Cadmium Exposure In Drinking Water Aggravates Kidney Fibrosis In Diabetic Mice

Objective:Cadmium pollution is a common environmental problem.Kidney is the main target organ of cadmium toxicity.The prevalence of diabetes is increasing year by year.Diabetic nephropathy is one of the complications of diabetes.It is worth further study whether kidney damage is aggravated when diabetic patients are exposed to cadmium polluted environment.Therefore,the purpose of this study was to investigate the effects of cadmium on kidney fibrosis and lipid metabolism in diabetic mice and human renal tubular epithelial cells(HK-2 cells)cultured with high glucose.Methods:1 Effects of cadmium exposure on kidney fibrosis in diabetic mice:Male C57BL/6 mice were randomly divided into four groups:control group(CON group,N=10),cadmium group(Cd group,N=10),diabetes group(DM group,N=25),diabetes and cadmium exposure group(DM+Cd group,N=20).Type 2 diabetic mice model was induced by high fat diet and Streptozotocin(STZ).Cd group and DM+Cd group were poisoned with 50 mg/L cadmium chloride by drinking water for 15 weeks.Cadmium content in mice kidney was detected by Inductively coupled plasma mass spectrometer.Kidney tissue changes in mice were observed by Hematoxylin and eosin(H&E)staining.Mice kidney fibrosis was observed by Masson staining.Morphology of glomerular basement membrane was observed by Periodic acid schiff(PAS)staining.Mice kidney expression levels of Epithelialmesenchymal transition(EMT)markers E-cadherin,Vimentin and Alpha smooth muscle actin(α-SMA)were observed by Immunohistochemical staining.The gene expression levels of E-cadherin,Vimentin,Fibroblast specific protein-1(FSP-1),Collagen1α1,Collagen3α1,Collagen4α1,Fibronectin,Transforming growth factor-beta 1(TGF-β1),Matrix metalloproteinase inhibitor-1(TIMP-1),Matrix metalloproteinase-9(MMP-9),Interleukin1 beta(IL-1β),Interleukin-6(IL-6),Tumor necrosis factor-alpha(TNF-α)and Kidney damage molecule-1(KIM-1)in mice kidney were determined by Real-time quantitative PCR(RT-qPCR).The protein expression levels of Collagen1,Collagen3α1,Fibronectin,TGF-β1,Smad3,p-Smad3,E-cadherin,Vimentin,α-SMA and MMP-9 in mice kidney were determined by Western blot.2 Effects of cadmium exposure on kidney lipid metabolism in diabetic mice:The contents of Triglyceride(TG),Total cholesterol(TC)and Lactic acid were measured by the kit.Kidney lipid drops were observed by Oil red O staining.The gene expression levels of Sterol regulatory element-binding protein-1(SREBP-1),Sterol regulatory element-binding protein-2(SREBP-2),Fatty acid synthase(FASN),Acetyl-CoA carboxylase alpha(ACCα),Hydroxy methyl glutaryl CoA reductases(HMGCR),ATP-binding cassette transporter A1(ABCA1),LDL receptors(LDL-R),Fatty acid translocase(CD36),Peroxisome proliferative activated receptor gamma coactivator 1 alpha(PGC-1α),Peroxisome proliferator activated receptor alpha(PPARα),Carnitine palmitoyl transferase 1(CPT1),Carnitine palmitoyl transferase 2(CPT2),Acyl-coa oxidase 1(ACOX1),Acyl-coa oxidase 2(ACOX2),Hexokinase-2(HK-2),Pyruvate kinase(PKM)and Lactate dehydrogenase A(LDHA)in mice kidney were determined by RT-qPCR.The protein expression levels of PPARα,CPT1,ACOX1,FASN and CD36 in mice kidney were determined by Western blot.3 Effects of cadmium exposure on fibrosis and lipid metabolism in HK-2 cells cultured with high glucose:On the basis of the CCK-8 experiment,selecting 10 μM cadmium chloride and 30 mM glucose cultured HK-2 cells for 24 h.Lipid drops in HK-2 cells were observed by Oil red O staining and Bodipy 493/503 staining.The contents of TG and TC in HK-2 cells were determined by the kit.The ROS content in HK-2 cells was determined by Reactive oxygen species kit.Mitochondrial membrane potential level in HK2 cells was determined by Mitochondrial membrane potential test kit.The gene expression levels of TGF-β1,E-cadherin,Vimentin,α-SMA,Collagenl,Fibronectin,PGC-1α,PPARα,CPT1,ACOX1,FASN,ACCα,SREBP-1,SREBP-2 and HMGCR in HK-2 cells were determined by RT-qPCR.The protein expression levels of Collagen1,Fibronectin,Ecadherin,Vimentin,α-SMA,TGF-β1,Smad3,p-Smad3,FASN,ACC,CPT1 and ACOX1 in HK-2 cells were determined by Western blot.Results:1 Cadmium exposure aggravated kidney fibrosis in diabetic mice:(1)Kidney histomorphology:The glomerular volume of diabetic mice increased significantly,the glomerular basement membrane thickened and collagen was deposited.After cadmium exposure intervention,kidney tubules were significantly dilated,a large amount of collagen was deposited and fibrosis was aggravated in diabetic mice.(2)Kidney inflammation factors and kidney tubular damage:Compared with DM group,cadmium exposure significantly increased the gene expression levels of kidney inflammation factors IL-1β,IL-6,TNF-α and kidney tubular injury marker KIM-1 in the kidney of diabetic mice.