Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment

Abstract

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

Figure 1

SBP and albuminuria in transgenic Ren2 rats. A, SBP in Ren2 rats at the end of treatment period. B, Albuminuria in the Ren2 rat as measured at the beginning of treatment (6 weeks of age or 0 weeks of treatment), middle of treatment period (7 to 8 weeks of age or 1.5 weeks of treatment), and end of treatment period (9 weeks of age or 3 weeks of treatment). *P<0.05 vs age-matched SD-C (n=6); #P=0.05 when Ren2-C (n=6) 3 week is compared with 0 weeks; **P<0.05 when Ren2-RSVs (n=4) are compared with age-matched SD-C rats.

Figure 2

Rosuvastatin improves indices of podocyte foot-process effacement on TEM. A, Representative TEM images at ×10 000 (left panel) and ×60 000 (right panel). B, Indices for glomerular filtration barrier integrity. *P<0.05 when Ren2-Cs (n=6) are compared with age-matched SD-Cs (n=6); **P<0.05 when Ren2-RSVs (n=4) or SD-RSVs (n=4) are compared with age-matched controls.

Figure 3

Rosuvastatin attenuation of NADPH oxidase in the transgenic Ren2 rat. A, Kidney cortical tissue NADPH oxidase activity in the Ren2 rat. B, Grayscale intensity measures of NADPH oxidase subunit expression for C. C, Representative images of glomerular sections immunostained for NADPH oxidase subunits NOX2 and Rac. (scale bar=50 μmol/L). *P<0.05 when Ren2-Cs (n=6) are compared with age-matched SD-Cs (n=6); **P<0.05 when Ren2-RSVs (n=4) or SD-RSVs (n=4) are compared with age-matched controls.

Figure 4

Rosuvastatin attenuation of Ang II stimulation of NADPH oxidase in podocytes. A, Dose response for Ang II stimulation of NADPH oxidase activity (n=6). B, Rosuvastatin reversal of Ang II stimulation of NADPH oxidase activity (n=7). C, Representative immunostaining of NADPH oxidase subunits in podocyte cell culture. D, mRNA expression of NADPH oxidase subunits in podocyte cell culture. *P<0.05 when Ang II was compared with control; for NOX4, Rac1 and p22^phox (n=14) and NOX2 (n=12). **P<0.05 when compared with Ang II stimulation alone; each (n=8).