Anees Ahmad Banday

Resveratrol prevents endothelial nitric oxide synthase uncoupling and attenuates development of hypertension in spontaneously hypertensive rats

Endothelial dysfunction is a hallmark of hypertension and vascular oxidative stress can contribute to endothelial dysfunction and hypertension development. Resveratrol is an antioxidant polyphenol which improves endothelium dependent relaxation, the mechanisms of which are unknown. Also, the role of resveratrol in hypertension remains to be established. The purpose of this study was to investigate the mechanisms of resveratrol induced improvement of endothelial function and establish its role in hypertension. SHR and WKY rats, 3-4 weeks old, were treated with resveratrol in drinking water for 10 weeks, untreated SHR and WKY rats served as controls. At the end of the treatment, control SHR exhibited increased blood pressure, oxidative stress and attenuated endothelium dependent relaxation in comparison to WKY rats. The impaired endothelium function in SHR was associated with lower nitrite/nitrate levels, elevated nitrotyrosine content and eNOS uncoupling. Resveratrol treatment attenuated hypertension development in SHR as indicated by lower blood pressure in resveratrol treated SHR (SHR-R) compared to control SHR. SHR-R also exhibited reduced H(2)O(2) content and elevated superoxide dismutase activity. Resveratrol treatment normalized endothelium dependent vasorelaxation in SHR. In parallel, resveratrol restored nitrite/nitrate levels and normalized nitrotyrosine content in SHR. SHR exhibited increased l-arginine dependent superoxide production which was blocked by NOS inhibitor l-NNA, suggesting eNOS uncoupling. eNOS uncoupling was prevented by resveratrol treatment. In conclusion, early treatment with resveratrol lowers oxidative stress, preserves endothelial function and attenuates development of hypertension in SHR. More importantly, prevention of eNOS uncoupling and NO scavenging could represent novel mechanisms for resveratrol-mediated antihypertensive effects.

Time dependent effects of gentamicin on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in rat kidney tissues

Gentamicin (GM), an antibiotic against life threatening bacterial infection, induces remarkable toxicity in the kidney. Histological studies have indicated that mitochondria, microsomes, lysosomes and plasma membranes of renal proximal convoluted tubules in particular are major GM targets. Despite numerous investigations, the biochemical/cellular basis of GM nephrotoxicity is not well understood. Recently reactive oxygen species (ROS) are considered to be important mediators of GM-induced nephrotoxicity. We hypothesize that GM causes damage to intracellular organelles and affects their structural integrity and alters metabolic and other functional capabilities. To address above hypothesis a long-term, time-dependent effect of GM has been studied on blood/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM) and basolateral (BLM), lysosomes and oxidative stress in renal tissues. A nephrotoxic dose of GM (80 mg/kg body weight) was administered to rats daily for 15 days. The long-term treatment with GM induced a significant increase in serum creatinine, blood urea nitrogen followed by massive proteinuria, glucosuria, enzymuria along with loss of electrolytes in the urine. The activities of the enzymes of carbohydrate metabolism, plasma membranes, lysosomes significantly declined. The activities of antioxidant enzymes e.g. superoxide dismutase, catalase and glutathione peroxidase were severely depressed and lipid peroxidation was significantly increased in the renal cortex and medulla. We conclude that GM administration induced oxidative damage to renal tissues that resulted in impaired carbohydrate metabolism and decreased activities of BBM, BLM and lysosomes associated with increased lipid peroxides.

Oxidative stress-induced renal angiotensin AT1 receptor upregulation causes increased stimulation of sodium transporters and hypertension

Reactive oxygen species have emerged as important molecules in cardiovascular dysfunction such as diabetes and hypertension. Recent work has shown that oxidative stress and angiotensin II signaling mutually regulate each other by multiple mechanisms and contribute to the development of hypertension. Most of the known biological actions of angiotensin II can be attributed to AT1 receptors. The present study was carried out to investigate the role of renal AT1 receptor signaling in oxidative stress-mediated hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol (an antioxidant) for 2 wk. Compared with control rats, BSO-treated rats exhibited increased oxidative stress and reduced antioxidant levels and developed hypertension. BSO treatment also caused increased renal proximal tubular AT1 receptor protein abundance, message levels, and ligand binding. In these rats, angiotensin II caused significantly higher accumulation of inositol trisphosphate (IP3) and phospholipase C (PLC) activation which was sensitive to blockade by AT1 but not to AT2 antagonist. Also, angiotensin II-mediated, AT1-dependent MAP kinase, Na-K-ATPase, and Na/H exchanger 3 activation was higher in BSO-treated rats than in control rats. Tempol supplementation of BSO-treated rats restored redox status, normalized AT1 receptor expression, and decreased blood pressure. Tempol also normalized the angiotensin II-mediated, AT1-dependent IP3 accumulation and PLC, MAP kinase, Na-K-ATPase, and Na/H exchanger 3 stimulation. These data suggest that oxidative stress leads to AT1 receptor upregulation, which in turn causes overstimulation of sodium transporters and subsequently contributes to sodium retention and hypertension. Tempol, while reducing oxidative stress, normalizes AT1 receptor signaling and decreases blood pressure.

