Comparative effect of direct renin inhibition and AT1R blockade on glomerular filtration barrier injury in the transgenic Ren2 rat

Abstract

Renin-angiotensin system (RAS) activation contributes to kidney injury through oxidative stress. Renin is the rate-limiting step in angiotensin (ANG II) generation. Recent work suggests renin inhibition improves proteinuria comparable to ANG type 1 receptor (AT1R) blockade (ARB). Thereby, we investigated the relative impact of treatment with a renin inhibitor vs. an ARB on renal oxidative stress and associated glomerular structural and functional changes in the transgenic Ren2 rat, which manifests hypertension, albuminuria, and increased tissue RAS activity. Young Ren2 and age-matched Sprague-Dawley (SD) controls (age 6-9 wk) were treated with a renin inhibitor (aliskiren), an ARB (irbesartan), or vehicle for 21 days. Ren2 rats exhibited increases in systolic pressure (SBP), albuminuria, and renal 3-nitrotyrosine content as well as ultrastructural podocyte foot-process effacement and diminution of the podocyte-specific protein nephrin. Structural and functional alterations were accompanied by increased renal cortical ANG II, AT1R, as well as NADPH oxidase subunit (Nox2) expression compared with SD controls. Abnormalities were attenuated to a similar extent with both aliskiren and irbesartan treatment. Despite the fact the dose of irbesartan used caused a greater reduction in SBP than aliskerin treatment (P < 0.05), the effects on proteinuria, nephrin, and oxidative stress were similar between the two treatments. Our results highlight both the importance of pressor-related reductions on podocyte integrity and albuminuria as well as RAS-mediated oxidant stress largely comparable between ARB and renin inhibition treatment.

Fig. 1.

Systolic blood pressure (SBP) and albuminuria in the Ren2 rat. SBP (A) as measured by tail cuff and albuminuria (B) as determined by urine albumin-to-creatinine ratio, both determined at the end of the treatment period of 21 days. Values presented as means ± SE. *P < 0.05 when Ren2 controls (Ren2-C; n = 5) are compared with age-matched Sprague-Dawley controls (SD-C; n = 5). **P < 0.05 when aliskiren- or irbesartan-treated Ren2 rats (Ren2-A and Ren2-I; n = 5) are compared with age-matched Ren2-C. #P < 0.05 when Ren2-I are compared with Ren2-A.

Fig. 2.

Podocyte foot process integrity in the Ren2. A: Western blot analysis of the podocyte-specific protein nephrin from kidney cortical tissue homogenates. Each lane was loaded with 50 μg of protein and equal loading was ensured by both Ponceau staining and the housekeeping protein, β-actin. B: ultrastructural analysis of the glomerular filtration barrier on transmission electron microscopy at ×10,000 (top) and ×60,000 (bottom). The Ren2 (Ren2-C; n = 4) rat displays loss of glomerular filtration barrier integrity with podocyte foot process effacement and loss of the slit pore diaphragm (arrows) compared with Sprague-Dawley (SD-C; n = 5) controls. Podocyte foot process effacement is improved with both aliskiren and irbesartan treatment in the Ren2 (Ren2-A and Ren2-I; n = 5) rats. *P < 0.05 when Ren2-C (n = 5) are compared with SD-C (n = 5). **P < 0.05 when Ren2-A and Ren2-I (n = 5) are compared with age-matched Ren2-C.

Fig. 3.

Semiquantitative analysis of oxidative stress in Ren2 cortical tissue. Representative images of immunohistochemistry analysis of kidney cortical tissue NADPH oxidase subunit Nox2 (A) and 3-nitrotyrosine (3-NT; B), as a marker for lipid peroxidative and oxidative stress, with corresponding average gray scale intensities below. Values presented as means ± SE. *P < 0.05 when Ren2-C (n = 5) are compared with SD-C (n = 5). **P < 0.05 when Ren2-A and Ren2-I (n = 5) are compared with age-matched Ren2-C. #P < 0.05 when Ren2-I are compared with Ren2-A. Scale bar = 50 μm.

Fig. 4.

Semiquantitative analysis of ANG II and AT₁R in Ren2 cortical tissue. Representative images of immunohistochemistry analysis of kidney cortical tissue for ANG II and AT₁R with corresponding average gray scale intensities below. Values presented as means ± SE. *P < 0.05 when Ren2-C (n = 5) are compared with SD-C (n = 5). **P < 0.05 when Ren2-A and Ren2-I (n = 5) are compared with age-matched Ren2-C. Scale bar = 50 μm.