Cadmium Exposure Exacerbates Kidney Injury In Diabetic Rats By Inhibiting Autophagy

In recent years,the incidence rate of diabetes(DM)is gradually rising,which seriously threatens people’s health.Cadmium(Cd)is a toxic heavy metal that is widely present in the environment.With the development of industry and agriculture,the risk of its toxicity gradually increases.Studies have shown that cadmium exposure may be related to the occurrence of diabetes nephropathy(DN).However,the mechanism of cadmium induced its occurrence is still unclear.Therefore,this experiment took diabetes rats and high glucose cultured rat renal tubular epithelial cells(NRK-52E cells)as the research object,explored the mechanism of cadmium’s influence on diabetes kidney damage through in vivo and in vitro experiments,and provided new ideas for revealing the mechanism of cadmium’s nephrotoxicity.1.Effect of cadmium exposure on renal autophagy in diabetes ratsIn order to explore the effect of cadmium exposure on renal autophagy in diabetes rats,SD rats fed with high fat and high sugar for 4 weeks were intraperitoneally injected with 35 mg/kg STZ(streptozotocin)to build a diabetes model,and stable diabetes rats were exposed to 25 mg/L cadmium chloride through free drinking water for 20 weeks.Detection of cadmium accumulation in the kidneys using flame atomic absorption spectrometry;Calculate the body weight and relative kidney weight of rats;Automatic biochemical analyzer for detecting blood lipids and blood sugar content;The content or activity of 24 h urinary microalbumin,urinary creatinine and renal tissue antioxidant enzymes were detected with the kit;HE,PAS,and Masson staining were used to observe renal pathological damage;Immunohistochemical detection of the expression levels of COL-Ⅰ,COL-Ⅳ,CTSB,and CTSD proteins in renal tissue;Immunofluorescence detection of the fluorescence intensity of p62 and LC3 proteins in renal tissue;Western blot was used to detect the expression levels of fibrosis,autophagy,and lysosomal related proteins in renal tissue;Transmission electron microscopy was used to observe the ultrastructure of mitochondria and the number of autophagosomes in renal tissue.The results showed that compared with the control group,the cadmium accumulation in the kidneys of the Cd group and DM+Cd group was significantly increased(P<0.01),and the cadmium accumulation in the DM+Cd group was further increased compared to the Cd treated group alone(P<0.01).Compared with the control group,the body weight and endogenous creatinine clearance rate of the Cd and DM groups of rats were significantly reduced(P<0.05),while the relative kidney weight and 24-hour urine microalbumin/creatinine value were significantly increased(P<0.01).Pathological damage occurred to the kidneys,and the body weight and endogenous creatinine clearance rate of the DM+Cd group were further reduced compared to the DM group(P<0.05).The relative kidney weight and 24-hour urine microalbumin/creatinine value were further increased(P<0.01),and the pathological damage to the kidneys was exacerbated.Compared with the control group,the contents of serum triglyceride,total cholesterol,low-density lipoprotein and blood sugar in the Cd group and DM group were significantly higher(P<0.05),the content of high-density lipoprotein was significantly lower(P<0.01),and the contents of triglyceride,total cholesterol and low-density lipoprotein in the DM+Cd group were further higher than those in the DM group(P<0.05),and the contents of high-density lipoprotein and blood sugar were significantly lower(P<0.05).Compared with the control group,the MDA content in the renal tissue of the Cd and DM groups was significantly increased(P<0.05),while the GSH content and CAT,SOD,GSH-PX activity were significantly reduced(P<0.05).The DM+Cd group further decreased the GSH content and CAT,SOD,GSH-PX activity compared to the DM group(P<0.05),and the MDA content was further increased(P<0.05).Compared with the control group,the expression level of fibrosis protein in renal tissue in the Cd and DM groups was significantly increased(P<0.05),with an increase in blue purple collagen fibers.The expression level of fibrosis protein in the DM+Cd group was further increased compared to the DM group(P<0.05),and the positive rate of collagen fibers was further increased.Compared with the control group,the expression levels of p-mTOR,LC3-Ⅱ/LC3-Ⅰ,and p62 proteins in the kidney tissue of the Cd and DM groups were significantly increased(P<0.05),while the expression levels of mature-CTSB,mature-CTSD,LAMP1,and LAMP2 proteins were significantly reduced(P<0.01).Autophagy accumulated and the expression levels of p-mTOR,LC3-Ⅱ/LC3-Ⅰ,and P62 proteins in the DM+Cd group were further increased compared to the DM group(P<0.01)The expression levels of LAMP1 and LAMP2 proteins further decreased(P<0.05),and the accumulation of autophagosomes increased.2.The effect of cadmium exposure on autophagy of rat renal tubular epithelial cells cultured with high glucoseTo further explore the effect of cadmium exposure on renal autophagy in diabetes rats,renal tubular epithelial cells of rats were cultured in a medium with 30 mmol/L sugar concentration for 24 hours to simulate the hyperglycemic environment of diabetes rats in vivo,and on this basis,10 μmol/L cadmium chloride for 24 hours.CCK-8 method was used to detect cell survival rate;Flow cytometry was used to detect cell ROS content and apoptosis rate;Immunofluorescence detection of fluorescence intensity of p62 and LC3 proteins;Western blot was used to detect the expression levels of apoptosis,fibrosis,autophagy,and lysosomal related proteins in cells;Transmission electron microscopy was used to observe the ultrastructure of cell mitochondria and the number of autophagosomes.The results showed that compared with the control group,the cell survival rate of the Cd and HG groups was significantly reduced(P<0.01),and the ROS content was significantly increased(P<0.05).Mitochondria were significantly damaged,and the survival rate of the HG+Cd group was further reduced(P<0.01)compared to the HG group.The ROS content was further increased(P<0.01),and mitochondrial damage was exacerbated.Compared with the control group,the apoptosis rate of the Cd and HG groups was significantly increased(P<0.05),the expression level of BAX protein was significantly increased(P<0.01),and the expression level of BCL-2 protein was significantly reduced(P<0.01).The apoptosis rate of the HG+Cd group was further increased compared to the HG group(P<0.01).Compared with the control group,the expression levels of fibrosis proteins in the Cd and HG groups were significantly increased(P<0.05),and the protein expression levels in the HG+Cd group were further increased compared to the HG group(P<0.05).Compared with the control group,the expression levels of p-mTOR,LC3-Ⅱ/LC3-Ⅰ,and p62 proteins in the Cd and HG groups were significantly increased(P<0.05),while the expression levels of mature-CTSB,mature-CTSD,LAMP1,and LAMP2 proteins were significantly reduced(P<0.05).Autophagy accumulated and the expression levels of p-mTOR,LC3-Ⅱ/LC3-Ⅰ,and p62 proteins in the HG+Cd group were further increased compared to the HG group(P<0.01)The expression levels of LAMP1 and LAMP2 proteins further decreased(P<0.05),and the accumulation of autophagosomes increased.In conclusion:①Through drinking water exposure,diabetes rats are more sensitive to cadmium nephrotoxicity;②In vivo experiments show that cadmium exposure aggravates renal damage in diabetes by inducing autophagy inhibition;③In vitro experiments showed that cadmium exposure aggravated renal injury in diabetes rats by inducing autophagy inhibition of renal tubular epithelial cells.