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Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell–derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry -/-Rag2 -/- mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system.

Chronic activation of the renin-angiotensin system promotes hypertension, renal microvascular dysfunction, tissue hypoxia, and inflammation. Despite similar hypertension, an injurious response to excess angiotensin II is greater in F344 than in Lewis rats; the latter displaying renoprotection. Here we studied whether p2rx7, encoding the P2X7 receptor (P2X7R), is a candidate gene for the differential susceptibility to vascular dysfunction under high angiotensin II tone. A 14-day infusion of angiotensin II into F344 rats increased blood pressure by about 15 mm Hg without inducing fibrosis or albuminuria. In vivo pressure natriuresis was suppressed, medullary perfusion reduced by half, and the corticomedullary oxygenation gradient disrupted. Selective P2X7R antagonism restored pressure natriuresis, promoting a significant leftward shift in the intercept and increasing the slope. Sodium excretion was increased sixfold and blood pressure normalized. The specific P2X7R antagonist AZ11657312 increased renal medullary perfusion, but only in angiotensin II-treated rats. Tissue oxygenation was improved by P2X7R blockade, particularly in poorly oxygenated regions of the kidney. Thus, activation of P2X7R induces microvascular dysfunction and regional hypoxia when angiotensin II is elevated and these effects may contribute to progression of renal injury induced by chronic angiotensin II.

Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression.

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 minute injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 minute unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury blood flow was decreased in the inner cortex of wild type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 hours after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMC) to secrete the macrophage chemoattractant, RANTES; a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared to transgenic and non-transgenic mice after the 10 minute injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.

To explore the potential therapeutic effects of angiotensin(1–7) (Ang(1–7)), an endogenous ligand of the Mas receptor, on streptozotocin-induced diabetic nephropathy, male Wistar rats were randomly divided into two groups: a control group and a diabetic model group. After 12 weeks, the diabetic rats were divided into subgroups for 4-week treatments consisting of no-treatment group, small-, moderate-, and large-dose Ang(1–7) groups, a valsartan group, a large-dose Ang(1–7) plus valsartan group, and an A779 (antagonist of the Mas receptor) group, each with 15 rats. Ang(1–7) improved renal function, attenuated glomeruli sclerosis, oxidative stress, and cell proliferation, decreased the expression of collagen IV, TGF-β1, VEGF, NOX4, p47phox, PKCα, and PKCβ1, and the phosphorylation of Smad3. In the rat mesangial HBZY-1 cell line, Ang(1–7) decreased high-glucose-induced oxidative stress, the proliferation and expression of NOX4, p47phox, and TGF-β1, the phosphorylation of Smad3, collagen IV, and VEGF, and the membrane translocation of PKCα and PKCβ1. A779 blocked the effects of Ang(1–7) both in vivo and in vitro. The effects of large-dose Ang(1–7) alone and in combination with valsartan were superior to valsartan alone, but the combination had no significant synergistic effect compared with Ang(1–7) alone. Thus, Ang(1–7) ameliorated streptozotocin-induced diabetic renal injury. Large-dose treatment was superior to valsartan in reducing oxidative stress and inhibiting TGFβ1/Smad3- and VEGF-mediated pathways.

Angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers (ARBs) may induce an acute decrease of glomerular filtration rate (GFR) in the stenotic kidney in renal artery stenosis, but most patients tolerate these drugs well. We hypothesized that angiotensin-converting enzyme inhibitors/ARBs stabilize stenotic kidney function during prolonged treatment by conferring protective effects. We tested this in control domestic pigs and pigs with renal artery stenosis untreated or treated with Valsartan, or triple therapy (seven pigs in each group) for 4 weeks starting 6 weeks after stenosis induction. Renal function, oxygenation, tubular function, and microcirculation were assessed by multi-detector computed tomography (CT), blood oxygen level-dependent magnetic-resonance imaging, and micro-CT. Valsartan and triple therapy decreased blood pressure similarly; however, Valsartan did not change the GFR of the stenotic kidney compared with renal artery stenosis and was similar to triple therapy. Both Valsartan and triple therapy stimulated microvascular density and improved tubular function. Valsartan also caused a greater increase of angiogenic factors and a decrease in oxidative stress, which were related to higher cortical perfusion and tubular response than triple therapy. Thus, Valsartan did not decrease stenotic kidney GFR, but improved cortical perfusion and microcirculation. These beneficial effects may partly offset the hemodynamic GFR reduction in renal artery stenosis and preserve kidney function.

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that degrades angiotensin II with high efficiency leading to the formation of angiotensin-(1–7). ACE2 within the kidneys is largely localized in tubular epithelial cells and in glomerular epithelial cells. Decreased glomerular expression of this enzyme coupled with increased expression of ACE has been described in diabetic kidney disease, both in mice and humans with type 2 diabetes. Moreover, both ACE2 genetic ablation and pharmacological ACE2 inhibition have been shown to increase albuminuria and promote glomerular injury. Studies using recombinant ACE2 have shown the ability of ACE2 to rapidly metabolize Ang II in vivo and form the basis for future studies to examine the potential of ACE2 amplification in the therapy of diabetic kidney disease and cardiovascular disease.

Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers are considered the standard of care for treatment of cardiovascular disease and chronic kidney disease. Combination therapy with both angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers effectively inhibits the renin-angiotensin system as well as potentiates the vasodilatory effects of bradykinin. It has been advocated that this dual blockade approach theoretically should result in improved clinical outcomes in both cardiovascular disease and chronic kidney disease. Clinical trial evidence for the use of combination therapy with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in cardiovascular disease has provided conflicting results in hypertension, congestive heart failure, and ischemic heart disease. Clinical trial evidence to support combination therapy with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in chronic kidney disease has largely been based on proteinuria reduction as a surrogate marker for clinically meaningful outcomes. Recent large-scale randomized clinical trials have not been able to validate protection in halting progression in chronic kidney disease with a dual blockade approach. This review serves as an appraisal on the clinical evidence of combination angiotensin-converting enzyme inhibition and angiotensin II receptor blockade in both cardiovascular disease and chronic kidney disease.

The present study was designed to test the hypothesis that hypoxia inducible factor (HIF)-1α mediates profibrotic effects of angiotensin (ANG) II and to determine whether HIF prolyl-hydroxylase, the enzyme that promotes the degradation of HIF-1α, is involved in the profibrotic action of ANG II. In cultured renal medullary interstitial cells, ANG II (10−6 M) treatment for 20 hours remarkably increased HIF-1α levels, which was accompanied by the significant upregulation of collagen I/III and tissue inhibitor of metalloproteinases (TIMP)-1. HIF-1α siRNA decreased HIF-1α levels and completely blocked the effects of ANG II on collagen I/III and TIMP-1. HIF-1α siRNA also abolished ANG II-induced elevation of proliferating cell nuclear antigen, a marker of cell proliferation, and vimentin, a marker of cell transdifferentiation. HIF-2α siRNA did not affect the action of ANG II on collagen I/III and TIMP-1. Overexpression of PHD2 transgene, the predominant renal HIF prolyl-hydroxylase, attenuated ANG II-induced profibrotic action and silencing of PHD2 gene enhanced ANG II-induced profibrotic action. Removal of H2O2 eliminated ANG II-induced profibrotic effects. Two week ANG II infusion (150 ng/Kg/min) increased the expression of HIF-1α and α-smooth muscle actin in the renal medullary interstitial cells in vivo. Our data suggest that HIF-1α mediates ANG II-induced profibrotic effects through activation of cell transdifferentiation and that redox regulation of PHD plays a critical role in ANG II-induced activation of HIF-1α and consequent cell proliferation, transdifferentiation and abnormal extracellular matrix metabolism in renal cells.

