Effects Of RNA Interference Targeting Gremlin On Extracellular Secretions Of FN And Col Ⅳ In Rat Mesangial Cells Cultured With High Glucose

[Objective]Diabetic nephropathy(DN)is a serious complication of diabetes mellitus and10%of the patients may lead to the end-stage of renal failure if there is no effective treatment.Specific therapies of diabetic nephropathy are not available and current treatment strategies are limited to management of controlment of blood glucose levels,hypertension and hyperproteinuria. Pathological changes of glomarular mesangial cells are the most apparent changes of diabetes mellitus, in the early stage of which proliferation of mesangial cells and increase of extracellular matrix such as fibronectin and type IV collagen could be observed. But the definite mechanism of this state is still not identified. Reseaches show that a seris of growth and development related factors play important roles in the pathogenesis of diabetic nephropathy, which have a re-activation or an increase in activity, including Transforming growth factor beta(TGF-β), bone morphogenetic proteins(BMPs) antagonist--gremlin, connective tissue growth factor(CTGF) and vascular endothelialcell growth factor (VEGF).Gremlin is a highly conserved,24-26kDa secreted glycoprotein member of the cysteine knot superfamily, with the ability to heterodimerize and antagonize bone morphogenetic proteins (BMPs), in particular BMP-2,-4, and-7. Gremlin regulates outgrowth, chondrogenesis, and apoptosis of the developing limb bud, as well as branching morphogenesis during kidney development. Researches also find that gremlin play a significant role in mesangial proliferation、glomerular sclerosis and interstitial fibrosis of renal tubules. We also have found that gremlin is a diabetes nephropathy related gene by gene microarray screening. In recent years researchers pay more attention to finding out the ideal theraputic targets against diabetes nephropathy. The re-activation of developmental programs in diabetes nephropathy suggests new potential as therapeutic targets.In this study, we focus on gremlin as a theraputic target to explore the effects of RNA interference targeting gremlin on extracellular secretions of fibronectin(FN) and collagen type IV(Col IV) in rat mesangial cells(RMCs) cultured with high glucose.[Methods] The lentivirus vector based small hairpin RNA (shRNA) targeting gremlin, GREMLIN-RNAi-LV,was constructed and deliverd to the high glucose cultured RMCs. Then the cells were treated for another48h with5.5mmol/L glucose,30mmol/L glucose,30mmol/L glucose plus GREMLIN-RNAi-LV or30mmol/L glucose plus NC-GFP-LV. Gremlin mRNA expressions were determined by real-time PCR, and Western blot was used for protein of Gremlin. FN and Col IV in cell culture fluids were determined by ELISA. The results were analyzed semi-quantitatively by the pathological image analysis. All the data were analysed by SPSS13.0statistic software, P value<0.05was considered to have statistical significance.[Results] Compared with5.5mmol/L glucose group, the expressions of gremlin and secretions of FN and Col IV in cell culture fluids showed obvious elements in the30mmol/L glucose group (P<0.05). Lentivirus mediated RNA interference successfully suppressed the high glucose induced overexpressin of gremlin (P<0.05), at the same time reduced the secretions of FN and Col IV (P<0.05)[Conclusion] By infection of gremlin-specific lentivirus vectors, gremlin mRNA and protein were efficiently inhibited and the secretions of extracellular matrixs were decreased, which were the significant factors of renal fibrosis. Administration of gremlin siRNA plasmid to diabetic mice alleviated renal hypertrophy, decreased cell proliferation and apoptosis, and subsequently suppressed collagen type IV accumulation and mesangial expansion, which indicate that there are beneficial effects of gremlin inhibition on diabetic nephropathy. We conclude that inhibition of gremlin expression exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 expression and activity and that gremlin may serve as a new therapeutic target in the management of diabetic nephropathy.