Helmy M. Siragy

Angiotensin Type 2 Receptor Mediates Valsartan-Induced Hypotension in Conscious Rats

Inhibition of the renin-angiotensin system is associated with vasodilation and reduction in blood pressure. We hypothesized that angiotensin type 1 (AT(1)) receptor (AT(1)R) blockade is associated with increased production of renal nitric oxide (NO) mediated by release of bradykinin (BK). By use of a microdialysis technique, changes in renal interstitial fluid (RIF) BK, NO end products nitrite and nitrate (NOX), and cGMP were monitored in response to intravenous infusion of the AT(1)R blocker valsartan (10 mg/kg), the angiotensin type 2 (AT(2)) receptor (AT(2)R) blocker PD123319 (50 microg x kg(-1) x min(-1)), and the BK B(2) receptor blocker icatibant (10 microg x kg(-1) x min(-1)) in conscious rats (n=10) during low sodium intake. RIF BK, NOX, and cGMP significantly increased during valsartan treatment, whereas AT(2)R blockade caused a significant decrease in these autacoids. During icatibant infusion, RIF NOX and cGMP decreased by 64% and 40%, respectively, whereas BK increased. Combined administration of valsartan and icatibant, of valsartan and PD123319, or of valsartan, PD123319, and icatibant prevented the increase in RIF cGMP and NOX in response to valsartan alone. These data demonstrate that AT(1)R blockade with valsartan is associated with release of renal BK, which in turn mediates NO production. The results suggest that increased angiotensin II, in response to sodium restriction and valsartan infusion, stimulates AT(2)R, which mediates a BK and NO cascade.

Role of the Intrarenal Renin-Angiotensin-Aldosterone System in Chronic Kidney Disease

The existence of local or tissue-based renin-angiotensin-aldosterone systems (RAAS) is well documented and has been implicated as a key player in the pathogenesis of cardiovascular and renal diseases. The kidney contains all elements of the RAAS, and intrarenal formation of angiotensin II not only controls glomerular hemodynamics and tubule sodium transport, but also activates a number of inflammatory and fibrotic pathways. Experimental and clinical studies have shown that the intrarenal RAAS is activated early in diabetic nephropathy, the leading cause of chronic kidney disease (CKD). Although angiotensin-converting enzyme inhibitors and angiotensin receptor blockers decrease the rate of decline in kidney function in patients with diabetic and non-diabetic nephropathy, many patients still progress to end-stage renal disease or die from cardiovascular events. There is still a clear need for additional strategies to block the RAAS more effectively to reduce progression of CKD. The focus of this paper is to review the importance of the intrarenal RAAS in CKD and recent findings in renin-angiotensin biology pertinent to the kidney. We also discuss additional strategies to inhibit the RAAS more effectively and the potential impact of direct renin inhibition on the prevention and management of CKD.

Renal Angiotensin Type 2 Receptors Mediate Natriuresis Via Angiotensin III in the Angiotensin II Type 1 Receptor–Blocked Rat

Whereas angiotensin (Ang) II is the major effector peptide of the renin–angiotensin system, its metabolite, des-aspartyl1-Ang II (Ang III), may also have biologic activity. We investigated the effects of renal interstitial (RI) administration of candesartan (CAND), a specific Ang II type 1 receptor (AT1) blocker, with and without coinfusion of PD-123319 (PD), a specific Ang II type 2 receptor (AT2) blocker, on Na+ excretion (UNaV) in uninephrectomized rats. We also studied the effects of unilateral RI infusion of Ang II or Ang III on UNaV with and without systemic infusion of CAND with the noninfused kidney as control. In rats receiving normal Na+ intake, RI CAND increased UNaV from 0.07±0.08 to 0.82±0.17 &mgr;mol/min (P<0.01); this response was abolished by PD. During Na+ restriction, CAND increased UNaV from 0.06±0.02 to 0.1±0.02 &mgr;mol/min (P<0.05); this response also was blocked by PD. In rats with both kidneys intact, in the absence of CAND, unilateral RI infusion of Ang III did not significantly alter UNaV. However, with systemic CAND infusion, RI Ang III increased UNaV from 0.08±0.01 &mgr;mol/min to 0.18±0.04 &mgr;mol/min (P<0.01) at 3.5 nmol/kg per minute, and UNaV remained elevated throughout the infusion; this response was abolished by PD. However, RI infusion of Ang II did not significantly alter UNaV at any infusion rate (3.5 to 80 nmol/kg per minute) with or without systemic CAND infusion. These results suggest that intrarenal AT1 receptor blockade engenders natriuresis by activation of AT2 receptors. AT2 receptor activation via Ang III, but not via Ang II, mediates the natriuretic response in the presence of systemic AT1 receptor blockade.