Osteopontin, a secreted glycoprotein has been implicated in several renal pathological conditions such as those due to ureteral obstruction, ischemia, and cyclosporine toxicity. We studied its possible role in angiotensin II-mediated renal injury by infusing wild-type and osteopontin knockout mice with angiotensin II and found that it raised blood pressure and increased urinary albumin/creatinine ratios in both strains of mice. However, while wild-type mice responded to the infusion by macrophage infiltration and increased expression of alpha-smooth muscle actin, fibronectin, and transforming growth factor-beta; the osteopontin knockout mice developed none of these. Further, the knockout mice had increased expression of monocyte chemoattractant protein-1; NADPH oxidase subunits such as NOX2, gp47phox, and NOX4; and plasminogen activator inhibitor-1 compared to the wild type animals. Proximal tubule epithelial cells in culture treated with recombinant osteopontin and angiotensin II had increased alpha-smooth muscle actin and transforming growth factor-beta expression. The effect of angiotensin II was blocked by an antibody to osteopontin. In addition, osteopontin attenuated angiotensin II-induced plasminogen activator inhibitor-1 expression. These studies show that osteopontin is a promoter and an inhibitor of inflammation, oxidative stress, and fibrosis that is capable of modulating angiotensin II-induced renal damage.

In the setting of high salt intake, aldosterone stimulates fibrosis in the heart, great vessels, and kidney of rats. We used uninephrectomized rats treated with angiotensin II and placed on a high salt diet to exaggerate renal fibrosis. We then tested whether mineralocorticoid receptor blockade by spironolactone or aldosterone synthase inhibition by FAD286 have similar effects on end-organ damage and gene expression. Individually, both drugs prevented the hypertensive response to uninephrectomy and high salt intake but not when angiotensin II was administered. Following 4 weeks of treatment with FAD286, plasma aldosterone was reduced, whereas spironolactone increased aldosterone at 8 weeks of treatment. Angiotensin II and high salt treatment caused albuminuria, azotemia, renovascular hypertrophy, glomerular injury, increased plasminogen activator inhibitor-1 (PAI-1), and osteopontin mRNA expression, as well as tubulointerstitial fibrosis in the kidney. Both drugs prevented these renal effects and attenuated cardiac and aortic medial hypertrophy while reducing osteopontin and transforming growth factor-beta mRNA expression in the aorta. The two drugs also reduced cardiac interstitial fibrosis but had no effect on that of the perivascular region. Although spironolactone enhanced angiotensin II and salt-stimulated PAI-1 mRNA expression in aorta and heart, spironolactone and FAD286 prevented renal PAI-1 mRNA protein expression. Our study shows that mineralocorticoid receptor antagonism and aldosterone synthase inhibition similarly decrease hypertrophy and interstitial fibrosis of the kidney and heart caused by angiotensin II and high salt.

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor beta, TGFbeta) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-beta mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-beta upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-beta however, Smad-dependent transcription after 1 day was TGF-beta mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-beta-mediated processes; however, early changes were independent of endogenous TGF-beta. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-beta-independent processes, in vivo and in vitro, causing renal fibrosis.

Renin secretion is regulated by coordinated signaling between the various cells of the juxtaglomerular apparatus. The renin-secreting cells (RSC), which play a major role in the control of blood pressure, are coupled to each other and to endothelial cells by Connexin40 (Cx40)-containing channels. In this study, we show that Cx40 knockout (Cx40-/-) mice, but not their heterozygous littermates, are hypertensive due to the increase in the number of RSC, renin biosynthesis, and plasma renin. Treatment with the angiotensin II receptor AT1 antagonist candesartan or the angiotensin II-converting enzyme inhibitor ramipril reduced the blood pressure of the Cx40-/- mice to the same levels seen in wild-type (WT) mice. The elevated blood pressure of the knockout mice was not affected by clipping one renal artery (2K1C, renin-dependent model of hypertension) or after a high salt diet. Under these conditions, however, Cx40-/- mice showed an altered production and release of renin. The renin mRNA ratio between the clipped and the non-clipped kidney was lower in the knockout than in the WT 2K1C mice. This indicates that the response to a change in blood pressure was altered. The RSC of the Cx40-/- mice did not have a compensatory increase in the levels of either Cx43 or Cx37. Our data show that renin secretion is dependent on Cx40 and suggest the Cx40-/- mice may be a genetic model of renin-dependent hypertension.

Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-beta1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin.

The accumulation of advanced glycation end products (AGE) is a key factor in diabetic nephropathy (DN). Pyridoxamine inhibits AGE formation and protects against type I DN. Herein we tested: (1) whether C57BL6 db/db mice as a model of established type II DN resembled patients treated with drugs which inhibit angiotensin II action; (2) whether pyridoxamine was effective as a single therapy; and (3) whether pyridoxamine would add to the benefit of angiotensin-converting enzyme inhibition (ACEi) by enalapril. In first set of experiments mice were treated with ACEi (benazepril) and an angiotensin II receptor blocker (valsartan) combination for 16 weeks after the onset of diabetes. In second group, mice with established DN were treated with pyridoxamine for 8 weeks. In a third set, mice with established DN were treated with pyridoxamine and enalapril combination for 16 weeks. Benazepril and valsartan combination partially prevented the development and progression of DN. Pyridoxamine treatment, as single therapy, decreased the progression of albuminuria and glomerular lesions. The combination of pyridoxamine with enalapril reduced both mortality and the progression of DN. In conclusion, (1) C57 BL6 db/db mice are a model of progressive type II DN; (2) The combination of pyridoxamine with enalapril decreased progression of type 2 DN and overall mortality. Thus, pyridoxamine could be a valuable adjunct to the current treatment of established type II DN.

Information on the levels of angiotensin II (Ang II) and its receptors in the various renal tissue compartments is still incomplete. A model is presented describing the kinetics of Ang II production, distribution, and disposal in the renal cortex. Basic features are: (1) the model is designed to derive, from Ang II measurements in blood and in whole tissue, estimates of the local densities of the Ang II type 1 (AT(1)) and type 2 (AT(2)) receptors, and to calculate the concentrations of endocrine and paracrine Ang II they actually 'see'; (2) glomerular and peritubular tissue are conceived as separate regions (glomerular region (Glom), peritubular region (Pt)); (3) in Glom and in Pt, Ang II is homogeneously distributed in capillary blood and in interstitial fluid; (4) the model allows for local Ang II concentration gradients between interstitium and blood; (5) Ang II from the circulation diffuses into the interstitium of Glom after convective transcapillary transport; (6) Ang II produced in tubules or Pt enters the microcirculation through diffusive overflow from interstitium; (7) the presence of cell-surface-bound Ang II depends on the reaction with AT(1) and AT(2) receptors, and the presence of intracellular Ang II depends on the internalization of Ang II - AT(1) receptor complex; and (8) the model provides for glomerular filtration, vasopeptidase-mediated degradation, and intracellular degradation as mechanisms of elimination. This model can serve as a framework for detailed quantitative studies of the renin-angiotensin system in the kidney.

Information on the regional concentrations of angiotensin (Ang) II and its type-1 and -2 receptors (AT 1 R, AT 2 R) in the kidney is still incomplete. Published data on the levels of arterially delivered Ang I and II (Ang Ia, Ang IIa) and intrarenally produced Ang I and II (Ang Ii, Ang IIi) in the renal vein and in whole tissue were analyzed by using a kinetic model of Ang production and distribution in the glomerular and peritubular cortical tissue regions (Glom, Pt). (1) 90% of Ang II is cell-associated, due to its binding to AT 1 R and AT 2 R; (2) most Ang II in the renal cortex is Ang IIi; (3) Ang IIa is mainly localized in Glom; (4) Ang Ii rather than Ang Ia is a substrate of renal angiotensin-converting enzyme; (5) Ang IIi is localized in Pt and its concentration in interstitial fluid is 5–15 times the Ang II concentration in arterial plasma; and (6) in Glom the interstitial concentration of cell surface-bound AT 1 R is above 200 K d , and in Pt the AT 1 R and AT 2 R concentrations are above 10 K d . In conclusion, endocrine Ang II mainly acts in Glom, whereas Pt is exposed to paracrine Ang II generated by the conversion of intrarenally produced Ang I. High AT 1 R concentrations in Glom and Pt favor diffusion-limited binding, so that the apparent binding rate constant at sites closest to the source of Ang II delivery is greatly increased. Results may explain why the kidney is responsive to low levels of endocrine Ang II, despite its high content of paracrine Ang II.

