Quaisar Ali

Proximal Tubule Angiotensin AT2 Receptors Mediate an Anti-Inflammatory Response via Interleukin-10 Role in Renoprotection in Obese Rats

The angiotensin type 2 receptor (AT2R) has been shown to lower inflammation in the kidney. However, the role of the anti-inflammatory cytokine interleukin (IL)-10 in AT2R-mediated attenuation of inflammation has not been elucidated. We hypothesized that AT2R activation is renoprotective by directly increasing the levels of anti-inflammatory cytokine IL-10 in the kidney via nitric oxide (NO) signaling. For in vitro studies, the human proximal tubule epithelial cell-line (human kidney-2 [HK-2]) was activated with lipopolysaccharide (10 &mgr;g/mL) and AT2R agonist C21 (1 &mgr;mol/L) for 24 hours, and media cytokine levels were assessed. Lipopolysaccharide modestly downregulated AT2R expression. Treatment with C21 lowered lipopolysaccharide-induced levels of both tumor necrosis factor-&agr; and IL-6, but increased IL-10 levels. Treatment with neutralizing IL-10 antibody (1 &mgr;g/mL) or NO synthase inhibitor L-NAME (1 mmol/L) abolished this effect. For in vivo studies, prehypertensive obese Zucker rats and age-matched lean Zucker rats were treated for 2 weeks with C21 (300 &mgr;g/kg per day, IP) and AT2R antagonist (PD123319; 50 &mgr;g/kg per minute, SC infusion). Compared with lean Zucker rats, obese Zucker rats had higher levels of renal AT2R expression, tumor necrosis factor-&agr;, and IL-6. C21 treatment decreased levels of tumor necrosis factor-&agr; by 75% and IL-6 by 60%. Conversely, PD treatment lowered the renal IL-10 levels in obese Zucker rats by ≈60%. Renal morphometry revealed increased mesangial matrix expansion and glomerular macrophage infiltration, which was improved by C21 treatment in obese Zucker rats. Our findings suggest that proximal tubule AT2R activation is anti-inflammatory by increasing IL-10 production, which is largely NO dependent and thus offers renoprotection by preventing early inflammation–induced renal injury in obesity.

Chronic AT2 receptor activation increases renal ACE2 activity, attenuates AT1 receptor function and blood pressure in obese Zucker rats

Abnormal regulation of the renin angiotensin system such as enhanced renal AT1R function and reduced ACE2 activity contributes to obesity-related hypertension. Here we tested whether long-term AT2R activation affects renal function in obesity using lean and obese Zucker rats treated with the AT2R agonist CGP42112A for 2-weeks. This caused blood pressure to decrease by 13 mmHg which was associated with increased urinary sodium excretion in the obese rats. Cortical ACE2 expression and activity, the Mas receptor (MasR), and its ligand angiotensin-(1-7) were all increased in CGP-treated obese compared with control rats. Candesartan-induced natriuresis, a measure of AT1R function, was reduced but cortical AT1R expression and angiotensin II levels were similar in CGP-treated obese compared to control rats. Renin and AT2R expression in obese rats was not affected by CGP-treatment. In HK-2 cells in-vitro, CGP-treatment caused increased ACE2 activity and MasR levels but decreased AT1R levels and renin activity. Thus, long-term AT2R activation shifts the opposing arms of renin angiotensin system and contributes to natriuresis and blood pressure reduction in obese animals. Our study highlights the importance of AT2R as a target for treating obesity related hypertension.

Role of the angiotensin II AT2 receptor in inflammation and oxidative stress: opposing effects in lean and obese Zucker rats.

Inflammation and oxidative stress are believed to contribute to hypertension in obesity/diabetes. Recently, we reported a role for the AT(2) receptor in blood pressure control in obese Zucker rats. However, the role of AT(2) receptors in inflammation and oxidative stress in obesity is not known. Therefore, in the present study, we tested the effects of the AT(2) receptor agonist CGP-42112A on inflammation and oxidative stress in obese Zucker rats and compared them in their lean counterparts. Rats were systemically treated with either vehicle (control) or CGP-42112A (1 μg·kg(-1)·min(-1); osmotic pump) for 2 wk. Markers of inflammation (CRP, MCP-1, TNF-α, and IL-6) and oxidative stress (HO-1, gp-91(phox)) as well as an antioxidant (SOD) were determined. Control obese rats had higher plasma levels of CRP, MCP-1, TNF-α, IL-6, and HO-1 compared with control lean rats. Conversely, plasma SOD activity was lower in control obese than in control lean rats. Furthermore, the protein levels of TNF-α and gp-91(phox) were higher in the kidney cortex of control obese rats. Interestingly, CGP-42112A treatment in obese rats reduced the plasma and kidney cortex inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers and increased plasma SOD activity to the levels seen in lean control rats. However, CGP-42112A treatment in lean rats increased inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers in the plasma and kidney cortex. Our present studies suggest anti-inflammatory and antioxidative functions of AT(2) receptor in obese Zucker rats but proinflammatory and prooxidative functions in lean Zucker rats.

