The Role And Mechanism Of Endoplasmic Reticulum Stress-mediated Autophagy In The Formation Of Calcium Oxalate Renal Calculi

Part 1 Effect and mechanism of endoplasmic reticulum stress-mediated autophagy on renal tubular epithelial cell injury induced by calcium oxalate crystals Objective: This study was designed to observe whether calcium oxalate(CaO_x)crystals can activate endoplasmic reticulum stress(ERS)and autophagy in renal tubular epithelial cells,and to explore the regulatory mechanism of endoplasmic reticulum stress on autophagy in cell-crystal reaction.To elucidate the regulatory role of endoplasmic reticulum stress-mediated autophagy in renal tubular epithelial cell injury induced by calcium oxalate crystals.Methods: The cell-crystal reaction system was established by using calcium oxalate crystals and renal tubular epithelial cells.After intervention of different concentrations of calcium oxalate crystals(0,0.25,0.5,1.0,2.0,4.0 mmol/L)for24h and 2.0 mmol/L calcium oxalate crystals interfered with renal tubular epithelial cells at different time points(0,3,6,9,12,24 h),western blot technique was used to detect the expression of endoplasmic reticulum stress marker protein GRP78 and autophagy marker protein LC3 B in renal tubular epithelial cells of each group.Endoplasmic reticulum stress activator tunicamycin(TM)and endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA)were used to pretreatment renal tubular epithelial cells for 3h to regulate the level of endoplasmic reticulum stress.The cells were divided into four groups: control group(NC;cultured in complete medium),CaO_x crystal group(CaO_x;cultured in complete medium containing 2.0 mmol/L CaO_x crystal),CaO_x crystal + 4-PBA group(CaO_x+4-PBA;cultured in complete medium containing 2.0 mmol/L CaO_x crystal after 3h pretreatment with 2.0mmol/L 4-PBA),CaO_x crystal + TM group(CaO_x+TM;cultured in complete medium containing 2.0 mmol/L CaO_x crystal after 3h pretreatment with 2.0μg/mL TM).After 24 h,the activity of endoplasmic reticulum stress and autophagy was evaluated by Western blot and immunofluorescence techniques,and the mechanism of endoplasmic reticulum stress mediated by CaO_x crystal and its regulation on autophagy were clarified.To investigate the regulatory role of endoplasmic reticulum stress-mediated autophagy in renal tubular epithelial cell injury caused by CaO_x crystals.Results: with the increase of CaO_x crystal concentration and treatment time,the expression levels of endoplasmic reticulum stress marker protein GRP78 and autophagy marker protein LC3 B gradually increased,and reached the maximum at 2.0 mmol/L and 24 h.Endoplasmic reticulum stress activator TM and endoplasmic reticulum stress inhibitor 4-PBA can effectively regulate the endoplasmic reticulum stress induced by CaO_x crystals,and the change of autophagy activity is positively correlated with the endoplasmic reticulum stress level.At the same time,4-PBA pretreatment significantly reduced CaO_x crystal-induced crystal adhesion,secretion of IL-1β,secretion of LDH and apoptosis,while TM pretreatment further aggravated CaO_x crystal-induced crystal adhesion,secretion of IL-1β,secretion of LDH and apoptosis.Conclusion:1.The optimal concentration and time of endoplasmic reticulum stress and autophagy induced by CaO_x crystals were determined.2.CaO_x crystals can induce endoplasmic reticulum stress in renal tubular epithelial cells,and then mediate the activation of autophagy through endoplasmic reticulum stress.3.The application of endoplasmic reticulum stress activator TM and endoplasmic reticulum stress inhibitor 4-PBA can effectively regulate the level of endoplasmic reticulum stress induced by CaO_x crystals,and then regulate autophagy.4.Inhibition of autophagy mediated by endoplasmic reticulum stress can effectively reduce the injury of renal tubular epithelial cells induced by CaO_x crystals.Part 2 Regulation and mechanism of endoplasmic reticulum stress-mediated autophagy in the formation of calcium oxalate kidney stones in ratsObjective: This study was designed to explore the mechanis m of autophagy mediated by endoplasmic reticulum stress(ERS)in the formation of calcium oxalate kidney stone and apoptosis in rats.Methods: The rat kidney stone model of CaO_x was induced by ethylene glycol(EG),and the ERS inhibitor 4-phenylbutyric acid(4-PBA)and autophagy inhibitor chloroquine(CQ)were used to regulate the levels of ERS and autophagy.28 male Sprague-Dawley rats aged 6 weeks and weighing 180-200 g were selected.After one week of adaptive feeding,the experimental rats were randomly divided into four groups: control group(Control;fed with normal drinking water),stone model group(EG;fed with drinking water containing 1% EG),stone model + 4-PBA treatment group(EG+4-PBA;fed with drinking water containing 1% EG + intragastric administration of 4-PBA(100mg/kg/d)),stone model + CQ treatment group(EG+CQ;fed with drinking water containing 1% EG + intraperitoneal injection of CQ(60mg/kg/d)).Four weeks later,the renal tissues of rats in each group were collected.The technology of Western blot,RT-qPCR and immunohistochemistry were used to detect the expression of autophagy marker proteins LC3 B,BECN1,p62 and ERS marker proteins GRP78,CHOP,caspase12,p-PERK and p-eIF2α in renal tissue,and the number of autophagy vesicles in kidney were observed by transmission electron microscope.The 24 h urine of rats was collected by metabolic cage,and the contents of kidney injur y molecule-1(Kim-1)and neutrophil gelatinase-associated lipocalin(NGAL)in urine of rats in each group were detected by ELISA kit.The circulating blood of rats in each group was collected through the inferior vena cava,and the levels of serum creatini ne(CRE)and urea nitrogen(BUN)were measured by automatic biochemical instrument.Von Kossa silver nitrate staining was used to detect the intrarenal crystal deposition and the pathological changes of renal tissue in rats of each group.Results: we observed that EG induced CaO_x kidney stone formation in rats was accompanied by excessive activation of ERS and autophagy.CQ treatment can effectively inhibit the overactivation of autophagy and reduce EG-induced renal injury and apoptosis in rats.4-PBA treatment inhibited the activation of ERS and autophagy at the same time,and showed a nephroprotective effect similar to that of CQ,significantly reduced the contents of CRE and BUN,the excretion of NGAL and Kim-1,and the intrarenal crystal deposition,and alleviated EG-induced renal injury and apoptosis in rats.In addition,the autophagy inhibition of 4-PBA is accompanied by the low expression of PERK/eIF2α signal pathway.Conclusion: 1.EG-induced CaO_x kidney stone formation in rats is accompanied by excessive activation of ERS and autophagy.2.EG induces excessive activation of ERS,which can mediate autophagy activation and promote renal crystal formation and tissue damage.3.ERS inhibitor 4-PBA and autophagy inhibitor CQ can effectively regulate ERS and autophagy in rat model of EG-induced CaO_x kidney stone.4.ERS can mediate the activation of autophagy through PERK-eIF2α singal pathway.Inhibition of ERS-mediated autophagy can effectively protect renal function and reduce apoptosis of renal tubular epithelial cells and the formation of renal stones.