Weijian Shao

Intrarenal Angiotensin III Is the Predominant Agonist for Proximal Tubule Angiotensin Type 2 Receptors

In angiotensin type 1 receptor–blocked rats, renal interstitial (RI) administration of des-aspartyl1-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III–induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor–blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III–induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP–dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.

Augmentation of endogenous intrarenal angiotensin II levels in Val5-ANG II-infused rats

In angiotensin II (ANG II)-induced hypertension, intrarenal ANG II levels are increased by AT(1) receptor-mediated ANG II internalization and endogenous ANG II generation. The objective of the present study was to determine the relative contribution of de novo formation of endogenous ANG II. Male Sprague-Dawley rats were divided into three groups: sham operated (n = 6), Val(5)-ANG II infused (n = 16), and Ile(5)-ANG II infused (n = 6). Val(5)-ANG II and Ile(5)-ANG II were infused at 80 ng/min via subcutaneous osmotic minipump for 13 days, followed by harvesting of blood and kidney samples. In six Val(5)-ANG II-infused rats, urine was collected on the day before infusion and on day 12 of infusion. Extracted samples were subjected to HPLC to separate Val(5)-ANG II from Ile(5)-ANG II followed by RIA. Systolic blood pressure increased significantly from 121 +/- 2 to 206 +/- 4 mmHg in the Val(5)-ANG II-infused rats and from 124 +/- 3 to 215 +/- 5 mmHg in the Ile(5)-ANG II-infused rats. In the Val(5)-ANG II-infused rats, the plasma Ile(5)-ANG II levels increased 196.2 +/- 70.1% compared with sham plasma Ile(5)-ANG II concentration. Val(5)-ANG II levels were 150.0 +/- 28.2 fmol/ml which accounted for 53.5 +/- 10.1% of the total ANG II in plasma. The kidney Ile(5)-ANG II levels in the Val(5)-ANG II-infused rats increased 69.9 +/- 30.7% compared with sham kidney Ile(5)-ANG II concentrations. Intrarenal accumulation of Val(5)-ANG II accounted for 52.5 +/- 5.3% of the total kidney ANG II during Val(5)-ANG II infusion while endogenous Ile(5)-ANG II accounted for 47.5 +/- 8.6%. The urinary Ile(5)-ANG II excretion rate on day 12 increased 93.2 +/- 32.1% compared with preinfusion level indicating increased formation of endogenous ANG II. Thus, the increases in intrarenal ANG II levels during chronic ANG II infusions involve substantial stimulation of endogenous ANG II formation which contributes to overall augmentation of intrarenal ANG II.

Salt-induced renal injury in SHRs is mediated by AT1 receptor activation

Objective This study aimed to examine the effects of salt loading, with or without simultaneous angiotensin receptor blocker (ARB) treatment, on the systemic and tissue renin-angiotensin system (RAS) in spontaneously hypertensive rats (SHRs). Method Evaluation was performed early (4 weeks) in the course of salt loading in order to examine initial mediating events of cardiovascular and renal damage produced by salt excess. Four groups of rats were studied. Group 1 received regular rat chow (normal-salt diet); group 2 received normal-salt diet and an ARB (losartan, 30 mg/kg per day); group 3 received high-salt (8%) chow; and group 4 received high-salt diet and losartan. Results High-salt diet increased systolic pressure to 193 ± 1 mmHg compared to 180 ± 2 in normal-salt diet group. Losartan reduced SBP in SHRs fed normal-salt diet but did not reduce SBP in the SHRs fed high-salt diet (192 ± 2 mmHg). High-salt diet markedly increased urinary protein excretion from 27 ± 4 to 64 ± 13 mg/day and this increase was ameliorated by losartan (40 ± 9 mg/day). In SHRs on high-salt diet, plasma angiotensin II concentration increased three to four-fold, whereas urinary angiotensinogen excretion increased 10-fold; and these changes were significantly reduced by losartan. High-salt diet accelerated glomerular injury and interstitial fibrosis in SHRs which were reduced by losartan. Conclusion These results demonstrate that the activity of RAS was either not suppressed or, even augmented, after 4 weeks of salt loading despite high salt intake and increased SBP. The data suggest that an augmented intrarenal RAS during high-salt diet may contribute to the development of renal injury in this experimental model.