Mechanisms of Oxidative Stress-Induced Increase in Salt Sensitivity and Development of Hypertension in Sprague-Dawley Rats

High salt intake produces vascular changes that contribute to the development of hypertension in salt-sensitive individuals. Because reactive oxygen species play a role in the pathogenesis of cardiovascular diseases, we investigated whether oxidative stress contributes to salt-sensitive hypertension. Sprague–Dawley rats were divided in different groups and received tap water (vehicle), 30 mmol/L of l-buthionine sulfoximine ([BSO] an oxidant), high salt ([HS] 1% NaCl), and BSO plus HS without and with antioxidant tempol (1 mmol/L) in drinking water for 12 days. Compared with vehicle, BSO treatment caused oxidative stress and mild increase in blood pressure. Thoracic aortic rings from BSO–treated rats exhibited decreased response to endothelium–independent vasorelaxants. In HS–treated rats, the response to vasoactive agents, as well as blood pressure, was unaffected. Concomitant treatment of rats with BSO and HS produced a marked increase in blood pressure and a decreased response to both endothelium-dependent and endothelium-independent vasorelaxants with an increase in EC50. Incubation of aortic tissue from BSO-treated rats with sodium nitroprusside showed decreased cGMP accumulation, whereas HS rats had decreased basal NO synthase activity. Tempol decreased oxidative stress, normalized blood pressure, and restored NO signaling and responses to vasoactive compounds in BSO and BSO plus HS rats. We conclude that BSO increases oxidative stress and reduces NO signaling, whereas HS reduces NO levels by decreasing the NO synthase activity. These phenomena collectively result in reduced responsiveness to both endothelium -dependent and endothelium- independent vasorelaxants and may contribute to salt-sensitive hypertension.

Oxidative Stress Causes Renal Dopamine D1 Receptor Dysfunction and Salt-Sensitive Hypertension in Sprague-Dawley Rats

Renal dopamine plays an important role in maintaining sodium homeostasis and blood pressure (BP) during increased sodium intake. The present study was carried out to determine whether renal dopamine D1 receptor (D1R) dysfunction contributes to increase in salt sensitivity during oxidative stress. Male Sprague-Dawley rats, divided into various groups, received tap water (vehicle); 1% NaCl (high salt [HS]); l-buthionine sulfoximine (BSO), an oxidant; and HS plus BSO with or without Tempol, an antioxidant, for 12 days. Compared with vehicle, HS intake increased urinary dopamine production and decreased basal renal Na/K-ATPase activity but did not affect BP. BSO-treated rats exhibited oxidative stress and a mild increase in BP. In these rats, D1R expression and G protein coupling were reduced, and SKF38393, a D1R agonist, failed to inhibit Na/K-ATPase activity and promote sodium excretion. Concomitant administration of BSO and HS caused oxidative stress, D1R dysfunction, and a marked increase in BP. Although renal dopamine production was increased, it failed to reduce the basal Na/K-ATPase activity in these animals. Treatment of BSO plus HS rats with Tempol decreased oxidative stress and restored endogenous, as well as exogenous, D1R agonist-mediated Na/K-ATPase inhibition and normalized BP. In conclusion, during HS intake, the increased dopamine production via Na/K-ATPase inhibition prevents an increase in BP. During oxidative stress, D1R function is defective, and there is mild hypertension. However, in the presence of oxidative stress, HS intake causes marked elevation in BP, which results from a defective renal D1R function leading to the failure of dopamine to inhibit Na/K-ATPase and promote sodium excretion.