Local Renal Aldosterone System and Its Regulation by Salt, Diabetes, and Angiotensin II Type 1 Receptor

CYP11B2 is the enzyme responsible for aldosterone synthesis mainly in the adrenal gland. In this study, we hypothesized that CYP11B2 gene, protein, and aldosterone are produced locally in kidney and regulated by low salt intake, angiotensin II type 1 (AT1) receptor and insulin-deficient diabetes hyperglycemia. We used real-time RT-PCR, immunohistochemistry staining, and microdialysis techniques to monitor changes in renal CYP11B2 mRNA and protein and aldosterone production in normal, adrenalectomized, or streptozotocin-induced insulin-deficient diabetic hyperglycemic rats. In normal kidney, CYP11B2 mRNA and protein were localized mainly in the renal cortex and upregulated by angiotensin II and low salt intake. The angiotensin II effect was reversed by AT1 receptor blocker valsartan. Immunohistochemistry staining demonstrated presence of CYP11B2 in glomeruli. Although aldosterone was absent in plasma of adrenalectomized rats, it was present in renal interstitium and tissue. Diabetes increased renal cortical and total kidney CYP11B2 mRNA and protein. Lowering blood glucose with insulin decreased total renal CYP11B2 mRNA and protein. Despite lack of significant changes in blood glucose, valsartan treatment caused significant reduction in renal CYP11B2 mRNA and protein. In presence of diabetes, there was an increase in CYP11B2 immunostaining in glomeruli and proximal tubules. This expression was abrogated with insulin or valsartan treatment. These results demonstrate the presence of all components of local renal aldosterone system. This system is physiologically active because it is regulated by angiotensin II and low salt intake. In insulin-deficient diabetes hyperglycemia rat model, glucose, insulin, and AT1 receptor modulate CYP11B2 expression in the kidney.

Angiotensin AT2 Receptor Stimulation Inhibits Early Renal Inflammation in Renovascular Hypertension

Angiotensin II type 2 receptor (AT2R) counteracts most effects of angiotensin II type 1 receptor (AT1R). We hypothesized that direct AT2R stimulation reduces renal production of the inflammatory cytokines tumor necrosis factor-&agr; (TNF-&agr;), interleukin-6 (IL-6), and transforming growth factor-&bgr;1 (TGF-&bgr;1) and enhances the production of nitric oxide (NO) and cyclic guanosine 3′,5′-monophosphate (cGMP) in the clipped kidney of 2-kidney, 1-clip (2K1C) hypertension rat model. We used Sprague-Dawley rats to evaluate changes in renal interstitial fluid recovery levels of TNF-&agr;, IL-6, NO, and cGMP; renal expression of AT1R, AT2R, TGF-&bgr;1, TNF-&agr;, and IL-6 in sham and 2K1C rats treated for 4 days with vehicle, AT2R agonist compound 21 (C21), or AT2R antagonist PD123319 (PD), alone and combined (n=6, each group). Systolic blood pressure increased significantly in 2K1C and was not influenced by any treatment. Clipped kidneys showed significant increases in renal expression of AT1R, AT2R, TNF-&agr;, IL-6, TGF-&bgr;1 and decreases in NO and cGMP levels. These factors were not influenced by PD treatment. In contrast, C21 caused significant decrease in renal TNF-&agr;, IL-6, TGF-&bgr;1 and an increase in NO and cGMP levels. Combined C21 and PD treatment partially reversed the observed C21 effects. Compared to sham, there were no significant changes in TNF-&agr;, IL-6, TGF-&bgr;1, NO, or cGMP in the nonclipped kidneys of 2K1C animals. We conclude that direct AT2R stimulation reduces early renal inflammatory responses and improves production of NO and cGMP in renovascular hypertension independent of blood pressure reduction.

Angiotensin Subtype-2 Receptors Inhibit Renin Biosynthesis and Angiotensin II Formation

Renin is regulated by angiotensin subtype 1 (AT1) receptor, but it is unknown whether angiotensin subtype 2 (AT2) receptor contributes to this regulation. We hypothesized that AT2 receptors inhibit angiotensin II (Ang II) through inhibition of renin biosynthesis. We monitored changes in renal Ang II, renin mRNA and protein expression, and plasma renin concentration (PRC) in response to renal cortical administration of the AT1 receptor blocker valsartan or the AT2 receptor blocker PD 123319 (PD) in conscious rats administered low sodium intake (LS). Renal interstitial Ang II increased by 47-fold in response to LS and increased further in response to valsartan or PD by 67-fold and 61-fold from normal sodium diet (NS) and by ≈41% and 29% from LS, respectively. Renin mRNA increased 63% during LS, and either valsartan or PD increased it further by 600% and 250% from NS and 538% and 187% from LS, respectively. Similarly, renal renin content and PRC increased in response to LS and increased further in response to combined LS and valsartan or PD administration. Immunostaining for renal renin protein demonstrated an increase in its expression in juxtaglomular and tubular cells during LS and increased further during AT1 or AT2 receptor blockade. These data demonstrate for the first time to our knowledge that AT2 receptors regulate the renin-angiotensin system activity via inhibition of renin synthesis.

Pro)renin receptor contributes to diabetic nephropathy by enhancing renal inflammation

1.u2002(Pro)renin receptor (PRR) binding to renin or prorenin mediates angiotensin (Ang) II‐dependent and ‐independent effects. Expression of the PRR is increased in kidneys of diabetic rats, but its role in diabetic nephropathy is unknown. In the present study, we investigated the contribution of the PRR to the development of diabetic nephropathy through enhancement of renal production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β.

