Role Of NG2Proteoglycan On LPS-induced Glomerular Inflammation And Mesangial Cell Proliferation

Part Ⅰ The expression and localization of NG2proteoglycan in glomeruliAim:To observe the expression and localization of NG2Proteoglycan in glomeruli in vivo and in three kinds of glomerular intrinsic cell lines in vitro.Methods:Wistar rats with one day of birth were used in our study. Detected of the location of NG2in glomeruli was by immunohistochemical method. The conditional immortalized podocyte of mice, the mesangial cell of rats, and the endothelial cell of mice was cultured in vitro. The expression of NG2in these glomerularintrinsic cells was detected by immunofluorescent microscopy. The mRNA expression of NG2was measured by reverse transcription-polymerase chain reaction methods (RT-PCR).Results:Positive stainning of NG2protein was found in glomeruli, especially in the mesangial cells and the surrounding mesangial matrix. NG2protein was mainly distributed on the membrane of mesangial cells, but not in the nucleus. No NG2expression was found in podocytes and endothelial cells. The result of RT-PCR was consistent with it.Conclusion:NG2expressed in the mesangial cells of glomeruli, but not in the two other glomerular intrinsic cells. Part Ⅱ The expression of NG2proteoglycan and proinflammatory factors in the glomeruli of rats with LPS stimulationAims:To observe the effects of LPS stimulation on the expression of NG2, and proinflammatory cytokine TNF-α and IL-1β.Methods:Wistar rats with one day of birth were randomly divided into control group (n=10), LPS group (n=10) and LPS+Dexamethasone group (n=10). LPS and Dexamethasome was injected by i.p. The rats were sacrificed after12hours and the kidneys were removed. One kidney was used to make paraffin slices, and the other kidney was used to isolate glomeruli by mesh method. The expression of NG2in glomeruli was detected by immunohistochemistry(IHC). The mRNA expression of NG2, TNF-α and IL-1β was measured by real-time PCR. NG2, TNF-a and IL-1β in serum was detected by ELISA.Results:The result of IHC showed that in contrast to control, the expression of NG2protein was increased markedly with LPS stimulation. However, Dexamethasone attenuated the change induced by LPS. The result of RT-PCR suggested that the mRNA level of NG2,TNF-α and IL-1β was elevated in isolated glomeruli with LPS stimulation, but almost restored to the basal level after Dexamethasone treatment. The similiar result on protein level in isolated glomeruli was observed by ELISA.Conclusions:LPS stimulation induced the expression of NG2, TNF-α and IL-1β. However, Dexamethasone attenuated these changes. Part Ⅲ Role of NG2Proteoglycan on Glomerular Inflammation and Mesangial Cell ProliferationAims:To explore the role of NG2proteoglycan on the glomerular inflammation and mesangial cell proliferation.Methods:(1) In vitro, cultured RMC cells were divided to three groups:control group, LPS group and LPS+Dexamethasone group. After treatment for8hours, the RMC protein expression of NG2was detected by immunofluorescent staining, and the RMC mRNA expression of NG2, TNF-α and IL-1β was measured by real-time PCR. The protein expression of NG2, TNF-α and IL-1β in medium was detected by ELISA. The proliferation of RMC was detected by MTT.(2) NG2-siRNA was transfected into RMC by lipofectamine. The expression of Cy3was detected by fluorescence microscopy after24hours, and the inhibition efficiency of NG2was measured by real-time PCR after48hours.(3) RMC cells were divided into three groups:control group, LPS group, LPS+NG2-siRNA group. After treatment for8hours, the mRNA expression of NG2, TNF-α and IL-1β was measured by real-time PCR. The protein level of NG2, TNF-α and IL-1β in medium was detected by ELISA. The proliferation of RMC was detected by MTT.Results:(1) The result of IF showed that in contrast to control, NG2protein was increased markedly after LPS stimulation. And Dexamethasone could attenuate this change.(2) The mRNA expression of NG2,TNF-α,IL-1β was increased after LPS stimulated, and the similar result on protein level in supernate was detected by ELISA. However, these changes could partly reversed by Dexamethasone.(3) Cy3showed a high expression in fluorescence after NG2-siRNA transfected for24hours. The transfection efficiency was85%. In contrast to control and negative siRNA group, the expression of NG2was decreased markedly in NG2-siRNA transfection group. The suppression efficiency is73%.(4) In contrast to control group, the mRNA and protein expression of NG2,TNF-α,IL-1β was increased significantly with LPS stimulation for8hours. LPS induced RMC proliferation while NG2-siRNA can attenuate the proliferation.Conclusions:LPS induced the release of proinflammatory cytokines TNF-α and IL-1β from RMC and the proliferation of RMC. While knocking down NG2gene could reverse these effects. So NG2may be involved in the regulation of glomerular inflammation and mesangium expansion in some pathological condition.