The role of renin-angiotensin-aldosterone system in the progression of chronic kidney disease The renin-angiotensin-aldosterone system (RAAS) is a well known regulator of blood pressure (BP) and determinant of target-organ damage. It controls fluid and electrolyte balance through coordinated effects on the heart, blood vessels, and Kidneys. Angiotensin II (AII) is the main effector of the RAAS and exerts its vasoconstrictor effect predominantly on the postglomerular arterioles, thereby increasing the glomerular hydraulic pressure and the ultrafiltration of plasma proteins, effects that may contribute to the onset and progression of chronic renal damage. AII may also directly contribute to accelerate renal damage by sustaining cell growth, inflammation, and fibrosis. Interventions that inhibit the activity of the RAAS are renoprotective and may slow or even halt the progression of chronic nephropathies. ACE inhibitors and angiotensin II receptor antagonists can be used in combination to maximize RAAS inhibition and more effectively reduce proteinuria and GFR decline in diabetic and nondiabetic renal disease. Recent evidence suggests that add-on therapy with an aldosterone antagonist may further increase renoprotection, but may also enhance the risk hyperkalemia. Maximized RAAS inhibition, combined with intensified blood pressure control (and metabolic control in diabetics) and amelioration of dyslipidemia in a multimodal approach including lifestyle modifications (Remission Clinic), may achieve remission of proteinuria and renal function stabilization in a substantial proportion of patients with proteinuric renal disease. Ongoing studies will tell whether novel drugs inhibiting the RAAS, such as the renin inhibitors or the vasopeptidase inhibitors, may offer additional benefits to those who do not respond, or only partially respond, to this multimodal regimen.

Prevention of progression and remission/regression strategies for chronic renal diseases: Can we do better now than five years ago? The prevalence of chronic renal diseases is increasing worldwide. There is a great need to identify therapies that arrest disease progression to end-stage renal failure. Inhibition of renin-angiotensin system both by ACE inhibitors and angiotensin II receptor antagonists is probably the best therapeutic option available. Several large, multicenter studies have indeed shown a significant reduction in the risk of doubling baseline serum creatinine or progression toward end-stage renal failure in diabetic and nondiabetic patients with chronic nephropathies treated with ACE inhibitors or angiotensin II receptor antagonists. However, the number of patients that reach end-stage renal failure is still considerably high. Significant reduction of the incidence of end-stage renal disease is likely to be achieved in the next future for chronic nephropathies, provided that we can improve the degree of renoprotection. This goal may be attainable with a more complex strategy than with a single or dual pharmacologic intervention on the renin-angiotensin system. Strict control of blood pressure and protein excretion rate, lowering of blood lipids, tight glucose control for diabetics, and lifestyle changes form part of the future multimodal protocol for management of patients with chronic nephropathies.

Oxygen consumption in the kidney: Effects of nitric oxide synthase isoforms and angiotensin II. Background Oxygen mitochondrial effects consumption by the kidney (Qo 2 ), is linearly related to sodium reabsorption (T na ), but recent studies suggest this relationship is variable and that metabolic efficiency (Qo 2 /T na ) in kidney is regulated by hormonal factors. In the dog, nonselective inhibitors of nitric oxide synthase (NOS) increase Qo 2 and Qo 2 /T na . Glomerular hemodynamic and reabsorptive consequences of NOS inhibition require angiotensin II (Ang II), implying an antagonistic relationship between nitric oxide and Ang II. Effects of NOS inhibition in the rat, the role of Ang II and the responsible NOS isoform have not been elucidated. Methods Kidney blood flow [renal blood flow (RBF)], glomerular filtration rate (GFR), and Qo 2 /T na were measured before and during intravenous administration of N G -monomethyl-L-arginine (L-NMMA), a nonselective NOS inhibitor, in control and losartan (Ang II receptor blocker)-treated rats and rats administered S-methyl-L-thiocitrulline (SMTC), a NOS-1 inhibitor. Effects of SMTC on oxygen consumption were also examined in freshly harvested proximal tubules. Results L-NMMA and high-dose SMTC decreased RBF, but L-NMMA + losartan and low-dose SMTC did not. Qo 2 /T na increased in both L-NMMA groups. Both low- and high-dose SMTC also increased Qo 2 /T na . SMTC increased Qo 2 in proximal tubules in vitro at presumed lower levels of vectorial NaCl transport. Results suggest this effect was not mediated by influences on sodium transport alone. Conclusion Nonselective NOS inhibition increases the oxygen costs of kidney function independent of Ang II. Kidney NOS-1 is responsible for these in vivo and in vitro effects. In vitro observations suggest that NOS-1 acts in part via effects on basal metabolism and mitochondrial function.

Aberrant ETB receptor regulation of AT 1 receptors in immortalized renal proximal tubule cells of spontaneously hypertensive rats. Background The renin-angiotensin and endothelin systems interact to regulate blood pressure, in part, by affecting sodium transport in the kidney. Because angiotensin II type 1 (AT 1 ) receptor activation increases ETB receptor expression in renal proximal tubule cells from Wistar-Kyoto (WKY) rat, we hypothesize that ETB receptor activation may also regulate AT 1 receptor expression. Furthermore, ETB receptor regulation of the AT 1 receptor may be different in the WKY and spontaneously hypertensive rat (SHR). Method AT 1 and ETB receptors were studied in immortalized renal proximal tubule cells from WKY and SHRs, using immunoblotting, confocal microscopic colocalization, and immunoprecipitation. Results In WKY renal proximal tubule cells, an ETB receptor agonist, BQ3020, decreased AT 1 receptor protein in a time- and concentration-dependent manner [median effective concentration (EC 50 ) = 3.2 × 10 -10 mol/L, t 1/2 = 15 hours]. The inhibitory effect of BQ3020 (10 -8 mol/L/24 hours) on AT 1 receptor protein was blocked by an ETB receptor antagonist (BQ788). However, BQ3020 (10 -8 mol/L/24 hours) increased ETB receptor protein in WKY renal proximal tubule cells. In contrast, in SHR renal proximal tubule cells, BQ3020 (10 -8 mol/L/24 hours) no longer affected AT 1 or ETB receptor protein. AT 1 /ETB receptors colocalized and coimmunoprecipitated in WKY and SHRs. BQ3020 (10 -8 mol/L/15 minutes) treatment had no effect on AT 1 /ETB coimmunoprecipitation in WKY but decreased it in SHRs. BQ3020 (10 -8 mol/L/15 minutes) treatment increased AT 1 receptor phosphorylation in WKY, but decreased it in SHRs. Conclusion ETB receptors regulate AT 1 receptors by direct physical receptor interaction and receptor expression. An impaired ETB receptor regulation of the AT 1 receptor may participate in the pathogenesis of high blood pressure in the SHR.

Angiotensin II receptor blocker inhibits p27Kip1 expression in glucose-stimulated podocytes and in diabetic glomeruli.BackgroundDiabetic nephropathy is characterized by glomerular and tubular hypertrophy, and angiotensin II receptor blockers (ARBs) are known to prevent renal hypertrophy in diabetic patients.MethodsTo determine the effect of ARB on podocyte p27Kip1 mRNA and protein expression, podocytes were exposed to 5.6 mmol/L normal glucose or 25 mmol/L high glucose with or without ARB, 10-7 mol/L L-158,809. For animal studies, streptozotocin-induced diabetic rats were left untreated or were treated with 1 mg/kg/day L-158,809 for 3 months (diabetes mellitus + ARB). Competitive reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and morphometric analyses were performed.Resultsp27Kip1 mRNA and protein expression in podocytes exposed to high glucose and in 3-month diabetic glomeruli were significantly increased (P < 0.01). High glucose significantly increased angiotensin II levels both in cell lysates and in media compared with normal glucose (P < 0.05) and exogenous angiotensin II also increased p27Kip1 mRNA and protein expression in podocytes. L-158,809 treatment in podocytes inhibited the increase in p27Kip1 mRNA expression by 84%, and protein expression by 89% (P < 0.05). p27Kip1 mRNA and protein expression in diabetic + ARB glomeruli were also significantly reduced by 78% and 85%, respectively, compared with diabetic glomeruli (P < 0.01). ARB treatment also significantly ameliorated increased glomerular p27Kip1 expression in diabetes mellitus as assessed by immunohistochemistry (P < 0.01). The increase in glomerular volume in diabetes mellitus was also inhibited by 81% with ARB treatment (P < 0.05).Conclusionp27Kip1 mRNA and protein expression were increased in diabetic glomeruli as well as in high glucose–stimulated podocytes, and this increment in p27Kip1 expression was ameliorated by ARB treatment. These findings indicate that ARB treatment has an additional effect on preventing renal hypertrophy in diabetes mellitus.

