(Pro)renin receptor antagonist PRO20 attenuates nephrectomy‐induced nephropathy in rats via inhibition of intrarenal RAS and Wnt/β‐catenin signaling

FIGURE 1. Histological analysis of renal injury. Male Wistar rats were treated for 12 weeks with control, 5/6Nx, or 5/6Nx + PRO20. (a) Representative photomicrographs showing glomerular structures (periodic acid‐schiff staining, 400×) and (b) summarized glomerular damage index by semiquantitation of scores in different groups. (c) PAS staining of renal cortex. Original magnification, ×200. (d) Renal tubulointerstitial injury score from semi‐quantitative analysis of renal pathologies. Average of 10 fields of area per rat. N = 5 per group. (e, f, g, and h) Immunoblotting analysis of FN, α‐SMA, COL‐1, E‐cadherin. The protein abundance of FN, α‐SMA, COL‐1, E‐cadherin protein levels calculated by densitometry were normalized relative to β‐actin signals. (i) qRT‐PCR analysis of mRNA expression of FN, α‐SMA, COL‐1, E‐cadherin in renal cortex. The expression values were normalized to that of GAPDH. N = 6 per group. *p < 0.05 versus CTR; # p < 0.05 versus 5/6Nx

FIGURE 2. Analysis of the expression levels of renal oxidative stress and pro‐inflammatory cytokines. (a and B) Plasma and urinary TBARS. (C) qRT‐PCR analysis of mRNA expression of IL‐1β, IL‐6, IL‐8 and MCP‐1 in renal cortex. The values were normalized by GAPDH. (d) Immunoblotting analysis of IL‐8. The protein abundance of IL‐8 was analyzed by densitometry and the values were normalized by β‐actin and shown underneath the blot. (e) qRT‐PCR analysis of mRNA expression of TGF‐β1, PAI‐1 in renal cortex. The expression values were normalized to that of GAPDH. (f) Immunoblotting analysis of TGF‐β1. The densitometry of TGF‐β1 was normalized by β‐actin. Data are means ± SEM. N = 6 per group. *p < 0.05 versus CTR; # p < 0.05 versus 5/6Nx

FIGURE 3. Analysis of PRR/sPRR expression. (a) Representative DAB IHC‐stained sections of the glomeruli demonstrating the expression of PRR. DAB, diaminobenzidine, IHC, immunohistochemistry, original magnification ×400. (B) qRT‐PCR analysis of renal cortex PRR expression. The expression values were normalized to that of β‐actin. (c) Renal expression of full‐length PRR and sPRR by immunoblot analysis. The data shown is a representative image. The expression values were normalized to that of β‐actin. (d) ELISA analysis of urinary sPRR excretion. (e) ELISA analysis of plasma sPRR. Data are means ± SEM. N = 6 per group. *p < 0.05 versus CTR; # p < 0.05 versus 5/6Nx

FIGURE 4. Analysis of intrarenal renin levels in comparison with plasma renin activity. (a) Urinary renin activity. (b) Plasma renin activity. (c) ELISA analysis of urinary AGT. (d) ELISA analysis of plasma AGT. (e) ELISA analysis of urinary Ang II. (f) ELISA analysis of plasma Ang II. Data are means ± SEM. N = 6. *p < 0.05 versus CTR; # p < 0.05 versus 5/6Nx

FIGURE 5. (a) Immunofluorescence microscopy show Nx‐induced β‐catenin activation that was attenuated by PRO20. Scale bars indicate 50 μm. (b) qRT‐PCR analysis of mRNA expression of Wnt3A, β‐catenin, snail, MMP‐7, Fsp‐1 in renal cortex. The expression values were normalized to that of GAPDH. (c) Immunoblotting analysis of Wnt3A, β‐catenin in renal cortex. The data shown is a representative image. The expression values were normalized to that of β‐actin. (d) Diagram shows experimental design. Green bar shows the treatment schedule of PRO20 or vehicle. N = 6. *p < 0.05 versus CTR; # p < 0.05 versus 5/6Nx