Rosiglitazone Treatment Restores Renal Dopamine Receptor Function in Obese Zucker Rats

Abstract—Earlier we have reported a defective dopamine D1–like receptor function, which was accompanied by a decrease in D1 receptor numbers and the inability of dopamine to inhibit Na,K-ATPase and Na,H-exchanger in proximal tubules of hyperinsulinemic obese Zucker rats. The present study was designed to test the hypothesis that the defect in dopamine receptor function is a result of hyperinsulinemia in obese rats. We designed experiments to study D1 receptor function in obese Zucker rats treated with rosiglitazone, as it lowers plasma insulin by improving insulin sensitivity. A group of untreated lean and obese rats served as controls. Rosiglitazone treatment (10 mg/kg orally, 4 weeks) caused significant decreases in plasma insulin, blood glucose, and blood pressure while causing an increase in renal sodium excretion compared with untreated obese rats. In the isolated proximal tubules obtained from untreated lean rats, dopamine caused concentration-dependent inhibition of the Na,K-ATPase activity, but this inhibitory effect was absent in untreated obese rats. In rosiglitazone-treated obese rats, the inhibitory effect of dopamine on Na,K-ATPase was significantly restored. This was accompanied by a complete restoration of D1 receptor numbers in proximal tubular membranes of treated obese rats. In another set of experiments, treatment of primary proximal tubule epithelial cells in culture medium with insulin caused a significant decrease in the D1 receptor abundance, suggesting a direct role of insulin on D1 receptor regulation. We conclude that hyperinsulinemia causes downregulation of D1 receptor function and lowering of plasma insulin levels leads to restoration of renal D1 receptor function.

Loss of Biphasic Effect on Na/K-ATPase Activity by Angiotensin II Involves Defective Angiotensin Type 1 Receptor-Nitric Oxide Signaling

Oxidative stress causes changes in angiotensin (Ang) type 1 receptor (AT1R) function, which contributes to hypertension. Ang II affects blood pressure via maintenance of sodium homeostasis by regulating renal Na+ absorption through its effects on Na/K-ATPase (NKA). At low concentrations, Ang II stimulates NKA; higher concentrations inhibit the enzyme. We examined the effect of oxidative stress on renal AT1R function involved in biphasic regulation of NKA. Male Sprague-Dawley rats received tap water (control) and 30 mmol/L of l-buthionine sulfoximine (BSO), an oxidant, with and without 1 mmol/L of Tempol (antioxidant) for 2 weeks. BSO-treated rats exhibited increased oxidative stress, AT1R upregulation, and hypertension. In proximal tubules from control rats, Ang II exerted a biphasic effect on NKA activity, causing stimulation of the enzyme at picomolar and inhibition at micromolar concentrations. However, in BSO-treated rats, Ang II caused stimulation of NKA at both of the concentrations. The effect of Ang II was abolished by the AT1R antagonist candesartan and the mitogen-activated protein kinase inhibitor UO126, whereas the Ang type 2 receptor antagonist PD-123319 and NO synthase inhibitor NG-nitro-l-arginine methyl ester had no effect. The inhibitory effect of Ang II was sensitive to candesartan and NG-nitro-l-arginine methyl ester, whereas PD-123319 and UO126 had no effect. In BSO-treated rats, Ang II showed exaggerated stimulation of NKA, mitogen-activated protein kinase, proline-rich-tyrosine kinase 2, and NADPH oxidase but failed to activate NO signaling. Tempol reduced oxidative stress, normalized AT1R signaling, unmasked the biphasic effect on NKA, and reduced blood pressure in BSO-treated rats. In conclusion, oxidative stress–mediated AT1R upregulation caused a loss of NKA biphasic response and hypertension. Tempol normalized AT1R signaling and blood pressure.

Oxidative Stress Causes Renal Angiotensin II Type 1 Receptor Upregulation, Na+/H+ Exchanger 3 Overstimulation, and Hypertension