Protective Role of Angiotensin II Subtype 2 Receptor in Blood Pressure Increase in Obese Zucker Rats

Earlier, we reported that there was an increase in angiotensin II type 2 (AT2) receptor expression in the renal proximal tubule, and selective activation of the AT2 receptor by AT2 agonist inhibits Na,K-ATPase activity in the proximal tubules and increases urinary Na excretion in obese Zucker rats. We hypothesized that the AT2 receptor has a protective role against blood pressure increase in obese Zucker rats. To test this hypothesis, we treated obese Zucker rats with the AT2 receptor antagonist PD123319 (PD; 30 &mgr;g/kg per minute) using osmotic pumps. Age-matched lean rats and vehicle-treated obese Zucker rats served as controls. On day 15 of the treatment with PD, arterial blood pressure was measured by cannulation of the left carotid artery under anesthesia. Control obese rats exhibited higher mean arterial pressure (122.0±3.4 mm Hg) compared with lean control rats (97.0±4.8 mm Hg). The PD treatment of obese rats raised mean arterial pressure further by 13 mm Hg. The plasma renin activity was significantly increased in the PD-treated obese compared with control-obese or lean rats. Western blot analysis revealed that the PD treatment in obese rats caused an ≈3-fold increase in the renin expression in the kidney cortex but had no effect on the expression of the cortical angiotensin II type 1 and AT2 receptors. The present study suggests that the renal AT2 receptors provide a protective role against blood pressure increase in obese Zucker rats, and this protective effect, in part, could be because of the ability of the AT2 receptors to keep the kidney renin expression low in obese rats.

High Na intake increases renal angiotensin II levels and reduces expression of the ACE2-AT2R-MasR axis in obese Zucker rats

High sodium intake is known to regulate the renal renin-angiotensin system (RAS) and is a risk factor for the pathogenesis of obesity-related hypertension. The complex nature of the RAS reveals that its various components may have opposing effects on natriuresis and blood pressure regulation. We hypothesized that high sodium intake differentially regulates and shifts a balance between opposing components of the renal RAS, namely, angiotensin-converting enzyme (ACE)-ANG II-type 1 ANG II receptor (AT(1)R) vs. AT(2)-ACE2-angiotensinogen (Ang) (1-7)-Mas receptor (MasR), in obesity. In the present study, we evaluated protein and/or mRNA expression of angiotensinogen, renin, AT(1A/B)R, ACE, AT(2)R, ACE2, and MasR in the kidney cortex following 2 wk of a 8% high-sodium (HS) diet in lean and obese Zucker rats. The expression data showed that the relative expression pattern of ACE and AT(1B)R increased, renin decreased, and ACE2, AT(2)R, and MasR remained unaltered in HS-fed lean rats. On the other hand, HS intake in obese rats caused an increase in the cortical expression of ACE, a decrease in ACE2, AT(2)R, and MasR, and no changes in renin and AT(1)R. The cortical levels of ANG II increased by threefold in obese rats on HS compared with obese rats on normal salt (NS), which was not different than in lean rats. The HS intake elevated mean arterial pressure in obese rats (27 mmHg) more than in lean rats (16 mmHg). This study suggests that HS intake causes a pronounced increase in ANG II levels and a reduction in the expression of the ACE2-AT(2)R-MasR axis in the kidney cortex of obese rats. We conclude that such changes may lead to the potentially unopposed function of AT(1)R, with its various cellular and physiological roles, including the contribution to the pathogenesis of obesity-related hypertension.

High glucose up-regulates angiotensin II subtype 2 receptors via interferon regulatory factor-1 in proximal tubule epithelial cells

Earlier studies have reported an increase in the proximal tubule AT2 receptor (AT2R) expression in diabetes, with a beneficial role in kidney function and blood pressure regulation. Here, we demonstrate that the increase in AT2R protein expression is associated with an increased expression of transcription factor IRF-1 in hyperglycemic rat and in high glucose-treated HK2 cells. Knock-down of IRF-1 by siRNA in HK2 cells prevented high glucose-induced AT2R up-regulation. The data suggest that IRF-1 is a transcriptional regulator of AT2R expression in hyperglycemia, and warrant further studies to understand the physiological role of IRF-1 along with AT2R in diabetic kidney.

Angiotensin AT2 receptor agonist prevents salt-sensitive hypertension in obese Zucker rats.

