Effects of fungal statins on high-glucose-induced mouse mesangial cell hypocontractility may involve filamentous actin, t-complex polypeptide 1 subunit beta, and glucose regulated protein 78

Glomerular hyperfiltration is associated with mesangial cell hypocontractility. How 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) influence mesangial cell contraction is unclear. We investigated the effect of statins on mesangial cell hypocontractility and identified candidate proteins and filamentous/globular (F/G)-actin involved in this process. A high-glucose-induced mouse mesangial cell hypocontractility model was treated with fungal statins, simvastatin (Sim), lovastatin (Lov), and pravastatin (Pra). The optimum statin dose was determined by an 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and then applied to a cell model. A 2-dimensional gel/matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis was used to evaluate protein expression cells incubated in the presence of a normal level of glucose (N), a high level of glucose (H), and a high level of glucose plus Sim (H + S). Candidate proteins were analyzed. Finally, the ratio of F/G actin in groups N, H, and H+S was evaluated. The MTT assay showed that Sim and Lov exerted dose- and time-related inhibition of proliferation of mesangial cells at N, but Pra had no effect. The optimum doses selected for Sim was 1 μM and for Lov was 3 μM, which were 1 increment before significant proliferation inhibition. Both doses reversed cell hypocontractility significantly, but Sim was chosen for further proteomic and F/G actin analyses. Proteomic analysis of groups N, H, and H + S showed that 18 proteins were involved in hypocontractility. These proteins were grouped and analyzed based on their known functions. Two selected proteins, TCP-1β and GRP78, that were upregulated and downregulated, respectively, were confirmed by Western blot and immunohistochemistry. In regard to the F/G actin, group H had a significantly lower ratio than that of group N, and group H + S returned to a level similar to that of group N. In conclusion, Sim and Lov both seem to reverse mesangial cell hypocontractility. The process of Sim reversal of mesangial cell hypocontractility may involve F-actin, TCP-1β, and GRP78.

Abbreviations: 2-D gel/MALDI-TOF MS, two-dimensional polyacrylamide gel electrophoresis/matrix-assisted laser desorption ionization time-of-flight mass spectrometry, C, medium only group, ddH₂O, double distilled H2O, DM, diabetes mellitus, DMEM, Dulbecco's modified Eagle's medium, DN, diabetic nephropathy, ER, endoplasmic reticulum, F, filamentous, FBS, fetal bovine serum, FG, filamentous/globular, G, Globular, GRP78, glucose regulated protein 78, H, high level of glucose, H + S, high level of glucose plus Sim, HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A, HRP, Horseradish-peroxidase, IHC, immunohistochemistry, IPG, immobilized pH gradient, Lov, lovastatin, MTT, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, N, normal level of glucose, PBS, phosphate buffered saline, pI/MW, isoelectric point/molecular weight, PMA, phorbol 12-myristate 13-acetate, PMF, peptide mass finger, Pra, pravastatin, RT, room temperature, SDS, sodium dodecyl sulfate, Sim, simvastatin, statin, 3-hydroxy-3methylglutanyl coenzyme-A reductase inhibitor, TBST, tris-buffered saline tween-20, TCP-1β, t-complex polypeptide 1 subunit beta, V, vehicle only group, WB, Western blot