Preliminary Study On The Protective Effect Of Vitamin D On Diabetic Nephropathy In Mice

Aim:To investigate whether vitamin D can ameliorate diabetic nephropathy and the underlying mechanism.Methods:The mainly work of this study contains three parts:1)Establishment of diabetic mice model induced by streptozotocin?STZ?.C57BL/6J mice were injected intraperitoneally by 40 mg/kg STZ for 5 days continuously.The diabetic mice were identified based on fasting blood glucose?FBG?11.1 mmol/L?4 days after STZ injection.2)The impact of vitamin D supplement on renal injury in diabetic mice.The diabetic mice were randomly divided into four groups:model control group,vitamin D₃group?VD3,1000 IU/week,intramuscular injection once a week?,metformin group?Met,0.3 mg/ml,drinking water administration?,combined treatment group?VD3+Met,1000 IU/week+0.15 mg/ml?.Meanwhile,normal control group without STZ treatment was set up.The mice were continuously studied for 12 weeks.At the same time,the general situations in mice were observed including body weight and FBG.At the end of the 12th week,mice urine,serum and renal tissue samples were studied.Then24 h urinary protein,urine creatinine,blood urea nitrogen?BUN?and serum creatinine were measured.Mice renal histopathological changes with hematoxylin-eosin?HE?in the light microscope were observed.Western Blot was used to detect the expression levels of vitamin D receptor?VDR?,Slug,Snail,?-catenin,E-cadherin,Nephrin and Vimentin in renal tissue.3)Effects of 1?,25?OH?₂D₃ on epithelial-mesenchymal transition?EMT?induced by high glucose?HG?in podocyte.The cell survival rate was determined with Cell Counting Kit-8?CCK-8?.Western Blot analysis was performed to measure the protein levels of VDR,Slug,Snail,?-catenin,E-cadherin,Nephrin and Vimentin in podocyte.Results:?1?The mice injected with STZ showed polydipsia,polyphagia,polyuria,gradually emaciation,and reduced activity after 3 to 5 days.These symptoms were most distinguished in diabetic group.FBG was significantly increased?P<0.01?.?2?At the end of the experiment,the mice in the model group appeared slow weight gain and FBG remained at a high level.Vitamin D₃ group,metformin group and combination group also appeared similar symptoms of model group,but FBG was lower than the model group?P>0.05?.The heart,liver and renal coefficient?expect Met group?of the diabetic group with the injection of STZ was obviously larger than the control group?P<0.05?.The liver coefficient was distinctly lower in the VD3 and VD3+Met group?P<0.05?,and renal coefficient was decreased visibly in Met group?P<0.05?than the model group.Biochemical test results showed the serum creatinine,urinary protein excretion,and urinary protein creatinine ratio?ACR?in diabetic mice increased significantly than control group?P<0.05?.BUN levels have a certain reduction in all of intervention group,but there are no differences compared to control group?P>0.05?.Compared with the model group,vitamin D₃ alone intervention decreased clearly the levels of serum creatinine,urinary protein excretion and ACR?P<0.05?,Met alone intervention decreased the urinary protein excretion and ACR?P<0.05?.Vitamin D₃ and metformin joint intervention decreased significantly serum creatinine and urinary protein excretion?P<0.05?.The ACR level of the Met group was lower compared with VD3 group?P<0.05?.The light microscope HE staining revealed that,the model group showed glomerular volume increased,diffuse mesangial matrix and mesangial cells compared with the normal control group.Vitamin D₃ and metformin alone or combination administration both evidently improved pathological changes.The combination group showed synergistic effect.Western Blot showed the expression decreased obviously in model group compared with normal control group in renal tissue of mice with VDR,E-cadherin and Nephrin?P<0.01?,and the expression increased markedly with?-catenin,Snail,Slug and Vimentin?P<0.01?.Compared with normal control group,after vitamin D₃ intervention,the expression levels of VDR,Nephrin protein were significantly increased while?-catenin,Slug,Vimentin levels clearly decreased?P<0.01?and E-cadherin,Snail has no significant change.Metformin alone intervention obviously decreased the expression of Snail,Slug and Vimentin?P<0.01?,while no significant differences in VDR,?-catenin,E-cadherin and Nephrin.The expression of E-cadherin was improved after the vitamin D₃ and metformin combination,while the expressions of?-catenin and Snail were decreased?P<0.01?.?3?1?,25?OH?₂D₃ intervention effectively alleviated cell survival reduction induced by HG.Western Blot showed that compared with normal control group,HG obviously increased?-catenin,Snail,Slug and Vimentin expressions while decreased VDR,E-cadherin and Nephrin expressions in podocyte.However,1?,25?OH?₂D₃significantly inhibited?-catenin,Snail,Slug and Vimentin expression?P<0.01?and partially restored VDR,E-cadherin and Nephrin expression in HG-stimulated podocyte?P<0.01?.Conclusion:1.The STZ-induced diabetic mice model was successfully established,with exhibition of polydipsia,polyphagia,polyuria and FBG?11.1 mmol/L.2.Vitamin D₃ significantly reduced serum creatinine,proteinuria,urinary protein creatinine ratio level,and improved renal pathological changes in diabetic mice.Meanwhile,the level of VDR expression and EMT-related protein were changed,which suggested that these molecular might play an important role in the development of DN.3.1?,25?OH?₂D₃ significantly alleviated the upregulation of?-catenin,Snail and Slug induced by HG,while improved the expression of VDR and reversed HG-induced EMT in podocyte.Thus,modulating VDR might be a target for the treatment and prevention of DN.