Oxidative stress modulates angiotensin (Ang) II type 1 receptor (AT1R) expression and function. Ang II activates renal Na+/H+ exchanger 3 (NHE3) to increase sodium reabsorption, but the mechanisms are still elusive. In addition, the upregulation of AT1R during oxidative stress could promote sodium retention and lead to an increase in blood pressure. Herein, we investigated the mechanism of Ang II–mediated, AT1R-dependent renal NHE3 regulation and effect of oxidative stress on AT1R signaling and development of hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mmol/L of l-buthionine-sulfoximine, an oxidant, with and without 1 mmol/L of Tempol, an antioxidant, for 3 weeks. l-Buthionine-sulfoximine–treated rats exhibited oxidative stress and high blood pressure. Incubation of renal proximal tubules with Ang II caused significantly higher NHE3 activation in l-buthionine-sulfoximine–treated rats compared with control. The activation of NHE3 was sensitive to AT1R blocker and inhibitors of phospholipase C, tyrosine kinase, janus kinase 2 (Jak2), Ca2+–dependent calmodulin (CaM), and Ca2+ chelator. Also, incubation of proximal tubules with Ang II caused Jak2-dependent CaM phosphorylation, which led to Jak2-CaM complex formation and increased Jak2-CaM interaction with NHE3. The activation of these signaling molecules was exaggerated in l-buthionine-sulfoximine–treated rats, whereas Tempol normalized the AT1R signaling. In conclusion, Ang II activates renal proximal tubular NHE3 through novel pathways that involve phospholipase C and an increase in intracellular Ca2+, Jak2, and CaM. In addition, oxidative stress exaggerates Ang II signaling, which leads to overstimulation of renal NHE3 and contributes to an increase in blood pressure.

Resveratrol restored Nrf2 function, reduced renal inflammation, and mitigated hypertension in spontaneously hypertensive rats

Compelling evidence supports the role of oxidative stress and renal interstitial inflammation in the pathogenesis of hypertension. Resveratrol is a polyphenolic stilbene, which can lower oxidative stress by activating the transcription factor nuclear factor-E2-related factor-2 (Nrf2), the master regulator of numerous genes encoding antioxidant and phase II-detoxifying enzymes and molecules. Given the role of oxidative stress and inflammation in the pathogenesis of hypertension, we conducted this study to test the hypothesis that long-term administration of resveratrol will attenuate renal inflammation and oxidative stress and, hence, progression of hypertension in the young spontaneously hypertensive rats (SHR). SHR and control [Wistar-Kyoto (WKY)] rats were treated for 9 wk with resveratrol or vehicle in their drinking water. Vehicle-treated SHR exhibited renal inflammatory injury and oxidative stress, as evidenced by glomerulosclerosis, tubulointerstitial injury, infiltration of inflammatory cells, and increased levels of renal 8-isoprostane and protein carbonylation. This was associated with reduced antioxidant capacity and downregulations of Nrf2 and phase II antioxidant enzyme glutathione-S-transferase (GST). Resveratrol treatment mitigated renal inflammation and injury, reduced oxidative stress, normalized antioxidant capacity, restored Nrf2 and GST activity, and attenuated the progression of hypertension in SHR. However, resveratrol had no effect on these parameters in WKY rats. In conclusion, development and progression of hypertension in the SHR are associated with inflammation, oxidative stress, and impaired Nrf2-GST activity in the kidney. Long-term administration of resveratrol restores Nrf2 expression, ameliorates inflammation, and attenuates development of hypertension in SHR. Clinical studies are needed to explore efficacy of resveratrol in human hypertension.

Effect of potassium dichromate on renal brush border membrane enzymes and phosphate transport in rats

Chromium is widely used in industry but exposure to chromium compounds in the workplace can result in nephrotoxicity. Various nephrotoxicants affect the brush border membrane (BBM) lining the epithelial cells of the proximal tubule, but there have been no studies regarding the effect of potassium dichromate (K2Cr2O7), a hexava-lent chromium compound, on renal BBM. In the present work, the effect of administering a single intraperitoneal dose (15 mg/kg body weight) of K2Cr2O7 on rat renal BBM enzymes and inorganic phosphate (Pi) transport was studied. The animals were administered normal saline (control) or K2Cr2O7 and sacrificed 1, 2, 4 and 8 days after treatment. K2Cr2O7 induced reversible damage to the rat kidney function as indicated by serum creatinine (Scr) and urea nitrogen levels. The activities of BBM marker enzymes were significantly decreased in isolated BBM vesicles (BBMV) and homogenates of cortex and medulla on 1, 2 and 4 days after administration of K2Cr2O7with complete recovery to control values after 8 days. The decrease in the activities of the enzymes was mainly due to changes in maximum velocity (Vmax) values, while the Michaelis constant (Km) remained unchanged. The sodium dependent Pi transport across BBMV was reduced by 50% after treatment with K2Cr2O7. Thus, the administration of a single dose of K2Cr2O7 leads to impairment in the functions of renal BBM. These results suggest that the nephrotoxicity of K2Cr2O7 may be mediated, at least in part, by its effect on renal BBM.