Activation of the renin-angiotensin system by a low-salt diet does not augment intratubular angiotensinogen and angiotensin II in rats

In angiotensin II (ANG II) infusion hypertension, there is an augmentation of intratubular angiotensinogen (AGT) and ANG II leading to increased urinary AGT and ANG II excretion rates associated with tissue injury. However, the changes in urinary AGT and ANG II excretion rates and markers of renal injury during physiologically induced stimulation of the renin-angiotensin system (RAS) by a low-salt diet remain unclear. Male Sprague-Dawley rats received a low-salt diet (0.03% NaCl; n = 6) and normal-salt diet (0.3% NaCl, n = 6) for 13 days. Low-salt diet rats had markedly higher plasma renin activity and plasma ANG II levels. Kidney cortex renin mRNA, kidney AGT mRNA, and AGT immunoreactivity were not different; however, medullary renin mRNA, kidney renin content, and kidney ANG II levels were significantly elevated by the low-salt diet. Kidney renin immunoreactivity was also markedly increased in juxtaglomerular apparati and in cortical and medullary collecting ducts. Urinary AGT excretion rates and urinary ANG II excretion rates were not augmented by the low-salt diet. The low-salt diet caused mild renal fibrosis in glomeruli and the tubulointerstitium, but no other signs of kidney injury were evident. These results indicate that, in contrast to the response in ANG II infusion hypertension, the elevated plasma and intrarenal ANG II levels caused by physiological stimulation of RAS are not reflected by increased urinary AGT or ANG II excretion rates or the development of renal injury.

Angiotensin II Type 1 Receptor-Mediated Augmentation of Urinary Excretion of Endogenous Angiotensin II in Val5-Angiotensin II-Infused Rats

Rats infused chronically with Val5-Angiotensin (Ang) II exhibit increased urinary excretion of endogenous Ile5-Ang II by the 12th day of infusion, suggesting the stimulation of endogenous Ang II formation by Val5-Ang II infusion. The present study determined the time course of increased urinary Ang II excretion and the effects of Ang II type 1 receptor blockade (candesartan, 2 mg/kg per day) on the urinary excretion rates of Ile5-Ang II in Val5-Ang II–infused (80 ng/min) rats. Ile5-Ang II was separated from Val5-Ang II by high-performance liquid chromatography and measured by radioimmunoassay. Systolic blood pressure increased progressively (215±2 mm Hg) in Val5-Ang II–infused rats (n=5), whereas the candesartan-treated group (n=6) remained normotensive (124±3 mm Hg). Candesartan treatment significantly increased the level of plasma Ile5-Ang II (24.0±7.6 versus 156.9±24.6 fmol/mL; P<0.01); in contrast, there was a markedly lower intrarenal Ile5-Ang II content (357.9±76.6 versus 21.1±2.8 fmol/g; P<0.01). Urinary Ile5-Ang II excretion rates were elevated by day 9 (2185.7±283.2 fmol/24 hours) in Val5-Ang II–infused rats but not in candesartan-treated rats (740.6±110.3 fmol/24 hours). Thus, Ang II type 1 receptor blockade prevents the increase in urinary excretion of endogenous Ang II in rats subjected to chronic Ang II infusion. These data indicate that the increased urinary excretion of endogenous Ang II in Val5-Ang II–infused rats is primarily attributed to Ang II type 1 receptor–dependent secretion into and/or de novo formation of Ang II within the tubular lumen.

Role of Stimulated Intrarenal Angiotensinogen in Hypertension

Experimental models of hypertension and patients with inappropriately increased renin formation due to a stenotic kidney, arteriosclerotic narrowing of the renal arterioles or a rare juxtaglomerular cell tumor have shown a progressive augmentation of the intrarenal/intratubular renin–angiotensin system (RAS). The increased intrarenal angiotensin II (Ang II) elicits renal vasoconstriction and enhanced tubular sodium reabsorption in proximal and distal nephron segments. The enhanced intrarenal Ang II levels are due to both increased Ang II type 1 (AT1) receptor mediated Ang II uptake and AT1 receptor dependent stimulation of renal angiotensinogen (AGT) mRNA and augmented AGT production. The increased AGT formation and secretion into the proximal tubular lumen leads to local formation of Ang II, which stimulates proximal transporters such as the sodium/hydrogen exchanger. Enhanced AGT production also leads to spillover of AGT into the distal nephron segments as reflected by AGT in the urine, which provides an index of intrarenal RAS activity. There is also increased Ang II concentration in distal nephron with stimulation of distal sodium transport. Increased urinary excretion of AGT has been demonstrated in patients with hypertension, type 1 and type 2 diabetes mellitus, and several types of chronic kidney diseases indicating an upregulation of intrarenal RAS activity.