Angiotensin II receptor blockade prevents acute renal sodium retention induced by low levels of orthostatic stress.BackgroundDepending on its magnitude, lower body negative pressure (LBNP) has been shown to induce a progressive activation of neurohormonal, renal tubular, and renal hemodynamic responses, thereby mimicking the renal responses observed in clinical conditions characterized by a low effective arterial volume such as congestive heart failure. Our objective was to evaluate the impact of angiotensin II receptor blockade with candesartan on the renal hemodynamic and urinary excretory responses to a progressive orthostatic stress in normal subjects.MethodsTwenty healthy men were submitted to three levels of LBNP (0, -10, and -20mbar or 0, -7.5, and -15mm Hg) for 1hour according to a crossover design with a minimum of 2days between each level of LBNP. Ten subjects were randomly allocated to receive a placebo and ten others were treated with candesartan 16mg orally for 10days before and during the three levels of LBNP. Systemic and renal hemodynamics, renal sodium excretions, and the hormonal response were measured hourly before, during, and for 2hours after LBNP.ResultsDuring placebo, LBNP induced no change in systemic and renal hemodynamics, but sodium excretion decreased dose dependently with higher levels of LBNP. At -20 mbar, cumulative 3-hour sodium balance was negative at –2.3 ± 2.3mmol (mean ± SEM). With candesartan, mean blood pressure decreased (76 ± 1mm Hg vs. 83 ± 3mm Hg, candesartan vs. placebo, P < 0.05) and renal plasma flow increased (858 ± 52mL/min vs. 639 ± 36mL/min, candesartan vs. placebo, P < 0.05). Glomerular filtration rate (GFR) was not significantly higher with candesartan (127 ± 7mL/min in placebo vs. 144 ± 12mL/min in candesartan). No significant decrease in sodium and water excretion was found during LBNP in candesartan-treated subjects. At –20 mbar, the 3-hour cumulative sodium excretion was + 4.6 ± 1.4mmol in the candesartan group (P = 0.02 vs. placebo).ConclusionSelective blockade of angiotensin II type 1 (AT1) receptors with candesartan increases renal blood flow and prevents the antinatriuresis during sustained lower body negative pressure despite a modest decrease in blood pressure. These results thus provide interesting insights into potential benefits of AT1 receptor blockade in sodium-retaining states such as congestive heart failure.

Angiotensinogen gene variation and renoprotective efficacy of renin-angiotensin system blockade in IgA nephropathy.BackgroundBlockade of the renin-angiotensin system (RAS) is well documented to be renoprotective; however, not all patients with glomerulonephritis respond well to this therapy. The interindividual variation in response to the RAS blockade may be in part genetically determined, whereas the results have been controversial.MethodsWe investigated whether the therapeutic efficacy of angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin receptor blocker on renal prognosis is modified by the angiotensinogen gene (AGT) polymorphism in immunoglobulin A nephropathy (IgAN). In total, 259 patients with histologically proven IgAN were analyzed for clinical manifestations, renal survival, and their associations with AGT A(-20)C and M235T.ResultsThe renal prognosis of 110 patients, who received ACE inhibitors/angiotensin receptor blocker during their clinical course, was significantly better than those without ACE inhibitors/angiotensin receptor blockers despite higher blood pressures and heavier proteinuria. The Cox proportional hazards regression model showed an increased hazard ratio (HR) for urinary protein (more than 1.0g/day) of 3.346 (P = 0.0001), hypertension of 1.949 (P = 0.01), deteriorated renal function of 3.040 (P < 0.0001), no ACE inhibitor/angiotensin receptor blocker administration of 2.725 (P = 0.0004), and the T235 and C(-20) haplotype of 1.608 (P = 0.0322). Only in patients carrying at least one M235 and A(-20) haplotype did the administration of ACE inhibitors/angiotensin receptor blockers have no significant effect on the prognosis of renal function (Kaplan-Meier, log rank test, χ2 = 0.700; P = 0.4028), whereas it was significant in patients who had other haplotypes of AGT (χ2 = 11.805; P = 0.0006).ConclusionThis study provides evidence that the M235T and A(-20)C genotype of AGT can influence the therapeutic efficacy of a RAS blockade on the renal survival in IgAN.

Angiotensin II regulates 11β-hydroxysteroid dehydrogenase type 2 via AT2 receptors. Background In preeclampsia, cortisol degradation by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is compromised, which enhances intracellular cortisol availability. This leads to vasoconstriction and renal sodium retention with volume expansion, thus increasing blood pressure. An augmented availability of angiotensin II (Ang II) predisposes to preeclampsia. Some effects of Ang II are mediated by the mitogen-activated protein kinase (MAPK) cascade, which also regulates 11β-HSD2 activity. Therefore, we hypothesized that Ang II regulates 11β-HSD2. Methods The human choriocarcinoma cell line JEG-3, which expresses the 11β-HSD2 isoenzyme, was used. 3 H-cortisol/cortisone conversion assays and mRNA analyses by reverse transcription-polymerase chain reaction (RT-PCR) were performed. Cells were stimulated with Ang II and the effect was modulated by Ang II type 1 (AT1) and AT2 receptor blockers DUP753 or L-158809 and PD-123319, respectively. In order to elucidate the signaling cascade, the MAPK kinase inhibitors PD-098059 and U-0126 were probed. The impact of a modulated 11β-HSD2 activity was assessed by determining the effect of cortisol on AT1 receptor mRNA. Results Ang II reduced mRNA and activity of 11β-HSD2 mainly by a post-transcriptional mechanism. This Ang II effect was abrogated by AT2, but not by AT1 receptor blockade. Mitogen-activated protein (MAP) kinase kinase (MAPKK) inhibitors reversed the Ang II effect. Dexamethasone augmented the mRNA expression of AT1 receptors. Cortisol enhanced AT1 receptor mRNA expression when the 11β-HSD2 activity was reduced either by Ang II or by glycyrrhetinic acid, an 11β-HSD2 inhibitor. Conclusion Ang II decreases the activity of 11β-HSD2 by an AT2 receptor– and MAPK-dependent mechanism. The decreased activity of 11β-HSD2 increases the intracellular availability of cortisol, which might be relevant for the pathogenesis of hypertension and preeclampsia.

Effects of combined ACE inhibitor and angiotensin II antagonist treatment in human chronic nephropathies.BackgroundProteinuria predicts renal disease progression, and its reduction by angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II receptor antagonists (ARA) is renoprotective.MethodsIn this prospective, randomized, cross-over study of 24 patients with nondiabetic, chronic nephropathies, we compared the effects on proteinuria, renal hemodynamics, and glomerular permselectivity of 8 weeks with comparable blood pressure control achieved by benazepril (10 mg/day) and valsartan (80 mg/day) combined therapy with those achieved by benazepril (20 mg/day) or valsartan (160 mg/day) alone.ResultsDespite comparable changes in blood pressure and glomerular filtration rate (GFR), combined therapy decreased proteinuria more than benazepril (-56% vs. -45.9%, P = 0.02) and valsartan (-41.5%, P = 0.002). Changes in urinary protein to creatinine ratio followed the same trend. Filtration fraction and renal vascular resistances (RVR) decreased more with combined (-14.7%,-23.7%) or benazepril (-12.4%, -20.5%) than with valsartan (-2.7%, -12.5%, P < 0.05 vs. both). RVR changes, adjusted for GFR changes, were associated with those in proteinuria (P < 0.05). Changes in glomerular permeability were comparable and did not predict different changes in proteinuria in the three groups.ConclusionAt comparable blood pressure, combined ACEi and ARA decreased proteinuria better than ACEi and ARA. The greater antiproteinuric effect most likely depended on an ACEi-related hemodynamic effect, in addition to glomerular size selectivity amelioration. Long-term combined ACEi and ARA therapy may be more renoprotective than treatment with each agent alone.