Renal (pro)renin receptor upregulation in diabetic rats through enhanced angiotensin AT1 receptor and NADPH oxidase activity

Recent studies have demonstrated the presence of the (pro)renin receptor (PRR) in the glomerular mesangium and the subendothelial layer of the renal arteries. We hypothesized that diabetes upregulates PRR expression through enhanced angiotensin subtype 1 (AT1) receptor–NADPH oxidase cascade activity. Using real‐time polymerase chain reaction, Western blot analysis and immunostaining, we studied renal localization of the PRR in the streptozotocin‐induced diabetic rat model and in response to 1 week of treatment with the AT1 receptor blocker valsartan (10 mg kg−1 day−1), the angiotensin AT2 receptor blocker PD123319 (0.5 mg kg−1 day−1) or the NADPH oxidase inhibitor diphenylene iodonium (DPI; 0.5 mg kg−1 day−1) 6 weeks post‐induction of diabetes. Both PRR mRNA and protein were expressed constitutively in the kidneys of normal rat renal cortex and medulla, mainly in glomerular mesangium, proximal, distal and collecting tubules. Compared with normal rats (100%), diabetic rats demonstrated an increase in renal PRR mRNA (184%), protein (228%) and immunostaining. Valsartan and DPI prevented the increase in the PRR mRNA (106 and 126%, respectively), protein (97 and 140%, respectively) and immunostaining that was seen in the kidneys of diabetic rats. The AT2 blocker PD123319 did not have significant effects on PRR mRNA (157%) or protein expression (200%) in the kidneys of diabetic rats. These results demonstrate that the PRR is constitutively expressed in renal glomeruli and tubules. Expression of the PRR is upregulated in diabetes via enhancement of AT1 receptor–NADPH oxidase activity.

Glucose Promotes the Production of Interleukine-1β and Cyclooxygenase-2 in Mesangial Cells via Enhanced (Pro)Renin Receptor Expression

(Pro)renin receptor (PRR) is present in renal glomeruli, and its expression is up-regulated in diabetes. Similarly, renal inflammation is increased in the presence of hyperglycemia. The linkage between PRR and renal inflammation is not well established. We hypothesized that glucose-induced up-regulation of PRR leads to increased production of the proinflammatory factors IL-1beta and cyclooxygenase-2 (COX-2). Studies were conducted in rat mesangial cells (RMCs) exposed to 30 mm D-glucose for 2 wk followed by PRR small interfering RNA knockdown, IL-1 receptor blockade with IL-1 receptor antagonist or angiotensin II type 1 receptor blockade with valsartan. The results showed that D-glucose treatment up-regulates prorenin, renin, angiotensin II, PRR, IL-1beta, and COX-2 mRNA and protein expression and increases phosphorylation of ERK1/2, c-Jun N-terminal kinase, c-Jun, and nuclear factor-kappaB (NF-kappaB) p65 (serine 276,468 and 536), respectively. PRR small interfering RNA attenuated PRR, IL-1beta, and COX-2 mRNA and protein expressions and significantly decreased angiotensin II production and phosphorylation of ERK1/2 and NF-kappaB p65 associated with high glucose exposure. Similarly, IL-1 receptor antagonist significantly reduced COX-2 mRNA and protein expression induced by high glucose. COX-2 inhibition reduced high-glucose-induced PRR expression. We conclude that glucose induces the up-regulation of PRR and its ligands prorenin and renin, leading to increased IL-1beta and COX-2 production via the angiotensin II-dependent pathway. It is also possible that PRR could enhance the production of these inflammatory cytokines through direct stimulation of ERK1/2-NF-kappaB signaling cascade.

Distribution of type-1 and type-2 angiotensin receptors in the normal human lung and in lungs from patients with chronic obstructive pulmonary disease

Abstract. This study was designed to examine the cellular distribution of the angiotensinxa0II type-1 (AT1) and type-2 (AT2) receptors in the normal human and pathological human lung. Riboprobes were prepared against specific portions of each receptor DNA and labelled with FITC for detection using an anti-FITC antibody in combination with the alkaline phosphatase–anti-alkaline phosphatase technique and new Fuchsin. These were used to detect the presence of receptor mRNA in the lung. Specific antibodies were used to detect receptor protein in cells by immunocytochemistry. Image analysis was used in order to semi-quantify receptor density. AT1 receptor mRNA and protein were localised on vascular smooth muscle cells, macrophages and in the stroma underlying the airways epithelium probably relating to underlying fibroblasts. The AT1 receptor protein was not expressed in the epithelium although there was a low level of mRNA. In contrast, AT2 receptor RNA and protein was observed in the epithelium, with strong staining on the bronchial epithelial cell brush border and also on many of the underlying mucous glands. The AT2 receptor was also present on some endothelial cells. These findings were supported by the presence of mRNA in each case. In patients with chronic obstructive pulmonary disease, there was a five- to sixfold increase in the ratio of AT1 to AT2 receptors in the regions of marked fibrosis surrounding the bronchioles. This correlated well with the reduced lung function as expressed by the forced expiratory volume