Ubiquitin-dependent Degradation Of SnoN/Ski Protein In Glomerular Mesangial Cells Induced By High Glucose

objective:TGF-βis the meeting point of many pathogenic factors of Diabetic Nephropathy(DN). It can induce the phenotypic transition of kidney cells and promote the accumulation of extra cellular matrix(ECM). It plays an important role in the fibrogensis of renal tubule-interstitium. SnoN/Ski are important negative regulators in transforming growth factor-β/Smad (TGF-β/Smad) signal pathway. Their interaction with Smad protein can inhibit the activiation of TGF-βtarget genes and biological functions of TGF-β. Recent studies have showed that transcriptional co-repressor SnoN is mainly degradated by ubiquitin-proteasome pathway. Now, there are few studies about the diabetic nephropathy-related ubiqutin-proteasome pathway. In diabetes, whether the kidney ubiquitin-proteasome pathway is activated, and whether the cell signaling protein ubiquitination is enhanced, have not been reported. Previous studies have demonstrated that SnoN/Ski mRNA expression is increased and SnoN/Ski protein expression is reduced obviously in the fibrotic kidney after unilateral ureteral obstruction (UUO);and the reduction of SnoN/Ski protein expression is caused by the increase of the ubiquitin degradation which is mediated by the E3 ubiquitin ligase. The purpose of study is to observe the protein expression changes of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells(GMC) which are exposed to the high-level glucose; and preliminarily investigate the effect of the ubiquitin degradation of SnoN/Ski protein on DN by adding proteasome inhibitor MG-132 in high glucose medium. Methods:Cultured HBZY-1 rat glomerular mesangial cells are divided into 5 groups. High glucose as a stimulating factor and MG132 as a proteasome inhibitor:①control group:culture medium contains 5.6mmol/L glucose;③20mmol/L gluclose group;③30mmol/L gluclose group;④mannitol group:culture medium contains 5.6mmol/L glucose and 24.4mmol/L mannitol, as an osmotic control;⑤30mmol/L gluclose +MG132 group:culture medium contains 30mmol/L glucose and 0.5μmol/L specific proteasome inhibitor MG132, and the two factors affect simultaneously.. The expression of SnoN,Ski and Arkadia are measured by Western-blot assay, immunofluorescence and laser scanning confocal microscope. Results:1. The expression level of SnoN/Ski protein were rich and persietent in control GMCs, but significantly,decreased in a dose and time dependent manner in GMCs stimulated with high glucose(P<0.01). Compared with high glucose group, the levels of SnoN/Ski are reverted mostly by adding the proteasome inhibitor MG132 (0.5umol/L, P<0.01). The expressions of SnoN/Ski are not changed significantly in mannitol group. (P>0.05).2. Arkadia was expressed weakly in control GMCs, however, the expression of Arkadia was gradually increased in GMCs stimulated with high glucose after 48 hours (P<0.01). Compared with high glucose group, the level of Arkadia is reverted mostly by adding the proteasome inhibitor MG132(0.5umol/L, P< 0.01). The expression of Arkadia is not changed significantly in mannitol group. (P>0.05). Conlusions:Our studies suggest that:1. High glucose can decrease the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski.2. The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathologensis of diabetic nephropathy.