Clinical renoprotection trials involving angiotensin II-receptor antagonists and angiotensin-converting-enzyme inhibitors. The leading cause of end-stage renal disease (ESRD) is diabetic nephropathy. Interventions that prevent or palliate renal disease have a positive impact by improving patient care and diminishing healthcare costs. The following review summarizes clinical trals that evaluated treatment in various patient populations including Type 2 diabetic patients with microalbuminuria, subjects with proteinuric non-diabetic nephropathies, and Type 1 and Type 2 diabetic patients with overt nephropathy. These clinical trials have demonstrated that agents directly affecting the renin-angiotensin-aldosterone system provide renal protection beyond that attributable to blood pressure control.

Effects of angiotensin II receptor antagonism on the renal hemodynamic response to cardiovascular stress.BackgroundTo elucidate the effect of the angiotensin type 1 (AT1) receptor antagonist (AT1RA) eprosartan (E) on renal hemodynamics in normotensive and borderline hypertensive subjects, we investigated the hormonal and renal hemodynamic responses during cardiopulmonary stress testing.MethodsIn a prospective, double-blind, randomized, placebo-controlled crossover study, the effects of E on renal plasma flow (RPF), renal blood flow (RBF), glomerular filtration rate (GFR), and the concentration of angiotensin II (Ang II) levels were measured with the subjects at rest and during perturbation of cardiopulmonary baroreceptors using lower body negative pressure (LBNP). Ten normotensive male subjects (NT) versus 14 males with mild hypertension (HT), matched for age and body mass index, who were all free of any medication, were randomly assigned to receive placebo or E 600 mg/day PO for seven days (intake phase 1). After a washout period of four weeks the subjects started the intake of the other substance for seven days in a crossover manner (intake phase 2). The measurements were taken on day 7 of both intake phases.ResultsDuring the LBNP test, RPF and RBF were reduced significantly in all subjects; GFR, however, decreased significantly during cardiopulmonary stress testing in the subjects taking the placebo (P < 0.05) and remained unchanged in those under treatment with AT1RA. Ang II levels increased significantly during cardiopulmonary stress test only in the subjects with hypertension who were on placebo, whereas the Ang II levels did not change in normotensive subjects or those treated with the AT1RA.ConclusionsThe data confirm that with cardiovascular stress simulating orthostasis or volume depletion, subjects with AT1RA can maintain their GFR level, suggesting that AT1RA potentially is renoprotective. Additionally, the neurohumoral system is activated after cardiovascular stress in subjects even at an early stage of hypertension.

Glomerular hemodynamics and the renin-angiotensin system in patients with type 1 diabetes mellitus.BackgroundMany studies have reported that blocking the renin-angiotensin-system (RAS) with an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker in the patient with diabetes mellitus leads to an increase in renal plasma flow (RPF), no change in glomerular filtration rate (GFR), and a fall in filtration fraction. This constellation is generally attributed to predominant efferent arteriolar dilation.MethodsThis study examined the renal hemodynamic response to blocking the RAS with both captopril and candesartan on separate days in 31 patients with type 1 diabetes mellitus.ResultsThere was a wide range of changes in RPF and GFR in response to the two agents, each administered at the top of its dose-response range. The RPF response to the two agents was strongly concordant (r = 0.65; P < 0.001), as was the GFR response (r = 0.81; P < 0.001). Moreover, there was a strong correlation between the RPF response and the change in GFR with each agent (r = 0.83 and 0.66; P < 0.01). A significant rise in RPF was followed by a rise in GFR. The RPF dependency of GFR in the type 1 diabetics suggests strongly that glomerular filtration equilibrium exists in the glomeruli of the diabetic kidney: Simple notions of local control based on afferent:efferent arteriolar resistance ratios are too simplistic.ConclusionOur data suggest that the intrarenal RAS is activated in over 80% of patients with type 1 diabetes mellitus. Abundant evidence suggests that this activation predisposes to diabetic nephropathy.

Renal damage associated with proteinuria. Experimental studies have demonstrated that proteins filtered by the glomerulus induce a proliferation of proximal tubular cells accompanied by an increased synthesis of many vasoactive and proinflammatory substances. The appearance of interstitial cellular infiltrates, a well-known finding in proteinuric diseases, precedes progressive tubulointerstitial fibrosis. Activation of the transcription factor κB (NF-κB) plays a pivotal role in the renal damage induced by proteinuria. In this scenario, any therapeutic intervention that reduces proteinuria should be beneficial for the kidney. Drugs that block the renin-angiotensin system [angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor antagonists (ARA)] have repeatedly shown striking antiproteinuric and renoprotective properties, both in experimental and clinical studies. Studies in patients with type 1 and type 2 diabetic nephropathy as well as in non-diabetic nephropathies have confirmed that the renoprotection obtained with ACEI/ARA is closely related with their antiproteinuric effect and is largely independent of blood pressure changes. However, resistance to the antiproteinuric effect of ACEI/ARA is a common clinical observation. Several therapeutic measures (that is, adequate blood pressure control, early introduction of ACEI/ARA, dietary protein restriction, low salt diets, weight loss in overweight patients, addition of a diuretic, increasing ACEI/ARA dose titrated against proteinuria levels, combined therapy ACEI plus ARA, addition of drugs with antiproteinuric effect such as non-dihydropiridine calcium channel blockers or NSAIDs) may increase the proteinuria reduction induced by ACEI and ARA.

High glucose down-regulates angiotensin II binding via the PKC-MAPK-cPLA 2 signal cascade in renal proximal tubule cells. Background It has been reported that renal renin-angiotensin system contributes to the development of diabetic nephropathy. However, the mechanism of angiotensin II receptor regulation in diabetic condition has not been elucidated. Methods The effects of high glucose on [ 3 H]-arachidonic acid (AA) release and angiotensin II (Ang II) binding and its related signal pathway were examined in primary cultured rabbit renal proximal tubule cells (PTCs). Results High glucose down-regulated 125 I-Ang II binding from 12 hours and this response was sustained over 48 hours. Thus, the treatment of 25 mmol/L glucose for 48 hours was used for this study. High glucose-induced down-regulation of 125 I-Ang II binding was reversed by the removal of extracellular glucose, suggesting a role for glucose specificity. The high glucose-induced down-regulation of 125 I-Ang II binding was blocked by mepacrine, AACOCF 3 , phospholipase A 2 inhibitors, indomethacin, ibuprofen, and cyclooxygenase inhibitors. Indeed, high glucose significantly increased prostaglandin E 2 synthesis. In addition, the high glucose-induced AA release was blocked by PD 98059, a p44/42 mitogen-activated protein kinase (MAPK) inhibitor. PD 98059 also prevented the down-regulation of 125 I-Ang II binding by high glucose, suggesting a role for p44/42 MAPK. Indeed, high glucose significantly increased p44/42 MAPK activity after the 15-minute time point. Protein kinase C (PKC) inhibitor blocked high glucose-induced activation of p44/42 MAPK, increase of the [ 3 H]-AA release, and down-regulation of 125 I-Ang II binding. W-7 and KN-62 also blocked the high glucose-induced increase of [ 3 H]-AA release and down-regulation of 125 I-Ang II binding. However, phospholipase A 2 inhibitor did not block high glucose-induced activation of p44/42 MAPK. Conclusion High glucose down-regulates 125 I-Ang II binding via the PKC-MAPK-cPLA 2 signal pathway.

Coordinate regulation of canine glomeruli and adrenal angiotensin receptors by dietary sodium manipulation. Background This study evaluated the effects of dietary sodium manipulation in dogs on the regulation of canine angiotensin receptors (cAT1 and cAT2) in the kidney and adrenal. Methods Isolated glomeruli and membranes from renal medulla and the adrenal gland were used in radioligand binding assays from two groups of dogs: dogs maintained on low-sodium diet for two weeks followed by a high-sodium diet for two weeks (H), and dogs were maintained on the reverse schedule (L). Results Analysis of the binding data showed that dietary sodium manipulation had no significant effects on cAT1 and cAT2 receptor binding affinities in glomeruli, renal medulla, and adrenal tissues. In contrast, dietary sodium loading induced a marked increase in cAT1 receptor expression in both the glomeruli and adrenal compared with receptor expression in salt-restricted animals [H/L ratio: glomeruli (1.5), renal medulla (1.1), adrenal (1.6)] that inversely correlated with the activity of the plasma renin angiotensin system. Conversely, adrenal cAT2 receptor expression was regulated in an inverse manner in the H and L animal groups [H/L ratio: 0.7]. Conclusions This study demonstrates that renal glomerular and adrenal AT1 receptors in the dog are coordinately down-regulated by dietary sodium restriction compared with sodium loading, which is distinctly different from the reciprocal regulation observed for rat AT1 receptors in these tissues. Collectively, these data suggest that postreceptor events in dogs are determinants of the aldosterone response observed during sodium restriction. These findings have important implications for the regulation of the renin-angiotensin system in humans, and suggest that coordinate regulation of AT1 receptors in the adrenal and glomeruli represent a negative feedback mechanism that when functioning normally prevents fluctuations of arterial blood pressure and development of arterial hypertension in response to changes in dietary sodium.

