| Backgrounds and aimsGlobally,type 2 diabetes mellitus(T2DM)is one of the main risk factors of chronic kidney disease(CKD)and end-stage renal disease(ESRD).Also,diabetic nephropathy(DN)not only greatly affects the quality of life but also brings significant additional health expenditure to the patients' family and society.Current therapies for DN include controlling blood pressure and blood glucose levels as well as inhibiting the renin angiotensin system(RAS)in order to reduce or abrogate proteinuria.Although these strategies have some effect with regard to managing DN,results are far from satisfactory and many patients have experienced a progressive decline in kidney function leading to ESRD.Increasing studies have revealed that vitamin D3 and its analogue have protective effects on DN,but the mechanism is still unclear.Our study explores the mechanism and effects of vitamin D3 on the novel molecular pathogenesis(autophagy and apoptosis)of DN,which provides new and effective therapeutic targets for prevention and control of DN.Methods1.Modeling and grouping:120 Sprague-Dawley(SD)male rats were randomly divided into normal group and model group.Model group received 5 weeks of high-fat and high-sugar diet,and then were given a single intraperitoneal injection of 35 mg/kg 1%streptozotocin(STZ).Rats with three times causal blood glucose levels?16.7 mmol/L at 48 h,72 h and 1 week after injection were considered diabetic rats.The diabetic rats were randomly divided into four groups:T2DM model,high-dose intervention group(T2DM-VitD3-H),middle-dose intervention group(T2DM-VitD3-M),low-dose intervention group(T2DM-VitD3-L).Normal group and T2DM model group were given 2 mL/kg/d corn oil by oral gavage,while vitamin D3 intervention groups were received three doses of 1,25(OH)2D3 suspended in equal volume corn oil by oral gavage(0.3,0.15,or 0.75 ?g/kg/d).2.The rats were weighed once a week.Fasting blood glucose(FGB),24 hours urine output and 24 hours water intake were measured before modeling,after successful modeling,as well as at the end of 4,8 and 12 weeks of interventions.Renal function indexes were evaluated after 4 and 12 weeks of treatments.3.Kidneys were harvested after 4 and 12 weeks of interventions.The tissue slices were stained with Periodic Acid-Schiff(PAS)solution.Superoxide dismutase(SOD)and Malondialdehyde(MDA)were detected with assay kits.The expression of vitamin D receptor(VDR),microtubule associated protein 1 light chain 3(LC3)and Cleaved Cysteinyl aspartate specific proteinase-3(C-caspase3)proteins were measured by using western blot.4.Data were analyzed using SPSS 25.0(IBM)statistical software.One-way ANOVA was used for hypothesis testing.If the difference was statistically significant,LSD method was used to further compare normal group with T2DM model group or 1,25(OH)2D3 intervention groups and T2DM model group with the normal group or 1,25(OH)2D3 intervention groups.The histograms and line charts were performed by using GraphPad Prism 7.00.P<0.05 by two sides were considered statistically significant.Results1.FGB and general metabolism:The FGB in T2DM model was significantly higher than in normal rats(P<0.05).With the intervention time prolonged,FGB in moderate and low dose groups was markedly lowered than T2DM model(P<0.05).Also,1,25(OH)2D3 can improve the symptoms of weight loss,polydipsia and polyuria in rats with T2DM.2.Renal function indexes:Organ coefficient of kidney was significantly higher in T2DM model than in the normal group(P<0.05).However,After 4 and 12 weeks of intervention,no changes were observed.After both 4 and 12 weeks of intervention,1,25(OH)2D3 caused a significant decrease in urine microalbumin(P<0.05).While,we found there was no difference between the normal group and model groups in the levels of serum cystatin C and serum creatinine(P>0.05).3.PAS staining:compared with the normal group,glomerular basement membrane thickened;extracellular matrix increased and renal tubular atrophied.After 4 and 12 weeks of intervention,the pathology improved.4.Oxidative stress and the expression of VDR,LC3 and C-caspase3 protein:Compared with T2DM model,the activity of SOD increased markedly and the level of MDA in renal tissue decreased significantly after 4 and 12 weeks of interventions(P<0.05)in 1,25(OH)2D3-treatment groups.In addition,1,25(OH)2D3 significantly up-regulated the expression of VDR and LC3 and down-regulated the expression of C-caspase3(P<0.05)in diabetic kidney.ConclusionsOur present study suggests that 1,25(OH)2D3 has the function of improving pathological morphology of renal tissue and reducing the level of urine microalbumin in T2DM rats.The renoprotective properties of vitamin D3 are associated with the mechanism including suppression of oxidative stress,stimulation of autophagy and up-regulation of VDR expression in diabetic kidney. |
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