Effect Of Calcium Binding Protein S100A4 On The Glomerularmesangialcell Proliferation

Background and objective : Mesangial proliferative glomerulonephritis(MsPGN) is the highest incidence of kidney disease,The main pathological changes is mesangial cells (MC) proliferation and increased mesangial matrix.Continuing proliferation and mesangial increase in mesangial matrix eventually leading to irreversible glomerulosclerosis. Inhibition of cell proliferation is an important treatment strategy of proliferative glomerular disease. Despite a large number of studies found that angiotensin II (Ang II),cytokines (such as PDGF, EGF) and inflammatory mediators (IL-6, TNF-α) are important factors which can promote the proliferation of mesangial cells,but the intervention of the target cannot completely suppressed mesangial cell proliferation,surgesting that there are other important factors involved in the pathogenesis of mesangial cell proliferation。S100A4 , a member of S100 gene family, is a calcium-binding protein, also known as fibroblast-specific protein -1(FSP-1).And in the field of kidney disease , S100A4 is considered the marker of fibroblasts。However, in oncology research found that S100A4 could promote tumor cell proliferation, Therefore, our study is observe S100A4 expression changes with mesangial proliferative in Thy1 mesangial proliferative glomerulonephritis in rats.and verified the control role that regulate mesangial cell proliferation in vitro.Methods:Using OX-7 hybridoma cells to produce the anti-Thy1 antibody,After purification,tail inject to SD rat to establishment the anti-Thy1 mesangial proliferative glomerulonephritis model. Then detecting renal function and urinary protein,getting kidney specimens, extracting total protein in different time points(0,3,5,7,10,14d),and detecting the expression of S100A4 protein on each time point by Western Blot. Rat mesangial cells (RMC) were cultured, S100A4 siRNA was designed, which inhibied the expression of S100A4 in RMC, irrelevant siRNA was used as contol (siCon). The experiment was divided into four goups, 1)serum-deprived, 2)normal control, 3)siCon transfected, 4)siS100A4 transfected. After 24-48 hours, the alteration of cellular proliferation and cellular cycle was detected by MTT assay and Flow Cytometry. The changes of CDK2, CyclinD1 and p21 were detected by Western blot and indirect immunofluorescence.Results: Compared with control, total urine protein quantitation during 24 hours of Anti-Thy1 glomerulonephritis group was significantly increased at 5d and 7d (p<0.05, n=6), which was decreased after 10d. It was shown by Western blot that the peak of S100A4 occurred in 7d, which was decresed subsequently, parralled with the alteration of mesangial cell proliferation.In vitro, compared with siCon transfected group, S100A4 siRNA inhibited the expressions of S100A4 mRNA and prtein after transfected into RMC (p<0.05). The number of cell in siS100A4 transfected group was signifiucantly decreased, compared with siCon transfected group, the number of cells in S phase was also decreased detected by Flow Cytometry (p<0.05, n=3). It was shown by Western blot and indirect immunofluorescence that cellular cycle related protein CDK2, cyclinD1and p21 were downregulaed (p<0.05, n=3).Conclusion:We found the chang of S100A4 protein expression level paralled with mesangial proliferative in the rat anti-Thy1 nephritis model. Inhibition of S100A4 expression could induced the alteration of cellular cycle protein CDK2, cyclinD1and p21 expression, followed by decrease of cells in S phase, suggested that S100A4 play an important role in process of mesangial proliferation, which provided new insight for regulation of mesangial proliferation.