3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: Implications for diabetic nephropathy

Abstract

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, also known as statins, are lipid-lowering agents widely used in the prevention of coronary heart disease. Recent experimental and clinical data, however, indicate that the overall benefits of statin therapy may exceed its cholesterol-lowering properties. We postulate that statins may ameliorate the detrimental effects of high glucose (HG)-induced proliferation of mesangial cells (MCs), a feature of early stages of diabetic nephropathy, by preventing Rho isoprenylation. Rat MCs cultured in HG milieu were treated with and without simvastatin, an HMG-CoA reductase inhibitor. Simvastatin inhibited HG-induced MC proliferation as measured by [(3)H]thymidine incorporation. This inhibitory effect was reversed with geranylgeranyl pyrophosphate, an isoprenoid intermediate of the cholesterol biosynthetic pathway. At the cell-cycle level, the HG-induced proliferation of MCs was associated with a decrease in cyclin dependent kinase (CDK) inhibitor p21 protein expression accompanied by an increase in CDK4 and CDK2 kinase activities. Simvastatin reversed the down-regulation of p21 protein expression and decreased CDK4 and CDK2 kinase activities. Exposure of MCs to HG was associated with an increase in membrane-associated Ras and Rho GTPase protein expression. Cotreatment of MCs with simvastatin reversed HG-induced Ras and Rho membrane translocation. Immunofluorescence microscopy revealed that the overexpression of the dominant-negative RhoA led to a significant increase in p21 expression. Our data suggest that simvastatin represses the HG-induced Rho GTPase/p21 signaling in glomerular MCs. Thus, this study provides a molecular basis for the use of statins, independently of their cholesterol-lowering effect, in early stages of diabetic nephropathy.

Figure 1

(A) Effect of simvastatin (1, 5, and 10 μM) on HG-induced MC proliferation, as assessed by [³H]thymidine incorporation (n = 10). *, P < 0.05, compared with the HG. (B) Effect of HG, HG in combination with simvastatin (SIMV), or l〈-mevalonate (MEV) on [³H]thymidine incorporation, as compared with NG (n = 18). *, P < 0.05, compared with NG.

Figure 2

(A) Immunoblot showing the time-dependent effect of HG (30 mM) and simvastatin (1 μM) on CDK inhibitor p21 after 6, 12, and 24 h of treatment. (B) Immunoblots showing the time-dependent effects of HG (30 mM) and simvastatin (1 μM) on the expression of cell-cycle regulatory proteins cyclin A and D1 and CDK inhibitor p27 after 6, 12, and 24 h of exposure. The blots in A and B are representative of three separate experiments.

Figure 3

Effects of HG (30 mM) and simvastatin (1 μM) on CDK4 (a) and CDK2 (b) after 24 h of exposure. The data reflect the results of duplicate experiments.

Figure 4

(A) Immunoblots showing the effect of HG and simvastatin on Ras GTPase protein expression assessed in the total cell lysate (a, cytosol) and cell membrane fraction (b, membrane) after 12 and 24 h of treatment. (B) Immunoblots showing the effect of HG and simvastatin on Rho GTPase protein expression assessed in the total cell lysate (a, cytosol) and cell membrane fraction (b, membrane) after 12 and 24 h of treatment.

Figure 5

Effect of SIMV (1 μM), FFP (5 μM), and GGPP (5 μM) on HG-induced DNA synthesis, as measured by [³H]thymidine incorporation (n = 10).

Figure 6

Immunofluorescence photomicrographs showing (A) expression of CDK inhibitor p21 in MCs transfected with wild-type Rho (wtRhoA) and (B) dominant-negative Rho mutant (N19Rho) in HG milieu (30 mM). Arrows indicate the expression of p21 in the nuclei.

Figure 7

Proposed HG-induced signaling leading to membrane translocation of Rho GTPase protein and down-regulation of CDK inhibitor p21. The schema is derived from the data included in Figs. 1, 2, 4, and 6. The results in Fig. 4 indicate that the HG milieu leads to the activation and translocation of Ras and Rho GTPases. The data in Figs. 1 and 2 suggest that HG induces proliferation of MCs via inhibition of p21. The results given in Fig. 6 indicate that MCs transfected with dominant-negative RhoA (N19RhoA mutant) show a significant increase in P21, suggesting a relationship between RhoA and p21 in HG-induced MC proliferation.