ROCK/NF-κB axis-dependent augmentation of angiotensinogen by angiotensin II in primary-cultured preglomerular vascular smooth muscle cells

In angiotensin II (ANG II)-dependent hypertension, the augmented intrarenal ANG II constricts the renal microvasculature and stimulates Rho kinase (ROCK), which modulates vascular contractile responses. Rho may also stimulate angiotensinogen (AGT) expression in preglomerular vascular smooth muscle cells (VSMCs), but this has not been established. Therefore, the aims of this study were to determine the direct interactions between Rho and ANG II in regulating AGT and other renin-angiotensin system (RAS) components and to elucidate the roles of the ROCK/NF-κB axis in the ANG II-induced AGT augmentation in primary cultures of preglomerular VSMCs. We first demonstrated that these preglomerular VSMCs express renin, AGT, angiotensin-converting enzyme, and ANG II type 1 (AT1) receptors. Furthermore, incubation with ANG II (100 pmol/l for 24 h) increased AGT mRNA (1.42 ± 0.03, ratio to control) and protein (1.68 ± 0.05, ratio to control) expression levels, intracellular ANG II levels, and NF-κB activity. In contrast, the ANG II treatment did not alter AT1a and AT1b mRNA levels in the cells. Treatment with H-1152 (ROCK inhibitor, 10 nmol/l) and ROCK1 small interfering (si) RNA suppressed the ANG II-induced AGT augmentation and the upregulation and translocalization of p65 into nuclei. Functional studies showed that ROCK exerted a greater influence on afferent arteriole responses to ANG II in rats subjected to chronic ANG II infusions. These results indicate that ROCK is involved in NF-κB activation and the ROCK/NF-κB axis contributes to ANG II-induced AGT upregulation, leading to intracellular ANG II augmentation.

Increased angiotensinogen expression, urinary angiotensinogen excretion, and tissue injury in nonclipped kidneys of two-kidney, one-clip hypertensive rats

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

Effects of Serelaxin on Renal Microcirculation in Rats under Control and High Angiotensin Environments

Serelaxin is a novel recombinant human relaxin-2 that has been investigated for the treatment of acute heart failure. However, its effects on renal function, especially on the renal microcirculation, remain incompletely characterized. Our immunoexpression studies localized RXFP1 receptors on vascular smooth muscle cells and endothelial cells of afferent arterioles and on principal cells of collecting ducts. Clearance experiments were performed in male and female normotensive rats and Ang II-infused male rats. Serelaxin increased mean arterial pressure slightly and significantly increased renal blood flow, urine flow, and sodium excretion rate. Group analysis of all serelaxin infusion experiments showed significant increases in GFR. During infusion with subthreshold levels of Ang II, serelaxin did not alter mean arterial pressure, renal blood flow, GFR, urine flow, or sodium excretion rate. Heart rates were elevated during serelaxin infusion alone (37 ± 5%) and in Ang II-infused rats (14 ± 2%). In studies using the in vitro isolated juxtamedullary nephron preparation, superfusion with serelaxin alone (40 ng/ml) significantly dilated afferent arterioles (10.8 ± 1.2 vs. 13.5 ± 1.1 µm) and efferent arterioles (9.9 ± 0.9 vs. 11.9 ± 1.0 µm). During Ang II superfusion, serelaxin did not alter afferent or efferent arteriolar diameters. During NO synthase inhibition (l-NNA), afferent arterioles also did not show any vasodilation during serelaxin infusion. In conclusion, serelaxin increased overall renal blood flow, urine flow, GFR, and sodium excretion and dilated the afferent and efferent arterioles in control conditions, but these effects were attenuated or prevented in the presence of exogenous Ang II and NO synthase inhibitors.

Intrarenal Angiotensin III Is the Predominant Agonist for Proximal Tubule Angiotensin Type 2 ReceptorsNovelty and Significance

In angiotensin type 1 receptor–blocked rats, renal interstitial (RI) administration of des-aspartyl1-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III–induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor–blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III–induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP–dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.