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Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells - PubMed

  • ️Sun Jan 01 2012

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

Jiandong Zhang et al. Am J Physiol Renal Physiol. 2012.

Abstract

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.

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Figures

Fig. 1.
Fig. 1.

SDS-PAGE analysis (A) and measurement of catalytic activity (B) of pure rat recombinant mutant prorenin (mProR) before or after incubation with agarose-immobilized TPCK trypsin for 6 h at 37°C digestion, compared with recombinant rat prorenin (ProR). A, from left to right: marker (Mr) and mProR before trypsin digestion (before) and after trypsin digestion (after). B: samples were collected from ProR and mProR, before and after trypsin digestion. The same volume of phosphate-buffered saline (buffer) was trypsin digested and used to control (Con) for an effect of trypsin on the catalytic activity of ProR and mProR. Catalytic activity was determined as the ability of a sample to generate angiotensin (ANG) I from nephrectomized rat serum substrate [containing angiotensinogen (AGT)]. Values are means ± SD.

Fig. 2.
Fig. 2.

(Pro)renin receptor [(p)RR] expression and production in rat mesangial cells (MC) and normal glomeruli (Glm). A: (p)RR mRNA expression by one-step RT-PCR described previously (17) in rat renal MC and normal Glm. PCR products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining under UV light to visualize the 378-bp (p)RR PCR product. Mr, DNA molecule size marker. B: Western blot analysis of (p)RR in cell membrane extracted from rat MCs and Glm described previously (44). Mr, protein molecule weight marker. C: immunofluorescent staining for (p)RR in normal renal cortex tissue. The antibodies used were described previously (44).

Fig. 3.
Fig. 3.

Effect of saralasin (Sara) and (p)RR on ProR or mProR activation after incubation with rat MCs. A: catalytic activity of 10−7 M recombinant inactive ProR or mProR incubated with MCs for 6 h in the absence or presence of 10−5 M Sara. *P < 0.05 vs. the no additive Con. B: catalytic activity of 10−7 M recombinant inactive ProR and mProR incubated with MCs for 6 h when the (p)RR was present or deleted by the (p)RR Stealth small interfering RNA (siRNA). *P < 0.05 vs. the Lipofectamine 2000 (Lip)-transfected, no ProR additive Con. C and D: (p)RR mRNA expression after treatment with Stealth siRNA determined by real-time RT-PCR. C: mRNA values are expressed relative to the Lip-transfected, no ProR additive Con, which was set at unity. Values are means ± SD. *P < 0.05 vs. Lip Con. D: the specificity and relative suppression of (p)RR mRNA expression were confirmed on a 1.5% agarose gel. Mr, DNA molecular size marker.

Fig. 4.
Fig. 4.

Effects of Sara and (p)RR on activation of native or mProR and subsequent angiotensin generation in cultured MCs when AGT is present. 10−5 M Sara had no effect on catalytic activity (A) and ANG I generation (B) in the culture supernatant when cellular (p)RR was present and treated with 10−7 M inactive ProR or mProR in the presence of 3.75% rat AGT serum for 6 h, detected by RIA. *P < 0.05 vs. no ProR or mProR additive but AGT-treated Con. Catalytic activity (C) and ANG I (D) generation in the culture supernatant when cellular (p)RR was deleted by Stealth siRNA, followed by treatment of 10−7 M inactive ProR or mProR in the presence of 3.75% rat AGT serum for 6 h, detected by RIA. *P < 0.05 vs. the Lip-transfected, no ProR or mProR additive, but AGT-treated Con. Values are means ± SD.

Fig. 5.
Fig. 5.

Effect of Sara (Sar) on ProR or mProR-induced mRNA expression of transforming growth factor (TGF)-β1 (A), plasminogen activator inhibitor-1 (PAI-1; B), and fibronectin (FN; C) by MCs. 10−5 M Sar had no effect on 10−7 M ProR or mProR induced TGF-β1 (A), PAI-1 (B), and FN (C) mRNA expression determined by real-time RT-PCR. mRNA values are expressed relative to the no-additive Con, which was set at unity. Values are means ± SD. *P < 0.05 vs. Con.

