The Role And Mechanism Of Endoplasmic Reticulum Stress In Renal Tubular Epithelial Cell Injury Induced By Calcium Oxalate Crystal

Objective: To observe whether endoplasmic reticulum stress is activated in renal tubular epithelial cell injury induced by calcium oxalate crystals(CaOx crystal),and interfering with endoplasmic reticulum stress inhibitor 4-phenylbutyric acid to confirm endoplasmic reticulum stress The role and mechanism of calcium oxalate crystals in inducing renal tubular epithelial cell injury provide new ideas for preventing the formation and treatment of kidney stones.Methods: Renal tubular epithelial cells were treated with 5 different concentrations of calcium oxalate crystals(0,0.25,0.5,1.0,2.0,4.0 mM)for 24 h,cell viability was measured by CCK-8,and LDH in cell supernatant was detected by LDH kit.Activity,using superoxide anion fluorescent probe to detect intracellular ROS levels and DAPI staining to detect apoptosis to comprehensively evaluate renal tubular epithelial cell damage.The expression levels of BIP and CHOP in endoplasmic reticulum stress were detected by Western blot and indirect immunofluorescence.The microscopic changes of endoplasmic reticulum were observed by transmission electron microscopy of calcium oxalate crystals after intervention of renal tubular epithelial cells for 24 hours.Endoplasmic reticulum stress,comprehensive evaluation of endoplasmic reticulum stress levels in renal tubular epithelial cells by the above experimental results.To further investigate the role and mechanism of endoplasmic reticulum stress in renal tubular epithelial cell injury induced by calcium oxalate crystal,we added endoplasmic reticulum stress-specific inhibitor 4-benzolidine to kidney in this experiment.The tubular epithelial cells were intervened and the relevant indicators after 4-benzolidine intervention were examined.The experimental group was the renal tubular epithelial cell group(Control group),renal tubular epithelial cells + calcium oxalate crystal group(CaOx group),renal tubular epithelial cells + calcium oxalate crystal + 4-phenylbutyric acid group(CaOx+4-PBA group).)and renal tubular epithelial cells + 4-phenylbutyric acid group(4-PBA group).After intervention,Western blotting was used to detect the changes of endoplasmic reticulum stress marker protein BIP and CHOP expression,and the endoplasmic reticulum stress level was evaluated.The ratio of autophagy marker proteins LC3-II,LC3-II/LC3-I was detected.BECLIN-1 was used to evaluate autophagy levels;DCFH-DA fluorescent probes were used to evaluate intracellular ROS levels,and finally cell damage was assessed by LDH,CCK8,and DAPI and TUNEL double staining.Result: With the increase of the concentration of calcium oxalate crystal,the effect of calcium oxalate crystal on renal tubular epithelial cells was more obvious;compared with the normal group,the cell viability showed significant inhibition at 1.0 mM calcium oxalate crystal intervention concentration(P<0.01).The LDH in the supernatant of the cells increased significantly at the concentration of O.5 mM calcium oxalate crystals(P<0.01);while the intracellular ROS increased at the concentration of 0.25 mM calcium oxalate crystals(P<0.01);In addition,DAPI staining indicated that the renal tubular epithelial nucleus was significantly concentrated and deformed at 1.0 mM.Western blot analysis showed that the expression of BIP and CHOP in the endoplasmic reticulum increased gradually with the increase of the concentration of calcium oxalate crystal.The expression of BIP in the 1.0 mM calcium oxalate crystal intervention concentration was statistically significant(P<0.01),while CHOP showed a statistically significant difference in the expression of 2.0 mM calcium oxalate crystals(P<0.01).All of the above indicators increased with the increase of crystal concentration of calcium oxalate,showing a dose-dependent effect.Indirect immunofluorescence of cells showed that the intracellular BIP fluorescence intensity was significantly increased after intervention of 4.0 mM calcium oxalate crystals in renal tubular epithelial cells compared with normal cells.Under transmission electron microscopy,we can see that compared with the control group,after 4.0 mM calcium oxalate crystals stimulated renal tubular epithelial cells for 24 hours,the endoplasmic reticulum of the cells showed obvious swelling and vacuolization.After interfering with the cells with 4-phenylbutyrate,the relevant indicators were tested.Western blot analysis showed that compared with CaOx group,the expression of BIP and CHOP in endoplasmic reticulum stress in CaOx+4-PBA group was decreased(P<0.05),autophagy key proteins LC3-II,LC3-II/LC3-The ratio of I and the expression level of BECLIN-1 were both decreased(P<0.05).In addition,after 4-phenylbutyric acid intervention,intracellular ROS levels decreased;cell viability increased(P<0.01);lactate dehydrogenase activity in cell supernatants decreased(P<0.01);The apoptotic condition was significantly improved.Conclusion: Calcium oxalate crystal may induce renal tubular epithelial cell injury and apoptosis by promoting endoplasmic reticulum stress-related pathway.4.Phenylbutyric acid can reduce the injury of renal tubular epithelial cells induced by calcium oxalate crystals by relieving the endoplasmic reticulum stress of renal tubular cells induced by calcium oxalate crystals and reducing the ROS and autophagy produced by endoplasmic reticulum stress.