Chronic potassium depletion induces renal injury, salt sensitivity, and hypertension in young rats. Background Chronic hypokalemia has been associated with renal hypertrophy, interstitial disease, and hypertension in both adult animals and humans. However, the effects of potassium (K + ) depletion on the rapidly growing infant have not been well studied. The purpose of this study was to determine the effects of severe chronic dietary K + depletion on blood pressure (BP) and renal structural changes in young rats. Methods Sprague-Dawley rats (50 ± 5g) were fed either a control or a potassium-deficient diet ( + ) for 14 to 21 days. At the end of this period, the blood pressure (BP) was measured in all rats, and six rats in each group were sacrificed to determine changes in renal histology and renin-angiotensin system (RAS) activity. The remaining rats in each group were then switched to a high-salt (6% NaCl)-normal-K + (0.5%) diet or were continued on their respective control or K + -deficient diet for an additional six days. Blood pressure measurements were done every three days until the end of the study. Results K + -depleted animals had significant growth retardation and increased RAS activity, manifested by high plasma renin activity, recruitment of renin-producing cells along the afferent arterioles, and down-regulation of angiotensin II receptors in renal glomeruli and ascending vasa rectae. K + -depleted kidneys also showed tubulointerstitial injury with tubular cell proliferation, osteopontin expression, macrophage infiltration, and early fibrosis. At week 2, K + -depleted rats had higher systolic BP than control rats. Switching to a high-salt (6% NaCl)-normal-K + diet resulted in further elevation of systolic BP in K + -depleted rats, which persisted even after the serum K + was normalized. Conclusion Dietary potassium deficiency per se increases the BP in young rats and induces salt sensitivity that may involve at least two different pathogenic pathways: increased RAS activity and induction of tubulointerstitial injury.

ACE inhibition or angiotensin receptor blockade: Impact on potassium in renal failure. Background Inhibition of the renin-angiotensin system is known to raise serum potassium [K + ] levels in patients with renal insufficiency or diabetes. No study has evaluated the comparative effects of an angiotensin-converting enzyme (ACE) inhibitor versus an angiotensin receptor blocker (ARB) on the changes in serum [K + ] in people with renal insufficiency. Methods The study was a multicenter, randomized, double crossover design, with each period lasting one month. A total of 35 people (21 males and 14 females, 19 African Americans and 16 Caucasian) participated, with the mean age being 56 ± 2 years. Mean baseline serum [K + ] was 4.4 ± 0.1 mEq/L. The glomerular filtration rate (GFR) was 65 ± 5 mL/min/1.73 m 2 , and blood pressure was 150 ± 2/88 ± 1 mm Hg. The main outcome measure was the difference from baseline in the level of serum [K + ], plasma aldosterone, and GFR following the initial and crossover periods. Results For the total group, serum [K + ] changes were not significantly different between the lisinopril or valsartan treatments. The subgroup with GFR values of ≤60 mL/min/1.73 m 2 who received lisinopril demonstrated significant increases in serum [K + ] of 0.28 mEq/L above the mean baseline of 4.6 mEq/L ( P = 0.04). This increase in serum [K + ] was also accompanied by a decrease in plasma aldosterone ( P = 0.003). Relative to the total group, the change in serum [K + ] from baseline to post-treatment in the lisinopril group was higher among those with GFR values of ≤60 mL/min/1.73 m 2 . The lower GFR group taking valsartan, however, demonstrated a smaller rise in serum [K + ], 0.12 mEq/L above baseline ( P = 0.1), a 43% lower value when compared with the change in those who received lisinopril. This blunted rise in [K + ] in people taking valsartan was not associated with a significant decrease in plasma aldosterone ( P = 0.14). Conclusions In the presence of renal insufficiency, the ARB valsartan did not raise serum [K + ] to the same degree as the ACE inhibitor lisinopril. This differential effect on serum [K + ] is related to a relatively smaller reduction in plasma aldosterone by the ARB and is not related to changes in GFR. This study provides evidence that increases in serum [K + ] are less likely with ARB therapy compared with ACE inhibitor therapy in people with renal insufficiency.

The angiotensin type 2 receptor tonically inhibits angiotensin- converting enzyme in AT2 null mutant mice.BackgroundPharmacologic inhibition of the angiotensin-converting enzyme (ACE) limits angiotensin II (Ang II)-induced vasoconstriction and cellular proliferation. There is emerging evidence that some of the beneficial effects of ACE inhibitors may be endogenously available through the angiotensin receptor type 2 (AT2).MethodsTo evaluate whether AT2 modulates ACE activity, we used an high-performance liquid chromatography (HPLC)-based enzymatic assay in tissues from AT2 knockout mice (Agtr2-/y) and cultured cells. These studies were complimented by physiologic studies of pharmacologic inhibition of AT2.ResultsCirculating (C) and tissue ACE activities in heart (H), lung (L), and kidney (K) were doubled in Agtr2-/y mice compared with wild-type mice [162.9 ± 17.6 mU/mL (C), 97.7 ± 20.7 (H), 6282.1 ± 508.3 (L), and 2295.0 ± 87.0 (K) mU/g tissue for Agtr2-/y vs. 65.3 ± 35.4 mU/mL (C), 44.5 ± 8.7 (H), 3392.4 ± 495.2 (L), and 1146.1 ± 217.3 (K) mU/g tissue for wild-type mice, P ≤ 0.05, 0.025, 0.002, and 0.0001, respectively]. Acute pharmacologic inhibition of AT2 [PD123319 (PD), 50 μg/kg/min, i.v.] significantly increased ACE activity in kidneys of wild-type mice (1591.2 ± 104.4 vs. 1233.6 ± 88.0 mU/g tissue in saline-infused mice, P < 0.05; P < 0.01 vs. uninfused, wild-type mice). Moreover, ACE activity increased in A10 cells exposed to PD (10-6 mol/L) together with Ang II (10-7 mol/L), but not with an AT1 antagonist (losartan, 10-6 mol/L). This heightened ACE activity appears functionally relevant because infusion of angiotensin I caused more prompt hypertension in Agtr2-/y mice than in wild-type littermates. Likewise, infusion of bradykinin, also a substrate for ACE, caused significantly less hypotension in Agtr2-/y mice than controls.ConclusionsThese studies indicate that AT2 functions to decrease ACE activity tonically, which may, in part, underlie AT2's increasingly recognized attenuation of AT1-mediated actions.

Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Background Angiotensin II is strongly incriminated in progressive renal injury. There is recent evidence that angiotensin II induces oxidative stress in vitro. We examined the capacity of angiotensin II to induce oxidative stress in vivo and the functional significance of such stress. The capacity of angiotensin II to induce the oxidant-sensitive gene heme oxygenase (HO) in vivo and in vitro was also examined. Methods Angiotensin II was administered via mini-osmotic pumps to rats maintained on standard diets. Indices of oxidative stress, including thiobarbituric acid reactive substance, carbonyl protein content, and HO activity, were determined. Indices of oxidative stress and functional markers were also determined in the DOCA salt model. The effect of angiotensin II was studied in rats maintained on antioxidant-deficient diets so as to examine the functional significance of oxidative stress induced by angiotensin II. We also explored the inductive effect of angiotensin II on HO in vivo and whether such actions occur in vitro. Results Angiotensin II administered in vivo increased kidney content of thiobarbituric acid reactive substances protein carbonyl content, and HO activity. These indices were not present in the kidney of rats treated with DOCA salt for three weeks. Such oxidative stress was functionally significant, since the administration of angiotensin II to rats maintained on a prooxidant diet demonstrated increased proteinuria and decreased creatinine clearance. The stimulatory effect on HO activity was due to induction of HO-1 mRNA, with HO-2 mRNA remaining unchanged. Expression of HO-1 was localized to the renal proximal tubules in vivo. We also demonstrate that angiotensin II at concentrations of 10 -8 and 10 -7 mol/L induces expression of HO-1 mRNA in LLC-PK1 cells. Conclusions Angiotensin II induces oxidative stress in vivo, which contributes to renal injury. This study also demonstrates that angiotensin II induces renal HO activity caused by up-regulation of HO-1 in renal proximal tubules. Finally, angiotensin II directly induces HO-1 in renal proximal tubular epithelial cells in vitro.