High-sodium intake is a risk factor for the pathogenesis of hypertension, especially in obesity. The present study is designed to investigate whether angiotensin type 2 receptor (AT2R) activation with selective agonist C21 prevents high-sodium diet (HSD)-induced hypertension in obese animals. Male obese rats were treated with AT2R agonist C21 (1 mg·kg(-1)·day(-1), oral) while maintained on either normal-sodium diet (NSD; 0.4%) or HSD (4%) for 2 wk. Radiotelemetric recording showed a time-dependent increase in systolic blood pressure in HSD-fed obese rats, being maximal increase (∼27 mmHg) at day 12 of the HSD regimen. C21 treatment completely prevented the increase in blood pressure of HSD-fed rats. Compared with NSD controls, HSD-fed obese rats had greater natriuresis/diuresis and urinary levels of nitrates, and these parameters were further increased by C21 treatment. Also, C21 treatment improved glomerular filtration rate in HSD-fed rats. HSD-fed rats expressed higher level of cortical ANG II, which was reduced to 50% by C21 treatment. HSD feeding and/or C21 treatment had no effects on cortical renin activity and the expression of angiotensin-converting enzyme (ACE) and chymase, which are ANG II-producing enzymes. However, ANG(1-7) concentration and ACE2 activity in the renal cortex were reduced by HSD feeding, and C21 treatment rescued both the parameters. Also, C21 treatment reduced the cortical expression of AT1R in HSD-fed rats, but had no effect of AT2R expression. We conclude that chronic treatment with the AT2R agonist C21 prevents salt-sensitive hypertension in obese rats, and a reduction in the renal ANG II/AT1R and enhanced ACE2/ANG(1-7) levels may play a potential role in this phenomenon.

Estimation of angiotensin peptides in biological samples by LC/MS method.

The low abundance of angiotensin peptides in biological tissues such as the kidney cortex, adipose tissue, urine and plasma makes their detection and quantification a challenge. A few available methods used to quantify these peptides involve lengthy processes of sample preparation and are hardly quantitative. Here, we report a mass spectrometry approach for quantifying angiotensin peptides [Ang II, Ang-(1-7)] in the kidney cortex, epididymal white adipose tissue (eWAT), urine and plasma of male mice. Tissue homogenates, urine and plasma samples were solid-phase extracted with C18 Sep-Pak cartridges and eluted off proteinaceous compounds. These extracted peptide samples were separated on C18 column with a linear acetonitrile gradient and detected by Q-ToF mass analyzer in ESI+-MS ion mode based on their retention time, accurate mass measurement of peptides, the isotope pattern of doubly charged molecular ion, and quantitation of peak area (or ion count) when referencing to the angiotensin peptide standards. The lower limit of quantitation for each angiotensin peptide was 10 pgmg-1 with the percent recovery at 100.6%. The intra-batch precision for Ang-(1-7) and Ang II were 24.0 and 12.7%, accuracy 84.0-123.0% and 100.2-116.0% respectively. Using this method, we determined the levels of Ang II and Ang-(1-7) in the kidney cortex, eWAT, urine and plasma. Quantification of angiotensin peptides could help target subtle therapeutics changes against pathophysiological conditions such as obesity, kidney disease and hypertension.

Chronic angiotensin AT2R activation prevents high-fat diet-induced adiposity and obesity in female mice independent of estrogen

OBJECTIVE Obesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that female angiotensin AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,β-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. METHODS Female C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5 μg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10 days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12 weeks. RESULTS Ovi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the β-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. CONCLUSION We suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Angiotensin II Type 2–Receptor Agonist C21 Reduces Proteinuria and Oxidative Stress in Kidney of High-Salt–Fed Obese Zucker Rats

Oxidative and nitrosative stress have been implicated in high-sodium diet (HSD)–related hypertensive renal injury. In this study, we investigated angiotensin II type 2-receptor–mediated renoprotection in obese Zucker rats fed HSD. Obese Zucker rats were fed normal sodium diet or HSD 4%, for 14 days, with/without angiotensin II type 2-receptor agonist C21, delivered subcutaneously via osmotic pump, 1 mg/kg per day. Compared with normal sodium diet controls, HSD rats exhibited increase in cortical nicotinamide adenine dinucleotide phosphate oxidase activity, urinary H2O2, and 8-isoprostanes, which were associated with severe glomerulosclerosis, interstitial fibrosis, decline in estimated glomerular filtration rate, and an increase in urinary leak and activity of N-acetyl-&bgr;-D-glucosaminidase, a lysosomal enzyme and a marker of tubular damage. These changes were improved by C21 treatment. Cortical expression of endothelial nitric oxide synthase, phospho-endothelial nitric oxide synthase (Ser1177), and plasma nitrites were reduced after HSD intake, whereas nitrosative stress (3-nitrotyrosine) and enzymatic defense (superoxide dismutase-to-catalase activity) remained unaltered. However, C21 preserved plasma nitrites in HSD-fed obese Zucker rat. C21 treatment reduced protein-to-creatinine, albumin-to-creatinine, as well as fractional excretion of protein and albumin in HSD-fed obese Zucker rat, which is independent of changes in protein recycling receptors, megalin, and cubilin. HSD intake also altered renal excretory and reabsorptive capacity as evident by elevated plasma urea nitrogen-to-creatinine and fractional excretion of urea nitrogen, and reduced urine-to-plasma creatinine, which were modestly, but insignificantly, improved by C21 treatment. Together results demonstrate that angiotensin II type 2-receptor activation protects against HSD-induced kidney damage in obesity plausibly by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and rescuing nitrites.