(3)Kidney TGFβ1/Smad3 signaling pathways:Compared with CON group,the protein expression levels of TGF-β1,p-Smad3,Vimentin,α-SMA,Collagen1,Collagen3α1 and Fibronectin were significantly increased in the kidney of diabetic mice.The protein expression levels of Ecadherin and MMP was significantly inhibited in the kidney of diabetic mice.Cadmium significantly increased the protein expression levels of TGF-β1,p-Smad3 and downstream proteins.Cadmium significantly inhibited the protein expression levels of E-cadherin and MMP in the kidney of diabetic mice.The results suggest that the TGF-β1/Smad3 signaling pathway is involved in the process of cadmium aggravating kidney fibrosis in diabetic mice2 Cadmium exposure aggravated kidney lipid metabolism disorder in diabetic mice:(1)Kidney lipid contents:Compared with CON group,diabetic mice kidney lipid droplets,TC and TG contents increased significantly.Cadmium exposure significantly reduced the contents of kidney lipid droplets,TG and TC in diabetic mice.(2)Kidney fatty acid synthesis and influx:Compared with CON group,the kidney gene expression levels of SREBP-1,FASN,ACCα and CD36 increased significantly and the protein expression levels of FASN and CD36 also increased significantly in diabetic mice.Cadmium exposure significantly inhibited the kidney gene expression levels of SREBP-1,FASN,ACCα,CD36 and the kidney protein expression levels of FASN and CD36 were also significantly inhibited in diabetic mice.(3)Kidney cholesterol metabolism:Compared with CON group,the kidney gene expression levels of SREBP-2,HMGCR and LDL-R increased significantly in the diabetic mice.There was no significantly difference in the gene expression level of ABCA1.Cadmium exposure significantly inhibited the kidney gene expression levels of SREBP-2,HMGCR,LDL-R and significantly increased the gene expression level of ABCA1 in diabetic mice.(4)Kidney fatty acid oxidation:Compared with CON group,the kidney gene expression levels of PGC-1α,PPARα,CPT1,CPT2 and ACOX2 increased significantly and the kidney protein expression levels of PPARa and CPT1 also increased significantly in diabetic mice.Cadmium exposure significantly inhibited the kidney gene expression levels of PGC-1α,PPARα and downstream genes and the kidney protein expression levels of PPARα,CPT1 and ACOX1 were also significantly inhibited in diabetic mice.(5)Kidney glycolysis and lactate contents:Compared with CON group,the kidney gene expression levels of HK-2 and Lactic acid contents increased significantly in diabetic mice.Cadmium exposure significantly increased the kidney gene expression levels of HK-2,PKM and LDHA in diabetic mice.Lactic acid contents increased slightly,but the difference was not statistically significant.3 Cadmium exposure aggravated fibrosis and lipid metabolism disorder in HK-2 cells cultured with high glucose:(1)HK-2 cells fibrosis markers and TGF-β1/Smad3 signaling pathway:Compared with NG group(Normal glucose group),high glucose significantly increased the protein expression levels of TGF-β1,p-Smad3,Vimentin,α-SMA,Collagen1 and Fibronectin and inhibited the protein expression level of E-cadherin in HK-2 cells.Cadmium exposure significantly increased the protein expression levels of TGF-β1,pSmad3,Vimentin,α-SMA,Collagen1 and Fibronectin and significantly inhibited the protein expression level of E-cadherin in HK-2 cells cultured with high glucose.(2)HK-2 cells lipid contents:Compared with NG group,the contents of lipid droplets,TG and TC increased significantly in HK-2 cells cultured with high glucose.Cadmium exposure significantly reduced the contents of lipid droplets,TG and TC in HK-2 cells cultured with high glucose.(3)HK-2 cells lipid synthesis:Compared with NG group,high glucose significantly increased the gene expression levels of SREBP-1,SREBP-2,FASN,Accα and HMGCR and the protein expression levels of FASN and ACC in HK-2 cells.However,cadmium exposure significantly inhibited the gene expression levels of SREBP-1,SREBP-2,ACCα,HMGCR and the protein expression levels of FASN and ACC in HK-2 cells cultured with high glucose.(4)HK-2 cells fatty acid β oxidation:Compared with NG group,high glucose significantly increased the gene expression levels of PGC-1α,CPT1,ACOX1 and the protein expression levels of CPT1 and ACOX1 in HK-2 cells.However,cadmium exposure significantly inhibited the gene expression levels of PPARα,CPT1,ACOX1 and the protein expression level of CPT1 in HK-2 cells cultured with high glucose.(5)HK-2 cells mitochondria:Compared with NG group,ROS content increased significantly in HK-2 cells cultured with high glucose.Cadmium exposure significantly increased ROS content and decreased mitochondrial membrane potential levels in HK-2 cells cultured with high glucose.Conclusions:Cadmium exposure aggravated kidney fibrosis in diabetic mice.The mechanism may be related to the enhancement of kidney mitochondrial damage,inhibition of kidney fatty acid β oxidation and activation of kidney TGF-β1/Smad3 signaling pathway.At the same time,cadmium exposure reduced kidney lipid deposition in diabetic mice.The mechanism may be related to the reduction of kidney lipid synthesis and influx,but the specific mechanism is still unclear and needs further investigation.It is suggested that cadmium exposure may aggravate kidney fibrosis in diabetic patients.Therefore,it is of great significance for prevention and treatment of diabetic nephropathy to explore the pathological rules of diabetic patients in cadmium-polluted environment.