Effects of endothelin or angiotensin II receptor blockade on diabetes in the transgenic (mRen-2)27 rat. Background Endothelin (ET) and angiotensin II (Ang II) are vasoactive/trophic peptides that may contribute to the progression of diabetic nephropathy. The transgenic (mRen-2)27 rat exhibits overexpression of Ang II at sites of normal physiological expression. Unlike other rat strains, the streptozotocin-induced diabetic Ren-2 rat develops progressive renal pathology associated with a declining glomerular filtration rate (GFR) and provides a convenient model to evaluate the role of these vasoactive peptides in the nephropathic process. Methods and Results Oral administration of either the endothelin A (ET A ) and ET B receptor antagonist bosentan or the angiotensin type 1 (AT 1 ) receptor antagonist valsartan for 12 weeks reduced systolic blood pressure (SBP) of nondiabetic and diabetic Ren-2 rats to normotensive levels. Diabetic renal pathology was associated with intense renin mRNA and protein in the proximal tubules and juxtaglomerular cells along with overexpression of transforming growth factor-β1 (TGF-β1) and collagen IV mRNA in glomeruli and tubules. With valsartan but not bosentan, renin mRNA and protein in the proximal tubules were not detected. Valsartan but not bosentan reduced TGF-β1 and collagen IV mRNA and the severity of diabetic renal pathology. A declining GFR with diabetes was attenuated by both treatments. Albuminuria in diabetic rats rose further with bosentan but was reduced with valsartan. Conclusions Despite producing normotension, severe diabetic renal pathology was not prevented by bosentan, suggesting dissociation of ET, albuminuria, and hypertension from the structural injury in this diabetic model. The beneficial effects afforded by valsartan therapy strengthen the importance of the local renin-angiotensin system in mediating progressive diabetic renal injury.

The role of vasoactive compounds, growth factors and cytokines in the progression of renal disease. A number of kidney diseases, and their progression to end-stage renal disease, are driven, in part, by the effects of angiotensin II. Increasing levels of angiotensin II may in turn up-regulate the expression of growth factors and cytokines, such as transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), osteopontin, vascular cell adhesion molecule-1 (VCAM-1), nuclear factor-kB (NF-kB), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor. Most of these compounds promote cell growth and fibrosis. Angiotensin II also stimulates oxidative stress. This stress in turn may potentiate the vasoconstrictor effect of the peptide due, in part, to increased catabolism of nitric oxide (NO). Oxidative stress, fueled in part by angiotensin II, up-regulates the expression of adhesion molecules, chemoattractant compounds and cytokines. The angiotensinogen gene, which provides the precursor for angiotensin production, is stimulated by NF-kB activation. NF-kB is activated by angiotensin in the liver and in the kidney. This provides an autocrine reinforcing loop that up-regulates angiotensin production. Angiotensin II activates NF-kB through both AT1 and AT2 receptors. In addition, angiotensin-converting enzyme (ACE) inhibition markedly decreases NF-kB activation in the setting of renal disease.

Angiotensin II plays an important role in the regulation of blood pressure, body salt and fluid balance, and urine concentration. Mice with deletion of the AT1a receptor develop polyuria and urine concentration defects. We studied the mechanisms of these urine concentration defects by treating wild-type and AT1a-knockout mice with arginine vasopressin (AVP) for 2 weeks, controlling their water intake, or giving them an osmotic diuretic (sucrose) in order to determine whether central or nephrogenic mechanisms were involved. Under basal conditions, AT1a-knockout mice were hypotensive, had lower plasma AVP, and excreted more urine with a markedly reduced osmolality compared with wild-type mice. However, basal glomerular filtration rates were similar in both strains of mice. We isolated total lysate and membrane proteins from the inner medulla of wild-type and mutant mouse kidneys, and found that the amounts of aquaporin 2 (AQP2), adenylyl cyclases III and V/VI, and phosphorylated MAP kinases ERK 1/2 proteins were all reduced in the inner medulla of the knockout mice. Infusion of AVP raised plasma levels and blood pressure proportionally in both strains, but polyuria persisted and urine osmolality remained significantly lower in the knockout mice. Although AVP increased urine osmolality slightly in water-deprived knockout mice, this was well below the basal osmolality of wild-type mice. The diuretic response to the hyperosmotic sucrose was also impaired in the knockout mice. Neither AVP nor water rationing restored the levels of the inner medullary signaling proteins and membrane AQP2 proteins in the knockout mice. We suggest that AT1a receptor deletion causes polyuria and urine concentration defects by decreasing basal AVP release and impairing AVP-induced receptor signaling in the inner medulla.

Cyclosporine A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells. Background The most widely used immunosuppressive drug for preventing graft rejection and treating autoimmune diseases is currently cyclosporine A (CsA). However, CsA also causes vasoconstriction, which is considered to be at the origin of CsA-induced nephrotoxicity and hypertension. To evaluate the cellular basis for these side effects, we studied the influence of CsA on the regulation of the free cytosolic Ca 2+ concentration ([Ca 2+ ] c ) in cultured human vascular smooth muscle cells (SMCs). Methods SMCs were isolated from the medial layer of human aorta. [Ca 2+ ] c regulation was studied by fluorimetry with fura 2 and by measuring 45 Ca 2+ effluxes. Angiotensin II (Ang II) receptors were detected by [ 125 I]Ang II binding. Results Pretreatment of human SMCs for 24hours with CsA in its therapeutic concentration range (0.1 to 10.0 μm) had no effect on basal [Ca 2+ ] c , but increased the [Ca 2+ ] c elevation and 45 Ca 2+ efflux when cells were stimulated with Ang II. Half-maximal effects occurred at approximately 1 μm CsA. The CsA effects on [Ca 2+ ] c were accompanied by a nearly twofold increase in Ang II receptor number, whereas no change in affinity to Ang II was observed. CsA did not alter endothelin-1- or thapsigargin-induced 45 Ca 2+ efflux. Increases in both Ca 2+ responses and [ 125 I]Ang II binding were attenuated by the transcriptional inhibitor actinomycin D. The effects of CsA did not appear to be mediated by calcineurin inhibition because cyclosporine H, which is not immunosuppressive, also increased the Ang II-induced 45 Ca 2+ efflux. Conclusion These data suggest that CsA preferentially up-regulates the transcription of Ang II receptors, which very likely leads to vasoconstriction in vivo and could be at the origin of CsA-induced hypertension and nephrotoxicity in humans.

Angiotensin II, nitric oxide, and end-organ damage in hypertension. The adaptive changes that accompany hypertension and involve the kidney, heart, and vessels, namely, muscle hypertrophy/hyperplasia, endothelial dysfunction and extracellular matrix increase can, in fact, be maladaptive and eventually lead to end- organ disease, such as renal failure, heart failure, and coronary disease. However, these changes vary markedly between individuals with similar levels of hypertension. Nitric oxide (NO), an endogenous vasodilator and inhibitor of vascular smooth muscle and mesangial cell growth, is synthesized in the endothelium by a constitutive NO synthase (NOS). NO antagonizes the effects of angiotensin II on vascular tone and growth and also down- regulates the synthesis of angiotensin converting enzyme (ACE) and angiotensin II type 1 (AT-1) receptors. In hypertension, the physiologic response to the increased shear stress and cyclic strain is to upregulate NOS activity in endothelial cells. Upregulation of vascular NOS activity is a homeostatic adaptation to the increased hemodynamic workload that may help in preventing end-organ damage. Indeed, hypertension-prone salt-sensitive rats manifest a decrease (instead of an increase) in vascular NOS activity when hypertensive; these rats develop severe vascular hypertrophy, left ventricular hypertrophy, and renal injury. Studies in hypertensive humans suggest that, independent of the effects of salt on blood pressure, salt sensitivity may be a marker for susceptibility to the development of endothelial dysfunction as well as cardiovascular and renal injury. We hypothesize that in hypertension, recognition of markers of cardiovascular susceptibility to injury and the understanding of the pathophysiological mechanisms involved may open new opportunities for therapeutic intervention. In this context, only those antihypertensive agents that lower blood pressure and concomitantly restore the homeostatic balance of vasoactive agents such as angiotensin II and NO within the vessel wall would be effective in preventing or arresting end-organ disease.

