The Effect Of Inhibition And Protection Of 1,25-Dihydroxy Vitamin D3 On Mtor-Mediated Early Diabetic Nephropathy

Part one 1,25（OH）₂D₃ inhibit the proliferation of rat mesangial cells induced by high glucoseObjective: To observe the influence of high glucose on rat mesangial cells（MCs）, explore the optimum conditions of 1,25（OH）₂D₃ on MCs, confirm the inhibited effect of 1,25（OH）₂D₃ on proliferation of MCs induced by high glucose.Methods: The cultured rat mesangial cells（HBZY-1） were treated with medium containing different concentrations of glucose:（1）Normal glucose group（NG） with the glucose concentration of 5.5mmol/L;（2）High glucose group（HG15） with the glucose concentration of 15mmol/L;（3）High glucose group（HG25） with the glucose concentration of 25mmol/L;（4）High glucose group（HG35） with the glucose concentration of 35mmol/L. The diameters of MCs were detected under light microscope. All the MCs were divided into 5 groups: NG group, high-glucose group（HG）, HG + 1,25（OH）₂D₃ group（HV）, HG + lenti-sh VDR group（HL） and HG + lenti-sh VDR and 1,25（OH）₂D₃ group（HLV）. Cells in HL and HLV groups were transfected with lenti-sh VDR for further studies. The proliferations of MCs were tested by trypan blue dye, MTT and flow cytometry.Results:（1）The diameters of MCs in HG25 and HG35 groups were increased compared with that in NG group（P < 0.05）.（2）MTT assay showed that the inhibition of proliferation of MCs by 1,25（OH）₂D₃ relied on its concentration and time, and the 1μM/L, 24 hours were the best conditions of 1,25（OH）₂D₃.（3） The flow cytometry indicated that the S phase of HBZY-1 cells in HG group were significantly elevated compared with that in NC group, while the S phase were decreased in HV group compared with that in HG group, the S phase in HL and HLV groups were increased compared with that in HV group（P < 0.05）.Conclusion: 25mmol/L is the optimal glucose concentration for HBZY-1 to simulate the extracellular environment in diabetic nephropathy. 1,25（OH）₂D₃ could significantly inhibit the proliferation of MCs induced by high glucose.Part two 1,25（OH）₂D₃ prevent kidney damage in early diabetic nephropathy ratsObjective: To explore the influence of 1,25（OH）₂D₃ and（or） Lenti-sh VDR on renal functions of early diabetic nephropathy rats.Methods: The diabetic nephropathy rats were randomized into 5 groups: control group（NC）, diabetic nephropathy group（DN）, DN + 1,25（OH）₂D₃ group（DV）, DN + lenti-sh VDR group（DL）, and DN + lenti-sh VDR+ 1,25（OH）₂D₃ group（DLV）. Streptozotocin-induced diabetic nephropathy rats were injected intravenously with a recombinant lentivirus against the rat vitamin D receptor（VDR） gene（lenti-sh VDR）. Urinary albumin, fasting plasma glucose（FPG） and serum 25（OH）D levels were detected by Elisa assay, transfected liver and kidney tissue sections were observed by fluorescence microscope, mean glomerular volume（MGV） and total kidney volume were determined, glomerular basement membrane（GBM） thickness was measured by transmission electron microscopy.Results:（1）The body weight in DN group was decreased compared with that in NC group, while the kidney weight and the ratio of kidney weight to body weight were increased in DN group compared with that in NC group. There were no significant differences for blood pressure between five groups. The FPG, TG and urinary albumin were higher in DN group than that in NC group, while the urinary albumin in DV group was lower than that in DN group. The serum 25（OH）D levels were decreased in DN group compared with that in NC and DV groups. Meanwhile, the serum 25（OH）D levels in DL and DLV groups were increased compared with that in DV group（P < 0.05）.（2）The green fluorescence was observed in liver and kidney tissue sections in transfected groups（DL and DLV）, while there were no green fluorescence in NC, DN and DV groups.（3）The rats in DN group have higher total kidney volume than that in NC group, while the rat in DV group have lower total kidney volume than that in DN group. The MGVs and GBMs in DN, DL and DLV groups were increased compared with that in NC group, while the rats in DV group have lower MGVs and GBMs than that in DN group（P < 0.05）.Conclusion: 1,25（OH）₂D₃ could significantly decreased the urinary albumin, total kidney volume, MGV and GBM of early diabetic nephropathy rats, thus delay the pathological progress of diabetic nephropathy, and this effect of 1,25（OH）₂D₃ may rely on the combining with vitamin d receptor.Part three The mechanism of 1,25（OH）₂D₃ on mTOR-mediated early diabetic nephropathyObjective: To expore the mechanisms of 1,25（OH）₂D₃ improving early diabetic nephropathy.Methods: The diabetic nephropathy rats were randomized into 5 groups: control group（NC）, diabetic nephropathy group（DN）, DN + 1,25（OH）₂D₃ group（DV）, DN + lenti-sh VDR group（DL）, and DN + lenti-sh VDR+ 1,25（OH）₂D₃ group（DLV）. The m RNA expressions of VDR and DDIT4 were detected by RT-PCR, the protein expressions and phosphorylation of VDR, DDIT4, TSC2, Akt, mTOR and P70S6 K were detected by western blot.Results:（1）The m RNA expressions of VDR and DDIT4 in DN group were lower than those in NC group, while the m RNA expressions of VDR and DDIT4 in DV group were higher than those in DN group. The rats in DL and DLV groups have lower m RNA expressions of VDR and DDIT4 than those in NC group（P < 0.05）.（2）The protein expressions of VDR and DDIT4 were similar to its m RNA expressions. The phosphorylation of TSC2 was similar to the expression of DDIT4, while the phosphorylations of Akt, mTOR and P70S6 K were contrary to the phosphorylation of TSC2. VDR gene silencing blocked all of the above results（P < 0.05）.Conclusion: 1,25（OH）₂D₃ could effectively induce the expression of DDIT4, thus suppressing the activation of mTOR. This effect of 1,25（OH）₂D₃ may rely on the combining with vitamin d receptor.