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Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells - PubMed

Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells

Jiandong Zhang et al. Am J Physiol Endocrinol Metab. 2008 Oct.

Abstract

Although elevated plasma prorenin levels are commonly found in diabetic patients and correlate with microvascular complications, the pathological role of these increases, if any, remains unclear. Prorenin/renin binding to the prorenin/renin receptor [(p)RR] enhances the efficiency of angiotensinogen cleavage by renin and unmasks prorenin catalytic activity. We asked whether plasma prorenin could be activated in local vascular tissue through receptor binding. Immunohistochemical staining showing localization of the (p)RR in the aorta to vascular smooth muscle cells (VSMCs). After cultured rat VSMCs were incubated with 10(-7) M inactive prorenin, cultured supernatant acquired the ability to generate ANG I from angiotensinogen, indicating that prorenin had been activated. Activated prorenin facilitated angiotensin generation in cultured VSMCs when exogenous angiotensinogen was added. Small interfering RNA (siRNA) against the (p)RR blocked this activation and subsequent angiotensin generation. Prorenin alone induced dose- and time-dependent increases in mRNA and protein for the profibrotic molecule plasminogen activator inhibitor (PAI)-1, effects that were blocked by siRNA, but not by the ANG II receptor antagonist saralasin. When inactive prorenin and angiotensinogen were incubated with cells, PAI-1 mRNA increased a striking 54-fold, 8-fold higher than the increase seen with prorenin alone. PAI-1 protein increased 2.75-fold. These effects were blocked by treatment with siRNA + saralasin. We conclude that prorenin at high concentration binds the (p)RR on VSMCs and is activated. This activation leads to increased expression of PAI-1 via ANG II-independent and -dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may contribute to the progression of fibrotic disease.

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Figures

Fig. 1.
Fig. 1.

Prorenin/renin receptor [(p)RR] production in normal rat aorta tissue. A: immunofluorescent staining for (p)RR in normal rat aorta tissue. Original magnification ×250 (left) and ×400 (right). B: double immunofluorescent staining for (p)RR and α-smooth muscle actin (α-SMA) in normal rat aorta tissue. Red-stained (p)RR and green-stained α-SMA produced strong yellow staining when viewed with a double filter. Original magnification ×250.

Fig. 2.
Fig. 2.

Expression of renin-angiotensin system (RAS) components and (p)RR in rat vascular smooth muscle cells (VSMCs). A: mRNA for RAS components in rat liver and VSMCs by real-time RT-PCR. Top: PCR products (30 μl) were electrophoresed on 1.5% agarose gel, and 245-bp angiotensinogen (AGT), 266-bp angiotensin-converting enzyme (ACE), 274-bp renin, and 239-bp GAPDH PCR products were visualized by ethidium bromide staining under UV light. Bottom: changes in mRNA levels were determined by normalization of amplification of each sample to that of GAPDH. For comparison, this ratio was set at unity for rat VSMC sample, and level for liver tissue was expressed as fold increase over this value. B: (p)RR mRNA expression by 1-step RT-PCR described previously (15) in rat renal mesangial cells (MC), normal glomeruli (Glm), VSMCs, and artery. M, DNA marker. C: Western blot analysis of (p)RR in cell membrane extracts from rat MCs and VSMCs. Total protein was separated by SDS-PAGE and analyzed by immunoblotting with anti-rat prorenin receptor F(ab')2. β-Actin staining was used as a control for sample loading of gel wells.

Fig. 3.
Fig. 3.

SDS-PAGE analysis of pure rat recombinant prorenin. After purification, recombinant prorenin was subjected to SDS-PAGE. Bands were visualized by Coomassie blue staining. Mr, molecular weight marker.

Fig. 4.
Fig. 4.

Prorenin activation after incubation with rat VSMCs. A: catalytic activity of 0–10−7 M recombinant inactive prorenin incubated with VSMCs for 6 h. B: catalytic activity of culture supernatant from VSMCs incubated with 10−7 M recombinant inactive prorenin for 0–8 h.

Fig. 5.
Fig. 5.

Activated prorenin facilitates angiotensin generation when exogenous AGT is present. Catalytic activity (A) and ANG I and ANG II generation (B) of 10−7 M recombinant prorenin incubated with VSMCs for 0–8 h in the presence of 3.75% rat AGT serum were detected by RIA and ELISA, respectively.

Fig. 6.
Fig. 6.

Effect of (p)RR on activation of prorenin and subsequent angiotensin generation in cultured VSMCs. Dose (A) and time (B) responses of rat (p)RR mRNA expression are plotted against Stealth small interfering RNA (siRNA) targeting (p)RR. (p)RR mRNA was measured by real-time RT-PCR and standardized to GAPDH mRNA levels. Levels of mRNA and protein are expressed relative to nontransfected normal control (Con) and Lipofectamine 2000 (Lipo)-transfected values in A and to Stealth siRNA-transfected value at time 0 in B, 2 of which were set at 100%. Catalytic activity (C) and ANG I (D) and ANG II (E) generation in the culture supernatant when cellular (p)RR was deleted by Stealth siRNA followed by treatment of 10−7 M inactive prorenin in the presence of 3.75% rat AGT serum for 6 h were detected by RIA and ELISA, respectively.

