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These data indicate that urinary angiotensinogen is a powerful tool for determining intrarenal RAS status and associated renal derangement in patients with IgA nephropathy.

Recent studies have implicated a contribution of oxidative stress to the development of hypertension. Studies were performed to determine the effects of the superoxide dismutase (SOD) mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) on vascular superoxide production and renal sympathetic nerve activity (RSNA) in anesthetized Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Compared with WKY rats (n=6), SHR showed a doubled vascular superoxide production, which was normalized by treatment with Tempol (3 mmol/L, n=7). In WKY rats (n=6), Tempol (30 mg/kg IV) significantly decreased mean arterial pressure (MAP) from 108+/-5 to 88+/-6 mm Hg and HR from 304+/-9 to 282+/-6 beats/min. In SHR (n=6), Tempol significantly decreased MAP from 166+/-4 to 123+/-9 mm Hg and HR from 380+/-7 to 329+/-12 beats/min. Furthermore, Tempol significantly decreased RSNA in both WKY rats and SHR. On the basis of group comparisons, the percentage decreases in MAP (-28+/-4%), HR (-16+/-3%) and integrated RSNA (-63+/-6%) in SHR were significantly greater than in WKY rats (-17+/-3%, -9+/-2%, and -30+/-4%, respectively). In SHR, changes in integrated RSNA were highly correlated with changes in MAP (r=0.85, P<0.0001) during administration of Tempol (3, 10, and 30 mg/kg IV). In both WKY rats and SHR (n=4, respectively), intracerebroventricular injection of Tempol (300 micro g/1 micro L) did not alter MAP, HR, or RSNA. Intravenous administration of a SOD inhibitor, diethyldithio-carbamic acid (30 mg/kg), significantly increased MAP, HR, and integrated RSNA in both WKY rats and SHR (n=6, respectively). These results suggest that augmented superoxide production contributes to the development of hypertension through activation of the sympathetic nervous system.

Patients and animals with renal injury exhibit increased urinary excretion of angiotensinogen. Although increased tubular synthesis of angiotensinogen contributes to the increased excretion, we do not know to what degree glomerular filtration of systemic angiotensinogen, especially through an abnormal glomerular filtration barrier, contributes to the increase in urinary levels. Here, we used multiphoton microscopy to visualize and quantify the glomerular permeability of angiotensinogen in the intact mouse and rat kidney. In healthy mice and Munich-Wistar-Frömter rats at the early stage of glomerulosclerosis, the glomerular sieving coefficient of systemically infused Atto565-labeled human angiotensinogen (Atto565-hAGT), which rodent renin cannot cleave, was only 25% of the glomerular sieving coefficient of albumin, and its urinary excretion was undetectable. In a more advanced phase of kidney disease, the glomerular permeability of Atto565-hAGT was slightly higher but still very low. Furthermore, unlike urinary albumin, the significantly higher urinary excretion of endogenous rat angiotensinogen did not correlate with either the Atto565-hAGT or Atto565-albumin glomerular sieving coefficients. These results strongly suggest that the vast majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration. The renin-angiotensin system (RAS) is one of the most important regulatory mechanisms of body fluid, electrolyte homeostasis, and BP. 1-4 RAS in the kidney is independently regulated from RAS in the systemic circulation, and it has been implicated in the development of hypertension and kidney diseases. For example, renal epithelial cellspecific overexpression of human angiotensinogen (AGT) in human renin transgenic mice resulted in increased renal angiotensin II, hypertension, and renal fibrosis without any changes in circulating angiotensin II. 5 Also, Dahl salt-sensitive rats, which show low plasma renin activity under high salt feeding, had higher renal angiotensin II content in the kidney compared with normal salt-fed control animals. 6 Although all components that are necessary for angiotensin II production are expressed in the kidney, 3 AGT is, currently, the only component that is noninvasively measurable in the urine of patients. The level of urinary AGT reflects the activity of the intrarenal RAS, and it is associated with pathologic states in several experimental 7,8 and clinical studies. 9,10 Although the major source of the circulating AGT is the liver, we and others have previously shown that AGT is produced in the proximal tubules through a de novo pathway. 3,11

