JR. Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat. Am J Physiol Endocrinol Metab 295: E110 -E116, 2008. First published April 29, 2008 doi:10.1152/ajpendo.00258.2007.-Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6 -8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P Ͻ 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P Ͻ 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue. Ren2 rat; mineralocorticoid receptor blockade THE ROLES OF INSULIN RESISTANCE in the pathophysiology of type 2 diabetes mellitus (T2DM) and the metabolic syndrome are well established (33,38), and exploration of the mechanisms leading to diminished insulin sensitivity is a field of active research.

CM, Sowers JR. Mineralocorticoid receptor antagonism attenuates glomerular filtration barrier remodeling in the transgenic Ren2 rat. Am J Physiol Renal Physiol 296: F1013-F1022, 2009. First published March 4, 2009 doi:10.1152/ajprenal.90646.2008.-Recent evidence suggests that mineralocorticoid receptor (MR) antagonism has beneficial effects on tissue oxidative stress and insulin metabolic signaling as well as reducing proteinuria. However, the mechanisms by which MR antagonism corrects both renin-angiotensin-aldosterone system (RAAS) impairments in renal insulin metabolic signaling and filtration barrier/podocyte injury remain unknown. To explore this potential beneficial interactive effect of MR antagonism we used young transgenic (mRen2)27 (Ren2) rats with increased tissue RAAS activity and elevated serum aldosterone levels. Ren2 and age-matched Sprague-Dawley (SD) control rats (age 6 -7 wk) were implanted with a low dose of the MR antagonist spironolactone (0.24 mg/day) or vehicle, both delivered over 21 days. Albuminuria, podocyte-specific proteins (synaptopodin, nephrin, and podocin), and ultrastructural analysis of the glomerular filtration barrier were measured in relation to RAAS activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reactive oxygen species (ROS), and the redox-sensitive Rho kinase (ROK). Insulin metabolic signaling was determined via measurement of insulin receptor substrate-1 (IRS-1) phosphorylation, IRS-1 ubiquitin/proteasomal degradation, and phosphorylation of Akt. Ren2 rats exhibited albuminuria, loss of podocytespecific proteins, and podocyte foot process effacement contemporaneous with reduced renal IRS-1 and protein kinase B/Akt phosphorylation compared with SD control rats (each P Ͻ 0.05). Ren2 kidneys also manifested increased NADPH oxidase/ROS/ROK in conjunction with enhanced renal tissue levels of angiotensin II (ANG II), ANG-(1-12), and angiotensin type 1 receptor. Low-dose spironolactone treatment reduced albuminuria and tissue RAAS activity and improved podocyte structural and protein integrity with improvements in IRS-1/Akt phosphorylation. Thus, in this model of RAAS activation, MR antagonism attenuates glomerular/podocyte remodeling and albuminuria, in part through reductions in redox-mediated impairment of insulin metabolic signaling. renal mineralocorticoid receptor; reduced nicotinamide adenine dinucleotide phosphate oxidase; oxidative stress; podocyte ALDOSTERONE, like angiotensin II (ANG II), exerts nongenomic actions in cardiovascular and renal tissues including the generation of reactive oxygen species (ROS), increases in inflammation, and impairment of insulin metabolic signaling and endothelial function, processes that promote glomerular disease and albuminuria (5,6,9,29,31). These adverse effects may result, in part, from mineralocorticoid receptor (MR)-and angiotensin type 1 receptor (AT 1 R)-mediated activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and redox-sensitive serine (Ser) kinases, such a...

We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats, in part, by reducing oxidative stress. Six-week-old male Ren2 and Sprague-Dawley rats received RSV (10 mg x kg(-1) x day(-)1 ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitrotyrosine, superoxide, stable nitric oxide (NO) metabolites [nitrates and nitrites (NO(x))], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of age, PAH develops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Ren2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitrotyrosine, NO(x), NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxynitrite formation.

Renin‐angiotensin‐aldosterone system (RAAS) activation is associated with glomerular filtration barrier/podocyte injury & accompanying proteinuria. To explore potential MR mediated renal effects, Ren2 transgenic rats, which manifest increased tissue RAAS activity with elevated serum aldosterone levels, were used. Six week old Ren2 and Sprague‐Dawley rats were treated with a low dose of the MR antagonist spironolactone (SP)(0.24 mg/day) or vehicle for 21 days. Ren2 rats exhibited albuminuria, loss of podocyte‐specific proteins and podocyte foot‐process effacement on ultrastructural analysis, along with reduced expression of mediators of insulin metabolic signaling. Ren2 kidneys also manifested increased NADPH oxidase/ROS and redox‐sensitive Rho kinase activation in conjunction with enhanced renal tissue levels of MR, angiotensin type 1 receptor, angiotensin II and angiotensin‐(1‐12). SP treatment reduced albuminuria and tissue RAAS expression and improved podocyte structural and protein integrity along with improvements in insulin metabolic signaling. MR blockade directly provides protection in this model of renal RAAS activation, in part, through reductions in redox‐sensitive ROK impairment of insulin metabolic signaling and glomerular remodeling.Supported by NIH R01 HL73101‐01A1 (JRS) & HL‐51952 (CMF), VA Merit (JRS), VA VISN 15 (AWC), & Missouri Kidney Program (AWC).

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P<0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS & iNOS) by 183%, 293% and 372%, respectively (all P<0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P<0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.