Fig. 7.
Fig. 7.

Effect of prorenin on plasminogen activator inhibitor (PAI)-1 mRNA and protein expression. Time course of PAI-1 mRNA (A) and protein (B) induction by 10−7 M prorenin was detected by real-time RT-PCR and Western blot, respectively. Relative values are expressed relative to time 0 control, which was set at unity. *P < 0.05 vs. time 0 medium-only control (A) or time 0 control (B). C and D: effect of prorenin dose on PAI-1 mRNA (C) and protein (D) production. PAI-1 mRNA and protein values are expressed relative to no-additive control (con), which was set at unity. *P < 0.05 vs. control.

Fig. 8.
Fig. 8.

Effects of saralasin and (p)RR Stealth siRNA on prorenin-induced PAI-1 mRNA and protein synthesis by VSMCs. Saralasin (Sar, 10−5 M) had no effect on 10−7 M prorenin-induced PAI-1 mRNA (A) and protein (B) production determined by real-time RT-PCR and Western blot. PAI-1 mRNA and protein values are expressed relative to no-additive control (Con) in A or saralasin-alone control in B, which was set at unity. *P < 0.05 vs. control. C and D: effect of (p)RR Stealth siRNA on prorenin-induced PAI-1 mRNA expression and protein production. Measurement of PAI-1 mRNA (C) and protein (D) production in prorenin-treated VSMCs was measured when cellular (p)RR mRNA expression was suppressed by siRNA. PAI-1 mRNA and protein values are expressed relative to Lipofectamine 2000-transfected, no-prorenin control, which was set at unity. *P < 0.05 vs. Lipo control. #P < 0.05 vs. 10−7 M prorenin-treated cells without siRNA transfection.

Fig. 9.
Fig. 9.

Effect of prorenin on PAI-1 mRNA and protein expression by VSMCs in the presence of exogenous AGT. A: dose effect of rat AGT serum on prorenin-induced PAI-1 mRNA expression. Serum-free medium (medium) and 3.75% normal rat serum (NS) served as controls. PAI-1 mRNA levels are expressed relative to 10−7 M prorenin in serum-free medium-treated control, which was set at unity. *P < 0.05 vs. control. B and C: time course of PAI-1 mRNA and protein induction by 10−7 M prorenin in the presence of 3.75% AGT detected by real-time RT-PCR and Western blot. Values are expressed relative to time 0 control, which was set at unity. *P < 0.05 vs. time 0 control. D and E: effect of prorenin in the presence of 3.75% AGT on PAI-1 mRNA and protein production. Serum-free medium and 3.75% normal rat serum served as controls. PAI-1 mRNA and protein values are expressed relative to no-additive serum-free medium control in D and 3.75% normal rat serum control in E, which were set at unity. *P < 0.05 vs. control.

Fig. 10.
Fig. 10.

Effects of saralasin and (p)RR Stealth siRNA on prorenin-induced PAI-1 mRNA and protein synthesis by VSMCs in the presence of exogenous AGT. A: PAI-1 mRNA expressed relative to no-additive control, which was set at unity. *P < 0.05 vs. control. #P < 0.05 vs. prorenin. §P < 0.05 vs. prorenin + AGT. B: PAI-1 protein expressed relative to 10−5 M saralasin + 3.75% AGT control, which was set at unity. *P < 0.05 vs. control. #P < 0.05 vs. prorenin + AGT. C–E: effect of (p)RR Stealth siRNA on prorenin-induced PAI-1 mRNA expression and protein production in the presence of exogenous AGT. C: measurement of (p)RR mRNA expression by real-time RT-PCR after transfection with 250 pmol of (p)RR Stealth siRNA for 48 h followed by 6 h of prorenin and/or AGT treatment. (p)RR mRNA expression was suppressed >95% only by siRNA treatment. D: measurement of PAI-1 mRNA expression by real-time RT-PCR in prorenin-treated VSMCs with or without 3.75% AGT after siRNA suppression of cellular (p)RR mRNA expression. PAI-1 mRNA values are expressed relative to no-additive control, which was set at unity. *P < 0.05 vs. no-additive control. #P < 0.05 vs. prorenin alone. §P < 0.05 vs. AGT alone. ‡P < 0.05 vs. prorenin + AGT. E: measurement of PAI-1 protein production by Western blot in VSMCs treated with prorenin + AGT after siRNA suppression of cellular (p)RR mRNA expression. PAI-1 protein values are expressed relative to Lipofectamine 2000-transfected, non-prorenin-treated, but AGT-treated, control, which was set at unity. *P < 0.05 vs. control. #P < 0.05 vs. non-Stealth siRNA-transfected cells treated with 10−7 M prorenin + 3.75% AGT.

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