Fig. 6.
Fig. 6.

Effect of (p)RR Stealth siRNA (A) on ProR or mProR-induced mRNA expression of TGF-β1 (C), PAI-1 (B), and FN (D) by MCs. Measurement of TGF-β1 (C), PAI-1 (B), and FN (D) in ProR or mProR-treated MCs when cellular (p)RR mRNA expression (A) was suppressed by siRNA is shown. mRNA values are expressed relative to the Lip-transfected, no ProR additive Con, which was set at unity. Values are means ± SD. *P < 0.05 vs. Lip Con. #P < 0.05 vs. Stealth siRNA-transfected cells plus 10−7 M ProR or mProR.

Fig. 7.
Fig. 7.

Effect of Sar on ProR or mProR-induced mRNA expression of TGF-β1 (A), PAI-1 (B), and FN (C) by MCs in the presence of exogenous AGT. Cultured rat MCs were incubated with 10−7 M ProR or mProR in the presence of 3.75% AGT for 6 h, with or without 10−5 M Sar. mRNA expression of TGF-β1 (A), PAI-1 (B), and FN (C) was detected by real-time RT-PCR. mRNA values are expressed relative to the no-additive Con, which was set at unity. Values are means ± SD. *P < 0.05 vs. no additive Con. #P < 0.05 vs. ProR alone. §P < 0.05 vs. ProR or mProR plus AGT when (p)RR was present.

Fig. 8.
Fig. 8.

Effects of (p)RR Stealth siRNA on ProR or mProR-induced TGF-β1 (A), PAI-1 (B), and FN (C) mRNA expression by MCs in the presence of exogenous AGT. Expression of TGF-β1 (A), PAI-1 (B), and FN (C) was measured by real-time RT-PCR in ProR-treated MCs, with or without 3.75% AGT after cellular (p)RR mRNA expression was suppressed by siRNA. mRNA values are expressed relative to the Lip-transfected, no ProR or mProR additive, but AGT-treated Con, which was set at unity. *P < 0.05 vs. Con. #P < 0.05 vs. Stealth siRNA-transfected cells plus 10−7 M ProR or mProR and 3.75% AGT treated.

Fig. 9.
Fig. 9.

Effects of Sar and (p)RR Stealth siRNA on native or mProR-induced PAI-1 protein expression by MCs. A: effect of (p)RR Stealth siRNA on ProR/mProR-induced PAI-1 protein production. PAI-1 protein production was determined by Western blot in ProR or mProR-treated MCs when cellular (p)RR mRNA expression was suppressed by siRNA. PAI-1 protein values are expressed relative to the Lip-transfected, no ProR or mProR additive Con, which was set at unity. *P < 0.05 vs. Lip Con. #P < 0.05 vs. Stealth siRNA-transfected cells plus 10−7 M ProR. B: effect of Sar on ProR or mProR-induced PAI-1 protein production in the presence of exogenous AGT. PAI-1 protein values are expressed relative to the 3.75% normal rat serum (NS) treated Con, which was set at unity. *P < 0.05 vs. Con. #P < 0.05 vs. ProR and AGT cotreated. C: effect of (p)RR Stealth siRNA on ProR/mProR-induced PAI-1 protein production in the presence of exogenous AGT. PAI-1 protein production was determined by Western blot in ProR or mProR and AGT cotreated MCs after cellular (p)RR mRNA expression was suppressed by siRNA. PAI-1 protein values are expressed relative to the Lip-transfected, no ProR or mProR additive but AGT-treated Con, which was set at unity. *P < 0.05 vs. Con. # P < 0.05 vs. Stealth siRNA-transfected cells plus 10−7 M ProR and 3.75% AGT treated.

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