-minute coronary occlusion reduced infarct size as determined 24 hours after reperfusion. The protective effects of Ang II were eliminated by pretreatment with 5-HD or apocynin, similar to tempol. Both 5-HD and apocynin suppressed the enhanced cardiac lipid peroxidation and activation of the apoptosis signal-regulating kinase/p38, c-Jun NH2-terminal kinase (JNK) pathways, but not the Raf/MEK/extracellular signal-regulated kinase pathway, elicited by acutely administered Ang II. Apocynin but not 5-HD suppressed Ang II–induced augmentations of the NAD(P)H oxidase complex formation (p47phox, p22phox, and Rac-1) and its activity in the heart. Finally, 5-HD suppressed superoxide production by isolated cardiac mitochondria without any effect on their respiration. These results suggest that the preconditioning effects of Ang II for cardiac I/R injury may be mediated by cardiac mitochondria-derived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation.

Slowly Progressive, Angiotensin II–Independent Glomerulosclerosis in Human (Pro)renin Receptor–Transgenic Rats

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.

Enhancement of Intrarenal Angiotensinogen in Dahl Salt-Sensitive Rats on High Salt Diet

Abstract—This study was performed to examine whether there is an inappropriate regulation of intrarenal angiotensinogen in Dahl-salt sensitive rats (DS) fed a high salt diet (HS). Dahl salt-resistant rats (DR) and DS were maintained on HS (8% NaCl) or low salt diet (LS, 0.3% NaCl) for 4 weeks. Systolic blood pressure (SBP), measured by tail-cuff plethysmography, was unaltered in DR (DR+HS, 127±3 mm Hg, n=5; DR+LS, 126±3, n=5); however, SBP was significantly increased in DS+HS (208±7, n=9) compared with DS+LS (134±2, n=5). HS suppressed plasma renin activity in both strains (0.7±0.2 ng of angiotensin I/mL per hour in DS+HS, 3.1±0.5 in DS+LS, 0.8±0.2 in DR+HS, 5.1±0.7 in DR+LS). Plasma angiotensinogen levels, measured by Western blot analysis, were also suppressed by HS in both strains (36 919±2170 integrated densitometric unit in DS+HS, 53 028±2752 in DS+LS, 44 722±1721 in DR+HS, 55782±3785 in DR+LS). However, kidney angiotensinogen levels were significantly increased in DS+HS (75 850±4171, integrated densitometric unit) compared with DS+LS (47 232±3470), DR+HS (44 748±8236), and DR+LS (42 504±4052). Urinary excretion of angiotensinogen, measured by radioimmunoassay of angiotensin I after incubation with excess renin, had a similar profile. Urinary excretion of angiotensinogen was significantly increased in DS+HS (2958±531 pmol/d) compared with DS+LS (56±4), DR+HS (31±12), and DR+LS (21±7). These data indicate that intrarenal angiotensinogen is enhanced in DS+HS, which is reflected by the increased urinary excretion of angiotensinogen. The results suggest that DS on HS have an inappropriate augmentation of intrarenal angiotensinogen, which may contribute to impaired sodium excretion during a high salt diet and the development of hypertension in this strain.

Urinary Angiotensinogen as an Indicator of Intrarenal Angiotensin Status in Hypertension

Abstract—Angiotensin II (AngII) infusions augment renal angiotensinogen mRNA and protein and urinary angiotensinogen excretion (UAGT). Further experiments were performed in 4 groups of rats: normal salt diet with sham operation, NS+Sham, n=6; NS with AngII infusion at 40 ng/min via osmotic minipump, NS+AngII(40), n=9; NS with AngII infusion at 80 ng/min, NS+AngII(80), n=9; high-salt diet with deoxycorticosterone acetate salt pellet (100 mg), HS+DOCA, n=4. These experiments sought to determine whether enhanced UAGT is specifically associated with increased kidney AngII levels or is a nonspecific consequence of the hypertension. Systolic BP (SBP) was significantly increased to 131±2 and 162±2 mm Hg at day 11 in NS+AngII(40) and NS+AngII(80), respectively, compared with NS+Sham (110±1). Regression analysis demonstrated a positive relationship (R =0.49) between SBP and UAGT for NS+Sham (1.1±0.3 nmol AngI/d), NS+AngII(40) (2.5±0.9), and NS+AngII(80) (5.5±1.5). UAGT was also highly correlated (R =0.70) with kidney AngII content for NS+Sham (49±6 fmol/g), NS+AngII(40) (215±49), and NS+AngII(80) (347±47); but not with plasma AngII (R =0.12). HS+DOCA rats also exhibited increased SBP to 134±1 mm Hg, but UAGT (1.4±0.4 nmol AngI/d) and intrarenal AngII content (13±2 fmol/g) were not increased despite the hypertension. Infused human angiotensinogen could not be detected in urine of sham-operated or AngII-infused rats (n=4 each). These data demonstrate that UAGT increases in AngII-dependent hypertension in a dose- and time-dependent manner, but not in hypertension elicited by HS+DOCA. The results support the hypothesis that AngII-dependent hypertension results in elevated intrarenal AngII and angiotensinogen levels, reflected by increased UAGT, which does not occur in an AngII-independent hypertensive model.

Redox control of resistance to cis-diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against CDDP-induced cytotoxicity.

Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter-reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent.

Aldosterone Stimulates Reactive Oxygen Species Production through Activation of NADPH Oxidase in Rat Mesangial Cells

It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O(2)(-)) in intact cells. The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 +/- 18 to 346 +/- 30 cpm/5 x 10(4) cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 micromol/L), significantly attenuated aldosterone-induced O(2)(-) production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.