Mizoribine Inhibits Rat Mesangial Cell Proliferation Via Cell Cycle Regulatory Proteins-dependent Mechanism

Background. Mesangial cell proliferation is a key feature of several forms of glomerular inflammatory diseases. Continuous mesangial cell proliferation ultimately results in glomerular scarring. Searching for agents inhibiting mesangial cell proliferation is therefore of substantial clinical inmportance. Cell proliferation is dependent on the co-ordinated activation of a series of cell cycle regulatory proteins. Cyclins and cyclin-dependent kinases (CDKs) represent the main positive regulatory proteins, while CDK-inhibitors (CKI) are their counterpart. There are obvious changes in cell cycle regulatory proteins during mensangial cell proliferation in vivo and in vitro. Mizoribine (MZR) competitively inhibits the sequential enzymes IMPDH and GMP synthetase in the de novo purine biosynthetic pathway. Whether MZR could inhibit mesangial cell proliferation and its mechanism is unknown. In the present study, we observed the inhibitory effects of MZR on mesangial cell proliferation through clinical trial, experiments in vivo and in vitro and explored the mechanism via cell cycle regulatory proteins.Methods. A prospective, randomized, controlled clinical trial was performed in order to observe the treatment effects of MZR on IgA nephropathy. Renal biopy-proved IgA nephropathy patients aged from 14-70, urine protein excretion ranged 0.5g/d to 3.5g/d were randomly assigned to the following groups: MZR group, losartan group, MZR and losartan group. Clinic visits were done every 1-1.5 months and the entire study duration was 1 year. Influence of MZR on urine protein and renal function was observed. Male Wistar rats were divided into three groups, control group, anti-Thy1 nephritis group and MZR treatment group. At day 0, anti-Thy1 nephritis were induced by injection of OX-7 monoclonal antibody (100mg/kg) and control group rats received equal volume of saline only.At day 1,day 3, day 5 and day 7, samples of serum, urine, and kidneys of each group (n = 6) were collected. Serum creatinine and urine protein excretion were measured. Paraffin sections of renal tissues were stained by PAS. BrdU and p27^kip1 positive cells were detected by indirect immunofluorescence stain. The mRNA and protein expressions of cyclinD1, CDK2, p27^kip1 were examined by RT-PCR and Western Blot respectively. Primary cultured rat mesangial cells were growth-arrested by withdrawl FCS for 24h and then stimulated by 20% FCS with or without MZR. Different concentration MZR were added and cell numbers were detected by MTT. S phase entry was measured by BrdU labeling. Cell cycle profiles were determined by flow cytometric analysis. The protein expressions of cyclinD1, cyclinE, CDK2, p27^kip1 and Skp2 ( an F-box protein which play crucial role in p27^kip1 degradetion) were examined by Western Blot. Intracellular location of p27^kip1 was detected by immunofluorescence stain, p27^kip1 and Skp2 mRNA expression was quantified by real-time quantified PCR. pIRES-GFP and pIRES-GFP-Skp2 plasmids were transfected by Lipofectmine 2000.Results. MZR was effective in deseasing proteinuria in selected IgAnephropathy patients and the application is safe. In anti-Thy1 model of experimental mesangial proliferative glomerulonephritis, therapy of MZR resulted in a significant amelioration of mesangial cell proliferantion, assessed by glomerular histology, glomerular total cellularity and BrdU positive cell numbers. Rats treated with MZR showed an obvious decrease in urine protein excretion compared to nephritis rats. Compared to normal rats, the renal expression of cyclinD1, CDK2 upregulated and protein expression of p27^kip1 significantly downregulated. MZR treatment decreased expression of cyclinD1, CDK2, while significantly increased p27^kip1 protein expression. RT-PCR revealed that MZR lower the mRNA level of cyclinD1. Contrast to the increased protein level, MZR produced no changes on the abundance of the p27^kip1 mRNA in kidney. In vitro, MZR inhibited FCS-stimulated mesangial cell proliferation dose-dependently. Cell cycle analysis and BrdU labeling showed that MZR prevent FCS stimulated mesangial cell from entrying S phase and induce G1/S arrest. FCS stimulationdownregulated the protein expression p27^kip1 and upregulated the protein level of cyclinD1, cyclinE and CDK2, MZR significantly abbreviated these changes. MZR maintained the nucleus expression and fluorescence intensity of p27^kip1 but produced no effect on p27^kip1 mRNA abundence, suggesting a posttranscriptional mode of regulation or impairment of protein degradation. MZR decreased Skp2 expression and reciprocally increased p27^kip1 expression and Skp2 overexpression alliviated the p27^kip1 increasement of MZR.Conclusion. Mizoribine effectively inhibited mesangial cell proliferation in vivo and in vitro. MZR downregulated cyclinD1, cyclinE, CDK2 expression meanwhile dramatically upegulated p27^kip1 protein level and maintained its nucleus expression, ultimately resulted in mesangial cell G1/S arrest. MZR-induced p27^kip1 increasement was at least partly Skp2-dependent.