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Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis - PubMed

Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis

Jiandong Zhang et al. Am J Physiol Renal Physiol. 2011 Oct.

Abstract

The limited antifibrotic effect of therapeutic angiotensin blockade, the fact that angiotensin blockade dramatically elevates renin levels, and recent evidence that renin has an angiotensin-independent, receptor-mediated profibrotic action led us to hypothesize that combining renin receptor inhibition and ANG II blockade would increase the antifibrotic effect of angiotensin blockade alone. Using cultured nephritic glomeruli from rats with anti-Thy-1-induced glomerulonephritis, the maximally effective dose of enalaprilate was determined to be 10(-4) M, which reduced mRNAs for transforming growth factor (TGF)-β1, fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) by 49, 65, and 56% and production of TGF-β1 and FN proteins by 60 and 49%, respectively. Disease alone caused 6.8-fold increases in ANG II levels that were reduced 64% with enalaprilate. In contrast, two- and threefold disease-induced increases in renin mRNA and activity were further increased 2- and 3.7-fold with 10(-4) M enalaprilate treatment. Depressing the renin receptor by 80% with small interfering (si) RNA alone reduced fibrotic markers in a manner remarkably similar to enalaprilate alone but had no effect on glomerular renin expression. Enalaprilate and siRNA combination therapy further reduced disease markers. Notably, elevated TGF-β1 and FN production was reduced by 73 and 81%, respectively. These results support the notion of a receptor-mediated profibrotic action of renin, suggest that the limited effectiveness of ANG II blockade may be due, at least in part, to the elevated renin they induce, and support our hypothesis that adding renin receptor inhibitor to ANG II blockade in patients may have therapeutic potential.

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Figures

Fig. 1.
Fig. 1.

Graphic representation of time course of glomerular periodic acid-Schiff (PAS) staining scores in anti-Thy-1-induced experimental glomerulonephritis. d, Day. *P < 0.05 vs. nephritic rats at 0 h after OX-7 injection.

Fig. 2.
Fig. 2.

Urinary protein excretion and glomerular matrix protein accumulation in nephritic rats at day 4. A: 24-h urinary protein excretion from d3 to d4. NC, normal control rats; DC, nephritic rats. B: representative photomicrographs of glomeruli from NC (n = 4) and DC (n = 4) at d4. Original magnification ×400. C: graphic representation of glomerular matrix score. *P < 0.05 vs. NC.

Fig. 3.
Fig. 3.

Effect of ex vivo incubation on mRNA expression of plasminogen activator inhibitor-1 (PAI-1), transforming growth factor (TGF)-β1, and fibronectin (FN) and the protein content of TGF-β1 and FN in isolated nephritic glomeruli. A: glomerular mRNA expression of PAI-1, TGF-β1, and FN was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. B and C: glomerular protein content of TGF-β1 (B) and FN (C) determined by ELISA. NC and DC, glomeruli from rats at day 4; before or after, before or after culture in 2.5% FCS RPMI 1640 for 48 h; glm, glomerulus. *P < 0.05 vs. NC before culture. #P < 0.05 vs. DC before culture.

Fig. 4.
Fig. 4.

Representative example of isolated glomeruli and effect of ex vivo incubation on glomerular mRNA expression of renin and renin receptor \(p)RR]. A: isolated glomerulus that was counted for no attachment by an afferent arteriole. B: afferent arteriole attached to an isolated glomerulus. I–IV: different isolated glomeruli attached with a similar length of afferent arteriole. Arrows point to the afferent arteriole. The glomerular isolation contains >95% of glomeruli, and total glomeruli contain >87% of afferent arterioles (glomeruli were isolated and counted from 3 individual rats). Original magnification ×250. C and D: glomerular mRNA expression of renin and (p)RR was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. *P < 0.05 vs. NC before culture.

Fig. 5.
Fig. 5.

Effect of increasing doses of enalaprilate on ANG II generation (A), renin expression (B), and renin activity (C) in isolated nephritic glomeruli. Glomerular mRNA expression of renin was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. Enalaprilate (E) was given at 4 different doses (E1–E4) for 48 h. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4.

Fig. 6.
Fig. 6.

Effect of increasing doses of enalaprilate on mRNA expression of PAI-1 (A), TGF-β1 (B), and FN (C) in isolated nephritic glomeruli determined by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4.

Fig. 7.
Fig. 7.

Effect of increasing doses of enalaprilate on TGF-β1 (A) and FN (B) production in isolated nephritic glomeruli. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4.

Fig. 8.
Fig. 8.

Effect of combinations of (p)RR small interfering (si) RNA (RRi) and enalaprilate (Ena) on mRNA expression of (p)RR (A) and renin (B) and ANG II generation (C) in isolated nephritic glomeruli. Glomerular mRNA expression of (p)RR and renin was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. SC, scrambled control siRNA. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4. §P < 0.05 compared with enalaprilate-treated nephritic glomeruli.

Fig. 9.
Fig. 9.

Effect of combinations of RRi and enalaprilate on mRNA expression of PAI-1, TGF-β1, and FN in isolated nephritic glomeruli. A–C: glomerular mRNA expression of PAI-1 (A), TGF-β1 (B), and FN (C) was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Relative values are expressed relative to NC, which was set at unity. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4. §P < 0.05 compared with enalaprilate-treated nephritic glomeruli.

Fig. 10.
Fig. 10.

Effect of combinations of RRi and enalaprilate on the protein production of TGF-β1 (A) and FN (B) in isolated nephritic glomeruli. *P < 0.05 compared with normal glomeruli isolated from NC. #P < 0.05 compared with nephritic glomeruli isolated from DC at d4. §P < 0.05 compared with enalaprilate-treated nephritic glomeruli.

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