| Objective Nephrolithiasis is a common disease in urology,and its pathogenesis is related to many factors.Recent studies have shown that reactive oxygen species(ROS)can promote autophagy during the formation of kidney stones and aggravate kidney damage.Endoplasmic reticulum stress(ERS)is a key factor that regulates the homeostasis of the cell environment and is also directly related to the generation of ROS.Therefore,in this study,the establishment of animal and cell calcium oxalate(Ca Ox)stone models are builded.By regulating the activity of superoxide dismutase(SOD),we aimed to investigate the effects of SOD on autophagy-ERS response and apoptosis in the formation of Ca Ox kidney stones.Methods The rat model of Ca Ox kidney stones was established by drinking water with 0.75% ethylene glycol(EG).32 rats were randomly divided into 4 groups,8 in each group: normal control group,stone model group,stone model + atorvastatin(ATO)group,stone model +diethyldithiocarbamate(DETC)group.After 4 weeks of treatment,the kidney / body weight ratio was calculated to assess kidney enlargement,and the kidney function was assessed by measuring serum superoxide dismutase(SOD),creatinine(CRE)and blood urea nitrogen(BUN)levels.Immunohistochemical staining to detect the expression of autophagyrelated proteins LC3 B and BECN1,while using Western-blot and RTq PCR to detect the expression of autophagy-ERS response-related proteins LC3 B,BECN1,p62,GRP78 and CHOP,using ELISA for the determination of neutrophil gelatinase-associated lipocalin(NGAL)and kidney injury molecule-1(Kim-1).Terminal deoxynucleotidyl transferasemediated d UTP nick end labeling(TUNEL staining)and Western-blot were used to detect the apoptosis of renal tubular cells and the signature proteins expression of cleaved Caspase-3,Bax and Bcl-2.Using Von Kossa staining to observe the crystal deposition and tissue damage.In order to further verify the role of SOD activity in the autophagyERS reaction,we used 4m M Ca Ox crystals to stimulate the rat renal tubular epithelial cell line NRK-52 E to establish a rat renal tubular epithelial cell-calcium oxalate stone crystal model.The experimental group is: NRK-52 E group,NRK-52 E + Ca Ox group,NRK-52 E + Ca Ox +ATO group,NRK-52 E + Ca Ox + DETC group.First,the cell proliferation activity detection kit(Cell Counting Kit-8,CCK-8)was used to determine the optimal concentration of ATO and DETC,and the cell activity of NRK-52 E cells in each group was detected.The activities of SOD and Lactate Dehydrogenase(LDH)of NRK-52 E cells in each group were determined by enzyme-label assay,and the ROS expression was detected by ROS staining,so as to verify the oxidative stress and damage of ERK-52 E cells in each group.Immunohistochemical staining was used to detect the expression of autophagy-related proteins LC3 B and BECN1,and Westernblot was used to detect the expression of autophagy-ERS response-related proteins LC3 B,BECN1,p62,GRP78 and CHOP.TUNEL staining was used to observe the apoptosis of NRK-52 E cells in each group.The expression of transforming growth factor-β1(TGF-β1),a marker of renal fibrosis,was detected by immunohistochemical analysis and Western-blot.Finally,the cell crystal adhesion was observed by microscopic examination.Results In the study of the rat calcium oxalate kidney stone model,compared with the control group,the kidney/body weight ratio of the stone model group was higher(P <0.05),SOD activity was reduced(P <0.001).The expression of autophagy-ERS response proteins and apoptosis-related proteins LC3 B,BECN1,GRP78,CHOP,Bax,and cleaved Caspase-3 were significantly increased(P <0.05),while the expression of p62 and Bcl-2proteins were significantly decreased(P <0.05).In addition,the levels of serum CRE,BUN,NGAL and Kim-1 in the stone model group were significantly higher than those of the control group(P <0.001),and the deposition of Ca Ox stone crystals and kidney damage were also significantly higher than those of the control group.Compared with the stone model group,the kidney/body weight ratio in the ATO intervention group decreased significantly(P <0.001),SOD activity increased(P<0.001).The expression of autophagy-ERS response,apoptosis-related proteins LC3 B,BECN1,GRP78,CHOP,Bax and cleaved Caspase-3 were significantly decreased(P <0.05),while the expression of p62 and Bcl-2protein were significantly increased(P <0.05).Meanwhile,the serum CRE,BUN,NGAL and Kim-1 levels were significantly lower in the ATO intervention group than those in the stone model group(P <0.05),calcium oxalate stone crystal deposition and kidney damage were also significantly lower than in the stone model group.The DETC intervention group significantly reduced the activity of SOD compared with the stone model group(P <0.001),the result was exactly the opposite of the ATO intervention group,the cell autophagy-ERS response was enhanced and the apoptosis and kidney damage were increased(P <0.05),Calcium oxalate stone crystal deposition also increased significantly.In the study of rat renal tubular epithelial cell-calcium oxalate stone crystal model,the optimal concentration of ATO was selected by CCK-8as 20μM(P <0.001),and the optimal concentration of DETC was 0.25 m M(P <0.001).Compared with the control group,the NRK-52 E cell activity and SOD activity in the Ca Ox group were significantly reduced(P <0.001),LDH concentration was significantly increased(P <0.001),apoptosis was significantly increased in TUNEL detection(P <0.001).The expressions of autophagy-related proteins LC3 B and BECN1 in immunohistochemistry were significantly increased(P <0.001),and the expressions of autophagyERS-related proteins LC3 B,BECN1,GRP78 and CHOP were significantly increased by Western-blot(P <0.05),while the expression of P62 was significantly reduced(P <0.001).The expression of TGF-β1 was significantly increased by immunohistochemistry and Western-blot(P<0.001),and the adhesion of Ca Ox crystals was significantly increased by microscopic observation(P <0.001).Compared with the Ca Ox group,the NRK-52 E cell activity and SOD activity in the ATO intervention group were significantly increased(P <0.001),the LDH concentration was significantly reduced(P <0.001),TUNEL detected apoptosis was significantly reduced(P <0.001).The expressions of autophagy-related proteins LC3 B and BECN1 were significantly reduced by immunohistochemistry detection(P <0.001),and the expressions of autophagy-ERS-related proteins LC3 B,BECN1,GRP78 and CHOP were significantly reduced by Western-blot(P <0.01),while the expression of P62 was significantly increased(P <0.001).The expression of TGF-β1,a marker of renal fibrosis,was significantly decreased by immunohistochemistry and Western-blot(P <0.05),and the adhesion of Ca Ox crystals was significantly reduced by microscopic observation(P<0.001).On the contrary,the DETC intervention group significantly reduced the NRK-52 E cell activity and SOD activity,and increased the LDH concentration when compared with the Ca Ox group(P <0.01),the result was exactly the opposite of the ATO intervention group.Compared with Ca Ox group,autophagy-endoplasmic reticulum stress response was enhanced and cell apoptosis and fibrosis were aggravated(P <0.05),and Ca Ox stone crystal deposition was significantly increased(P <0.001).Conclusions Ca Ox nephrolithiasis can activate the autophagy-ERS response of rat kidney by reducing SOD activity,thereby promoting the apoptosis and fibrosis of renal tubular epithelial cells and increasing the deposition of calculus crystals in the kidney.ATO can reduce renal autophagy-ERS response by enhancing SOD activity,reduce the apoptosis and fibrosis of renal tubular epithelial cells,and then reduce the deposition of crystals in the kidney,and play a role in preventing calcium oxalate kidney stones. |
PDF Full Text Request