Role Of Active Vitamin D In Regulation Of SOCS And JAK/STAT Pathways In Diabetic Kidney Diseases Via VDR

Objective:To investigate the role and possible mechanism of active vitamin D?1,25-dihydroxy vitamin D₃,1,25-?OH?₂VD₃?in regulating Suppressor of cytokine signaling?SOCS?and Janus kinase signal transducer and activator of transcription?JAK/STAT?pathways through vitamin D receptor?VDR?in Diabetic kidney disease?DKD?.Methods:Twelve-week-old male KKay mice weighing 20-30g were fed with high-fat and high-sugar diets to construct DKD mice models and DKD mice models that silenced VDR genes,and then randomly divided into 6 groups:?1?DKD group?2?Low concentration VD3?L-VD3??3?High concentration VD3?H-VD3??4?Lentivirus against the vitamin D receptor?Lenti-shVDR??5?Low concentration VD3+lentivirus transfection group?Lenti-shVDR+L-VD3??6?High concentration VD3+lentivirus transfection group?Lenti-shVDR+H-VD3?.During breeding observe behavioral changes and general conditions every day,record the food intake and blood glucose of the mice every 3 days,and record the body weight,water intake and urine volume of the mice once a week.24-hour urine volume and single morning urine were measured for 24-hour urine microalbumin?24h mAlb?and albumin creatinine ratio?ACR?at the 1st,4th,8th and 10th weeks of rearing.Serums were taken from the 1st,8th and 10th weeks of breeding for serum creatinine?Scr?,blood urea nitrogen?BUN?and fasting insulin?FINS?and Alanine aminotransfease?ALT?.Mice were sacrificed at 22 weeks of age,and kidney tissues were removed quickly for subsequent experiments.Real-time polymerase chain reaction?RT-PCR?was used to detect mRNA expression differences of JAK1,JAK2,STAT1,STAT3,SOCS1,and SOCS3 in kidney tissues;Western blot?WB?was used to detect proteins expression differences of phosphorylated JAK1?p-JAK1?,JAK1,p-JAK2,JAK2,p-STAT1,STAT1,p-STAT3,STAT3,SOCS1,and SOCS3 in kidney tissues;Hematoxylin-eosin?HE?staining to observe renal pathological changes under light microscope;Electron microscope to observe glomerular basement membrane thickness;Immunohistochemical observation of collagen-IV?Col-IV?and fibronectin?FN?expression in kidney tissue.Results:?1?At 14 weeks of age,Kkay mice had three consecutive non-same-day caudal venous glucose>16.7 mmol/L and the diabetes mellitus model was successfully constructed.At 20 weeks of age,the 24h urine volume increased>50%,24h mAlb>30 mg and/or ACR>30 mg/g and renal histopathology showed glomerular enlargement with parietal stromal hyperplasia and basement membrane thickening,the DKD model was successfully constructed.?2?Compared with the DKD group,the blood glucose,24h mAlb,ACR,BUN,and Scr of the L-VD3 group and the H-VD3 group were all reduced?P<0.05?,while FINS and ALT were not significantly changed?P>0.05?.Blood glucose,24h mAlb,ACR,BUN and Scr were all lower than those in L-VD3 group,all of which were statistically significant?P<0.05?;In the Lenti-shVDR group,both 24h mAlb and ACR increased?P<0.05?,and there was no significant change in blood glucose,BUN,Scr,FINS and ALT?all P>0.05?.Compared with the Lenti-shVDR group,the blood glucose in the Lenti-shVDR+L-VD3 group and the Lenti-shVDR+H-VD3 group were reduced?P<0.05?,and the remaining indicators were not significantly changed?P>0.05?.?3?Compared with DKD group,the expression levels of JAK1,JAK2,STAT1,STAT3,SOCS1 and SOCS3 mRNA and p-JAK1,p-JAK2,p-STAT1,and p-STAT3 protein in L-VD3 and H-VD3 groups were reduced?P<0.05?.The above indicators were lower in the H-VD3 group than in the L-VD3 group,while the SOCS1 and SOCS3 protein levels were increased?P<0.05?,and the H-VD3 group was higher than the L-VD3 group.The expression levels of JAK1,JAK2,STAT1,STAT3,SOCS1 and SOCS3 mRNA and p-JAK1,p-JAK2,p-STAT1,and p-STAT3 protein in the Lenti-shVDR group were higher than those in the DKD group?all P<0.05?;and SOCS1 And SOCS3 protein levels decreased?P<0.05?.Compared with Lenti-shVDR group,Lenti-shVDR+L-VD3 group and Lenti-shVDR+H-VD3 group JAK1,JAK2,STAT1,STAT3,SOCS1 and SOCS3 mRNA and p-JAK1,p-JAK2,p-STAT1,p-STAT3 protein expression level decreased?all P<0.05?,the above indicators Lenti-shVDR+H-VD3group was lower than Lenti-shVDR+L-VD3 group,while SOCS1 and SOCS3protein levels increased?P<0.05?,and the Lenti-shVDR+H-VD3 group was higher than the Lenti-shVDR+L-VD3 group.?4?HE staining showed enlarged glomeruli with mesangial matrix hyperplasia in the DKD group,and the lesions were alleviated after intervention with 1,25-?OH?₂VD₃,and the H-VD3 group improved significantly compared with the L-VD3 group;Compared with the DKD group,the glomerular lesions in the Lenti-shVDR group were aggravated.After intervention with different concentrations of 1,25-?OH?₂VD₃,the lesions were slightly reduced compared with the Lenti-shVDR group.?5?Electron microscopy results showed that the glomerular foot process was fused and the basement membrane was thickened in the DKD group.The condition of the foot process and basement membrane was improved after intervention with 1,25-?OH?₂VD₃,and the H-VD3 group was significantly improved compared with the L-VD3 group.Compared with the DKD group,the Lenti-shVDR group saw more foot process fusions and significantly thickened basement membranes.After intervention with different concentrations of 1,25-?OH?₂VD₃,the lesions were slightly reduced compared with the Lenti-shVDR group.?6?Immunohistochemistry showed the same staining results for Col-IV and FN.Compared with the DKD group,the brown particles in the L-VD3 group and the H-VD3 group have a lower degree of coloration,a smaller staining area,and less renal fibrosis,and the H-VD3 group has a slightly better improvement than the L-VD3 group;Renal fibrosis aggravated in the Lenti-shVDR group and renal fibrosis was reduced in the Lenti-shVDR group after intervention with different concentrations of 1,25-?OH?₂VD₃.Conclusion:1,25-?OH?₂VD₃ has a protective effect on DKD,which may be achieved by VDR enhancing the expression of SOCS and thus affecting the JAK/STAT signaling pathway.