Effect Of ?Klotho On Renal Tubular Cell Injury In Diabetic Nephropathy Via Inhibited Caspase-1 Mediated Pyroptosis

Objective: To investigate the expression of pyroptosis and ?Klotho in a rat model of diabetic nephropathy and high glucose-induced renal tubular epithelial cell injury,and to explore the relationship between them in diabetic renal tubular epithelial cell injury.Methods:(1)In vivo: thirty SD rats were divided into model group(n=20)and control group(n=10).Low-dose streptozotocin(STZ)injection was used to establish diabetic nephropathy model after a high-fat diet.The blood and kidney tissues of the rats were collected after 12 weeks after diabetic modeling.Changes in body weight and blood glucose were recorded.Changes in renal function and blood lipids were evaluated by Serum biochemical analysis.Renal injuries were observed by HE staining,Masson trichrome and Transmission electron microscope.?Klotho and Csapase-1 were assessed by Western blotting assay and real time PCR.(2)In vitro: human renal tubular epithelial cells(HK-2)cultured in vitro were divided into control group(Con48h),hyper osmotic group(HO48h),high glucose group(HG1h,HG6 h,HG24h,HG48h),?Klotho plus high glucose group(HG/K0 pM,HG/K100 pM,HG/K200 pM,HG/K400pM).Spectrophotometry was used to detect the activity of lactate dehydrogenase in cell culture supernatants.The expression of ?Klotho,Csapase-1 and GSDMD were detected with Western blotting assay.The mRNA levels of ?Klotho,Csapase-1,GSDMD and IL-1? were detected with real time PCR.The concentration of IL-1? in cell culture supernatant and cell lysate were detected by ELISA assay.Results:(1)In vivo: we established the model of diabetic nephropathy and we found the glomerulus had only mild hypertrophy,but tubular injury was more serious.The expression of ?Klotho in the kidney of the model rats decreased,and the expression of Caspase-1 increased.Compared with the control group,the difference was statistically significant(p<0.05).(2)In vitro: we found that HK-2 cells incubation with high glucose induced an increased in activity of LDH and peaked at 48h(p<0.001).The expression of Caspase-1 increased gradually after high glucose stimulation,and peaked at 48h(p<0.05).Compared with Con48 h group,the expression of Caspase-1,IL-1? was increased in HG48 h group(p<0.05),and ?Klotho expression was decreased(p<0.05).The mRNA expression of GSDMD was increased(p<0.01),but there was no statistical difference in protein level(p=0.234).Exogenous supplement ?Klotho inhibited the expression of Caspase-1,the concentration of IL-1? and the activity of LDH(p<0.05),and peaked at 200pmol/L.Conclusion: ?Klotho inhibits diabetic renal tubular epithelial cell injury by inhibiting Caspase-1 mediated pyroptosis.