Abstract-Clinical reports indicate that patients with primary aldosteronism commonly have impaired glucose tolerance; however, the relationship between aldosterone and insulin signaling pathway has not been clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor substrate-1 expression and insulin signaling pathway including Akt phosphorylation and glucose uptake in rat vascular smooth muscle cells. Insulin receptor substrate-1 protein expression and Akt phosphorylation were determined by Western blot analysis with anti-insulin receptor substrate-1 and phosphorylated-Akt antibodies, respectively. Glucose metabolism was evaluated using 3 H-labeled 2-deoxy-D-glucose uptake. Aldosterone (1-100 nmol/L) dose-dependently decreased insulin receptor substrate-1 protein expression with a peak at 18 hours (nϭ4). Aldosterone-induced degradation of insulin receptor substrate-1 was markedly attenuated by treatment with the selective mineralocorticoid receptor antagonist eplerenone (10 mol/L; nϭ4). Furthermore, degradation was blocked by the Src inhibitor PP1 (20 mol/L; nϭ4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L), or ebselen (40 mol/L) also attenuated aldosterone-induced insulin receptor substrate-1 degradation (nϭ4). In addition, proteasome inhibitor MG132 (1 mol/L) prevented insulin receptor substrate-1 degradation (nϭ4). Aldosterone treatment abolished insulin-induced Akt phosphorylation (100 nmol/L; 5 minutes; nϭ4). Furthermore, aldosterone pretreatment decreased insulin-stimulated (100 nmol/L; 60 minutes; nϭ4) glucose uptake by 50%, which was reversed by eplerenone (10 mol/L; nϭ4). These data indicate that aldosterone decreases insulin receptor substrate-1 expression via Src and reactive oxygen species stimulation by proteasome-dependent degradation in vascular smooth muscle cells; thus, aldosterone may be involved in the pathogenesis of vascular insulin resistance via oxidative stress. Key Words: aldosterone Ⅲ oxidative stress Ⅲ insulin receptor substrate-1 Ⅲ insulin resistance Ⅲ type 2 diabetes mellitus Ⅲ metabolic syndrome Ⅲ eplerenone I nsulin resistance is a key attribute of type 2 diabetes and the metabolic syndrome. 1,2 Systemic glucose metabolism is maintained in the liver and skeletal muscle, and in the insulin resistant state, insulin-stimulated glucose uptake is attenuated and accompanied with subsequent increases in blood glucose concentration. High blood glucose concentration induces secretion of insulin from the pancreas and results in hyperinsulinemia. Both hyperglycemia and hyperinsulinemia affect the vasculature and are associated with microangiopathy, including retinopathy and nephropathy, and macroangiopathy, including cardiovascular disease and atherosclerosis. 3 Insulin resistance in the vasculature might also affect systemic glucose metabolism. However, the involvement of normal insulin signaling contributes to arteriosclerosis. 4,5 On the other hand, serine phosphorylation and degradation of insulin receptor substrate-1 (IRS-1) is a possible...

Objective-Angiotensin II has been implicated in the pathogenesis of the vascular complications of insulin resistance.Recently, serine phosphorylation and degradation of insulin receptor substrate-1 (IRS-1) were shown to inhibit Akt activation and reduce glucose uptake. Therefore, we examined the effects of chronic angiotensin II treatment on IRS-1 phosphorylation and protein expression in vascular smooth muscle cells (VSMCs). Methods and Results-Using Western analysis, we found that angiotensin II (100 nmol/L; 18 hours) caused a 61Ϯ5% degradation of IRS-1 and abolished insulin-induced activation of Akt. Phosphorylation of IRS-1 on Ser307, which leads to subsequent IRS-1 degradation, was stimulated by angiotensin II. This phosphorylation was blocked by the Src inhibitor PP1 and by the antioxidants N-acetylcysteine and ebselen. Stable overexpression of catalase abrogated angiotensin II-induced IRS-1 phosphorylation and IRS-1 degradation. Similarly, a mutant phosphoinositide-dependent kinase-1 (PDK1) that cannot associate with Src abolished IRS-1 phosphorylation and degradation induced by angiotensin II. Proteasome inhibitors also prevented IRS-1 degradation. Conclusions-Thus, angiotensin II decreases IRS-1 protein levels in VSMCs via Src, PDK1, and reactive oxygen species-mediated phosphorylation of IRS-1 on Ser307 and subsequent proteasome-dependent degradation. These events impair insulin signaling and provide a molecular basis for understanding the clinical observation that angiotensin II type 1 receptor antagonists improve insulin resistance and its associated vasculopathies. Key Words: vascular smooth muscle Ⅲ angiotensin II Ⅲ insulin Ⅲ IRS-1 Ⅲ insulin resistance Ⅲ phosphoinositide-dependent kinase-1 C linical manifestations of type 2 diabetes occur when pancreatic insulin production cannot compensate for target tissue insulin resistance. In the context of the vascular system, "insulin resistance" likely does not regulate systemic glucose levels, but rather, in conjunction with the accompanying hyperglycemia, it manifests as impaired vasodilation and myogenic responsiveness, 1,2 microvessel disease leading to retinopathy and nephropathy, 3 enhanced atherosclerotic lesion formation, 4 constrictive remodeling of small arteries, 2 medial hypertrophy, 2 and enhanced vascular inflammation. 5 Recent work has implicated the renin-angiotensin system and reactive oxygen species (ROS) in the complications of insulin resistance. 4,6,7 In vascular smooth muscle cells (VSMCs), angiotensin II (Ang II) activates NAD(P)H oxidases to produce ROS, which contribute to hypertension and atherosclerosis. Recent studies indicate that angiotensinconverting enzyme (ACE) inhibitors and angiotensin II type 1 (AT 1 ) receptor blockers improve insulin resistance 4,8 and diabetic vascular complications. 4 ACE inhibitors restore impaired endothelium-dependent vasodilation in patients with type 2 diabetes 9 and improve arterial structure in diabetic rats. 10 These studies raise the provocative possibility that Ang II may directly participat...

In DS hypertensive rats, some of the renoprotective effects of AT1 receptor blockade are accompanied by reductions in intrarenal Ang II contents and MAPK activity, which might not be mediated through arterial pressure changes.