Inhibition of (pro)renin Receptor Contributes to Renoprotective Effects of Angiotensin II Type 1 Receptor Blockade in Diabetic Nephropathy

Figure 1. Evidence for diabetic nephropathy (DN) in different stages of STZ-induced diabetic rats. (A) Body weight, (B) ratio of right kidney to body weight of the Control (n = 7) and diabetic rats (DM rats) (n = 6) in 24 weeks after STZ administration. The photographs are the representative for rats and kidneys. (C) Representative hematoxylin and eosin (H&E) staining and (D) periodic acid-Schiff (PAS) staining of glomeruli from Control and DM rats in the different diabetes stages (6, 12, and 24 weeks). Scale bar: 50 μm. (E,F) Electron microscopy of kidney sections from Control and DM rats in the different diabetes stages (6, 12, and 24 weeks after STZ administration). (E) Representative electron microscopy images of glomerular basement membranes and podocytes in glomeruli. Scale bar: 1 μm. (F) Representative electron microscopy images of mesangial areas in glomeruli. Scale bar: 2 μm. *P < 0.05 Control vs. DM rats.

Figure 2. RAS components in the plasma and kidney of Control and DM rats at the end of 6, 12, or 24 weeks after STZ induction. (A) Plasma renin activity measured by radioimmunoassay. (B) Kidney (pro)renin and (C) PRR mRNA level by real-time PCR. (D) Kidney PRR protein level by Western blotting. (E) Plasma and (F) kidney Ang II measured by radioimmunoassay. *P < 0.05 Control vs. DM rats in the same time point.

Figure 3. Histology analysis of kidneys from Control, DM and losartan treated DM (DM + losartan) rats. (A) Representative H&E staining and (B) PAS staining of glomeruli from Control, DM and DM + losartan rats. Scale bar: 50 μm. (C,D) Electron microscopy of kidney sections. (C) Representative electron microscopy images of glomerular basement membranes and podocytes in glomeruli. Scale bar: 1 μm. (D) Representative electron microscopy images of mesangial areas of glomeruli. Scale bar: 2 μm.

Figure 4. Expression of the components of RAS and TGF-β1 in the kidney of Control, DM and DM + losartan rats. (A) Kidney PRR mRNA level by real-time PCR. (B) Immunohistochemical staining for PRR in the kidneys of Control (n = 8), DM (n = 8) and DM + losartan (n = 8) rats (not counterstained with hematoxylin to compare the PRR expression better). The arrows indicated the representative area of PRR expression in the renal tissues. (C) TGF-β1, (D) (pro)renin, (E) AT1R, (F) AT2R mRNA level in the kidneys of DM (n = 8) and DM + losartan (n = 8) rats by real-time PCR. *P < 0.05 compared with Control rats, #P < 0.05 compared with DM rats.

Figure 5. Co-localization of PRR and FITC-HRP. (Aa) Immunofluorescence showing FITC-HRP was mainly located in the cytoplasm and membrane. (b) Immunofluorescence showing PRR was mainly located in the cytoplasm and membrane. (c) Analysis of the overlay of PRR and HRP. Yellow is the co-localization. (B) Triple fluorescence photograph with confocal microscopy reveals the co-localization of PRR and FITC-HRP (5 min). The co-localization (yellow staining) mainly focused in the membrane and cytoplasm. Scale bar: 20 μm.

Figure 6. HRP suppressed high glucose and Ang II-induced proliferative and fibrotic effects in MCs. Antagonistic action of HRP (10−7 M and 10−6 M) on high glucose-induced increase in phosphorylation of ERK1/2 (A) and TGF-β1 mRNA expression (B) by real-time PCR. (C) Abolition of the high glucose-induced decrease in MMP-2 activity by HRP by gelatin zymography. *P < 0.05 compared with control; #P < 0.05 compared with high glucose group. (D) BrdU ELISA shows suppression of the Ang II-induced increase in proliferation of MCs by HRP (10−7 M and 10−6 M). (E) Abolition of the Ang II-induced decrease in MMP-2 activity by HRP (10−7 M and 10−6 M) by gelatin zymography. Co-treatment of HRP and losartan leads to a further reduction in phosphorylation of ERK1/2 (F) and TGF-β1 expressions (H) compared with losartan or HRP individual treatment. (G) Ang II causes a dose-dependent increase in TGF-β1 mRNA expression. (I) The suppressing effect of Ang II on PRR expression is induced by activation of AT2R. HRP has no effect on the PRR expression. (J) Effects of AT2R activation and PRR inhibition by HRP on TGF-β1 expressions in MCs by real-time PCR. *P < 0.05 compared with control; #P < 0.05 compared with Ang…

Figure 7. Schematic diagram of a proposed mechanism for blockade of (pro)renin/PRR activation as a therapeutic strategy. Binding of (pro)renin and PRR activates ERK1/2 signaling, resulting in the production of profibrotic factors in diabetic nephropathy, such as TGF-β1. During diabetes development, high glucose and Ang II, as well as high level (pro)renin, lead to a downregulation of PRR expression, through the mediation of AT2R (He et al., 2010) or PLZF (Schefe et al., 2006), indicating a negative feedback mechanism. But, the activation of the downregulated PRR still involes in the diabetic nephropathy development. Besides blocking AT1R, losartan plays renoprotective roles through further suppressing PRR via upregulation of AT2R. Moreover, HRP may enhance losartan's anti-fibrotic effects through further or fully inhibiting PRR. The present findings highlight blockade of PRR as a possible new addition therapy to AT1R blockade for DN. Abbreviations: Ang II, angiotensin II; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; HRP, handle region peptide (HRP) of prorenin; PLZF, promyelocytic zinc finger; PRR,…