The Inhibitory Effect Of Mizoribine On Mesangial Cell Proliferation In Anti-Thy1 Mesangial Proliferative Nephritis

Background and ObjectiveMesangial cell proliferation and extracellular matrix accumulation (ECM) are common characters of many kinds of glomerular diseases, and they may induce renal interstitial fibrosis and glomerlosclerosis. Further, these may cause chronic kidney disease (CKD). The acute anti-Thyl nephritis which is induced by anti-Thyl.1 antigen is a well established rat model, it is characterized by a significant proliferation of mesangial cells and accumulation of ECM. This experimental model offers the unique opportunity to evaluate possible interferences of novel drugs on mesangial proliferation. Studies have showed that the regulation of cell proliferation mainly occurs in cell cycle levels, which correlates closely with the co-ordinated activation of a series of cell cycle regulatory proteins. Mizoribine (MZ) represents a novel immunosuppressant. It can inhibit the proliferation of T cell and B cell by inhibiting the inosin monophosphate dehydrogenase (IMPDH) and GMP synzyme. However, whether it could inhibit the proliferation of other cells or not is unclear. Our past studies suggested that MZ could inhibit the proliferation of mesangial cells and down-regulate the expressions of cyclins and cyclin-dependent kinases (CDK), and up-regulate the expression of CDK inhibitor (CKI) in vitro. Nevertheless, whether MZ has the same effect in vivo remains unknown. So we successfully established acute anti-Thyl nephritis rat models to observe the effect of MZ on mesangial proliferation in the acute anti-Thyl nephritis and investigate its possible mechanism.Materials and MethodsOne hundred and fifty male Wistar rats were investigated and allocated randomly to 5 groups: normal control rats (Group C, n=30), anti-Thyl nephritic rats (group T, n=30), treated nephritic rats with MZ (group M, n=30), treated nephritic rats with losartan (group L, n=30) and treated nephritic rats with MZ + losartan (group M+ L, n=30). The acute anti-Thyl model was induced by an injection of OX-7 cell supernatant liquid into a tail vein (1.5ml /kg body weight equal 100mg protein /kg body weight) after concentrated. Normal control rats were only injected with saline into a tail vein. From 6h to 6d after nephritis models were induced, two groups (group M and group M+ L) received MZ at the dose of 20mg/kg of body weight by gavage once daily, two groups (group L and group M+ L) received losartan at the dose of 200mg/kg of body weight by gavage once daily. Rats of group C and group T were given the distilled water once daily. Rats were monitored and sacrificed on days 1,2,3,4,5,7 after nephritis was induced. All rats received bromodeoxyuridine (Brdu) by intraperitoneal injection three times a day (100 mg/kg body wt, Sigma) one day before sacrificed. Blood and urine were harvested to determine 24h urinary protein (UP), serum creatinine (SCr) and blood urea nitrogen (BUN). Kidneys of rats were used for histological studies, RT-PCR and Western blot ananlysis.Results1. 24h urinary protein, SCr and BUN: The UP of group T was higher than group C (P<0.05). MZ or losartan could reduce the UP of group M, group L and group M+L, and MZ or MZ+ losartan was more effective than losartan (P<0.05). There was no significant difference in SCr and BUN between groups at each time point (P>0.05).2. Histological change: As compared to group C, the total cell number per glomerular cross section in group T significantly increased, and accumulation of ECM of group T was higher than group C (P<0.05). MZ or losartan significantly ameliorated the mesangial cell proliferation and accumulation of ECM, and MZ or MZ+ losartan was more effective than losartan (P<0.05).3. RT-PCR: The mRNA levels of cyclin D1, cyclin A of group T were higher than group C (P<0.05). MZ or losartan could reduce the mRNA levels of these indexes, and MZ or MZ+ losartan was more effective than losatan (P<0.05). There was no significant difference in p27 mRNA expression between groups at each time point (P>0.05).4. Western blot: The protein levels of cyclin D1, cyclin A, CDK2 and p21 in group T were higher than in group C (P<0.05). MZ or losartan reduced the protein levels of these indexes, MZ or MZ+ losartan was more effective than losatan (P<0.05). Whereas p27 protein level of group T was lower than that of group C, MZ or losartan could up-regulate p27 protein level (P<0.05).5. Immunofluorescence: The number of Brdu-positive cells in group T increased markedly in comparison with group C (P<0.05); MZ and losartan reduced Brdu-positive cells significantly compared to group T (P<0.05). The number of positive cells of Brdu, cyclin D1, cyclin A or CDK2 in group T was more than group C, whereas p27 expression of group T was fewer than group C (P<0.05). As compared to group T, group M, group L or group M +L had sharply decline in the number of Brdu, cyclin D1, cyclin A, CDK2 positive cells, and increase in the number of p27 (P<0.05).Conclusions1. MZ could inhibit mesangial cell proliferation and ECM accumulation, and reduce UP in the acute anti-Thyl nephritis rats.2. The mechanism through which MZ inhibited mesangial proliferation was at least partly by down-regulating mRNA expression in cyclin D1, cyclin A and protein expression in cyclin D1,cyclin A,CDK2 and p21, up-regulating protein expression in p27.