Role of angiotensin in renal vascular development. All components of the renin-angiotensin system (RAS) are expressed in the developing kidney in a temporospatial pattern that suggests a role for this system in kidney morphogenesis. Pharmacological blockade of angiotensin actions in fetal and newborn animals results in striking alterations in kidney architecture, including immature glomeruli and papillae, dilated tubuli, and arrested vascular development. Inactivation of angiotensinogen or angiotensin converting enzyme genes in mice results in similar anomalies that begin as subtle alterations in early life and become more pronounced as extrauterine life progresses. However, inactivation of each angiotensin receptor subtype does not result in obvious morphological abnormalities, suggesting functional redundancy at the receptor level. Crossing of mice lacking the various receptor subtypes should be revealing. Overall, the available information suggests that the RAS is necessary for the normal morphological and functional development of the kidney and the preservation of kidney architecture in adult life.

Physiological and pharmacological implications of AT1versus AT2receptors. Angiotensin II (Ang II) has diverse physiological actions that lead, for instance, to increases in extracellular volume and peripheral vascular resistance and blood pressure, and it has also been implicated in the regulation of cell growth and differentiation. Molecular cloning and pharmacological studies have defined two major classes of Ang II receptors, designated AT1 and AT2. Most effects of Ang II are mediated by AT1 receptors. Much less is known about the physiological role of AT2 receptors. Recent evidence suggests involvement of AT2 receptors in development, cell differentiation, apoptosis, and regeneration in various tissues. AT1 and AT2 receptors have been shown to exert counteracting effects on cellular growth and differentiation, vascular tone, and the release of arginine vasopressin. In each condition, the AT2 receptor appears to down-modulate actions mediated by the AT1 receptor, resulting in decreased cellular proliferation, decreased levels of serum arginine vasopressin levels, or decreased vasoconstrictor responses. In addition, in neuronal cell lines, the AT2 receptor exerts antiproliferative actions and promotes neurite outgrowth, an effect accompanied by significant changes in the expression pattern of growth/differentiation-related genes.

Dominant negative c-Src inhibits angiotensin II induced activation of NHE3 in OKP cells. Background Angiotensin II is a potent stimulator of the proximal tubule apical membrane Na/H antiporter, encoded by NHE3. The nonreceptor tyrosine kinase, c-Src, plays a key role in regulation of NHE3 by acidosis in the proximal tubule, and in signaling effects of angiotensin II in vascular smooth muscle. Methods The present studies examined the role of c-Src in mediating angiotensin II-induced NHE3 activation in cultured OKP cells. c-Src was inhibited with herbimycin A, a tyrosine kinase inhibitor, and expression of a dominant negative c-Src, c-Src K295M . Results Herbimycin A blocked angiotensin II induced increases in Na/H antiporter activity. In two clonal cell lines expressing vector alone, angiotensin II increased Na/H antiporter activity, while in three clones expressing c-Src K295M , angiotensin II had no effect. Cyclic AMP and protein kinase A have been proposed to be key mediators in regulation of NHE3 by angiotensin II. 10 -4 m 8-bromo cAMP induced a 40 to 50% inhibition of Na/H antiporter activity in cells expressing c-Src K295M , similar to that seen in wild-type OKP cells. In addition, cells expressing c-Src K295M responded normally to 10 -7 m dexamethasone with a 50 to 80% increase in Na/H antiporter activity. Conclusions These studies demonstrate that c-Src is required for angiotensin II-induced increases in NHE3 activity. Thus, c-Src plays a key role in antiporter activation by acidosis and angiotensin II.

Regulation of human renin gene promoter activity: A new negative regulatory region determines the responsiveness to TNF α . Background The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by β -adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression. Methods Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5kb of the hREN 5′ flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines. Results Transient transfection analysis showed that the 5kb hREN 5′ flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF α . Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF α . In contrast, neither interleukin (IL)-1 α , IL-1 β , IL-2, nor IL-6 exerted any significant effect. Conclusions Together the results suggest that TNF α is a negative regulator of the hREN expression in the adrenal cells, and that the TNF α responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.

Renin can induce renal disease by generating angiotensin II and, thereby, increasing fibrosis. Huang et al describe a new mechanism of action whereby the renin-angiotensin system can also exert this effect. Direct activation of the renin/prorenin receptor in mesangial cells induced synthesis of TGF-beta and profibrotic proteins. Hence, like other proteases such as thrombin, renin and prorenin are capable of receptor-mediated cellular signaling.

Selective antagonism of the AT1 receptor inhibits angiotensin II stimulated DNA and protein synthesis in primary cultures of human proximal tubular cells. The hypertrophy of renal proximal tubular cells occurs as an adaptive response to a variety of stimuli and may be involved with the progression of renal disease. Angiotensin II acting alone or in combination with other growth factors has been implicated in this process. The aims of this study were to identify the role of both angiotensin II and the angiotensin receptor subtypes in DNA synthesis and protein synthesis in human renal proximal tubular cells. Primary cultures of human renal M, proximal tubular cells were incubated with angiotensin II (10-10 m, 10-8 m, 10-10 m) for 24 to 120 hours either alone or in combination with losartan, PD123319 or 8-bromo-cAMP. Incubation of human proximal tubular cells with angiotensin II (10-10 m, 10-8 m) induced a significant early increase in [3H]thymidine uptake by 19% and 56% (P < 0.01), respectively, and a later increase in total protein content by 30% (P < 0.01). The effect of angiotensin II upon DNA and protein synthesis was inhibited by 8-bromo-cAMP and losartan but not by PD123319, indicating that the responses are mediated via the AT1 receptor and dependent upon the inhibition of adenylate cyclase.

Effect of glucose, pyruvate, and insulin on type 1 angiotensin II receptor expression in SV40-immortalized rabbit proximal tubule epithelial cells. Ambient glucose concentrations alter type 1 angiotensin II receptor (AT1R) expression in renal tissues. The direction of change in AT1R density may depend on the specific cell type and the capacity for that cell type to use glucose as an energy substrate. Given the effects of angiotensin II (Ang II) in proximal tubule epithelia, glucose-mediated fluctuations in AT1R expression could significantly alter tubular Na+-H+ exchange and volume reabsorption. To determine if glucose influenced AT1R expression in cultured proximal tubule epithelial cells, SV40-immortalized rabbit proximal tubule epithelial cells (RPTEC) were exposed to 25mmol (hi-glc) or 5mmol glucose-containing serum-free medium (lo-glc) for seven to nine days, with or without an alternative energy substrate, pyruvate. AT1R expression, assessed by quantitative reverse-transcription polymerase chain reaction and specific 125I-Ang II binding, decreased in lo-glc medium (% reduction AT1R mRNA expression: 52 ± 8%; N = 6; P < 0.005 vs. hi-glc; % reduction specific 125I-Ang II binding: 48 ± 12%; N = 12; P < 0.03 vs. hi-glc). AT1R mRNA expression and specific 125I-Ang II binding recovered to hi-glc levels following the addition of pyruvate [60 mmol] to lo-glc cells. To ascertain if a growth factor that increases glucose uptake in vivo also altered AT1R expression, RPTEC were cultured in hi-glc medium with or without exogenous insulin [100 nM], Insulin addition increased AT1R mRNA expression and specific 125I-Ang II binding in a concentration-dependent manner. However, insulin (100 nM) addition to lo-glc cells did not significantly increase specific 125I-Ang II binding. These results suggest that AT1R expression in SV40-immortalized rabbit proximal tubule cells is significantly affected by the availability of energy substrate. Ultimately, changes in proximal tubule AT1R expression, mediated by elevated glucose concentrations and insulin, could contribute to sodium-dependent hypertension in the setting of hyperinsulinemia and hyperglycemia.