| Part 1 The expression and role of autophagy in the kidneys of patients with calcium oxalate kidney stonesObjective: To observe the changes of autophagic levels in kidneys of patients with calcium oxalate kidney stones,and to explore the role of autophagy in the formation of calcium oxalate nephrolithiasis.Methods: Collection of renal papilla and its surrounding interstitial specimens in 28 patients patients with calcium oxalate nephrolithiasis as an experimental group.During the same period,the kidneys of 12 patients undergoing radical nephrectomy were collected,and the normal tissue samples away from the tumor were taken as the control group.The stone composition in patients with kidney stones was analyzed by the use of an automatic stone infrared spectrum analysis system.HE staining was used to observe the pathological changes of renal tissue and the deposition of crystals in kidneys of patients with calcium oxalate kidney stones.The expression of autophagy key proteins LC3-II,BECN1 and p62 were detected by Western blot and immunohistochemistry.The formation and quantitative changes of autophagic vacuoles in renal tissues of patients with calcium oxalate kidney stones were observed by transmission electron microscopy.Results: 1.The stone infrared spectrum automatic analysis system analysis shows that the stones in patients with kidney stones are mainly calcium oxalate monohydrate.2.Compared with the normal control group,the results of HE staining in the stone group showed that the expansion and swelling of the renal tubular lumen,and the necrotic substances or the detached epithelial cells in the lumen,and the crystalline deposition.3.Compared with the normal control group,Western blot and immunohisto chemistry results showed that the expression of autophagic key proteins LC3-II and BECN1 were significantly increased,and the expression of p62 protein was significantly decreased in the renal tissue of patients with calcium oxalate kidney stones(P < 0.01).4.In the stone group,the typical autophagosomes and autolysosomes were observed by transmission electron microscopy.Compared with the normal group,the mitochondria showed significantly swollen and the number of autophagic vacuoles significantly increased in the kidney tissue(P < 0.01).Conclusion: The levels of autophagy in kidneys of patients with calcium oxalate kidney stones increased significantly,which may play an important role in the formation of calcium oxalate kidney stones.Part 2 The effect and mechanism of autophagy in renal tubular epithelial cells injury induced by calcium oxalate crystalsObjective: To observe whether calcium oxalate crystals can activate the occurrence of autophagy after renal tubular epithelial cells being intervened,and to explore the mechanism of ROS-activated autophagy mediated by calcium oxalate crystals and to elucidate the role of autophagy in renal tubular epithelial cell damage induced by calcium oxalate crystals.Methods: To establish a cell model of calcium oxalate crystal-renal tubular epithelial cell intervention system,that is,different concentrations of calcium oxalate crystals(0,0.1,0.25,0.5,1.0,2.0,4.0 m M)intervene in renal tubular epithelial cells 24 h and the same concentration of calcium oxalate crystals intervening in renal tubular epithelial cells at different points of time(0,2,4,8,h),the expression of LC3-II,BECN1 and p62 of autophagy key proteins in each group were detected by Western blot.The plasmid containing GFP-LC3 was transfected into HK-2 cells using Lipo 3000 liposome.Confocal microscopy was used to observe the formation of autophagosome after 24 h of intervention of renal tubular epithelial cells with different concentration of calcium oxalate crystals.Transmission electron microscope was used to observe the typical structure and quantity of intracellular autophagosomes and autolysosomes after intervention with 4.0 m M CaOx crystals in renal tubular epithelial cells for 24 h.Different concentrations of calcium oxalate crystals(0,0.25,0.5,1.0,2.0,4.0 m M)were used to intervene in HK-2 cells for 24 h,and superoxide anion Dihydroethidium(DHE)fluorescent probe was used to detect the ROS levels of each group in intracellular.ROS inhibitors(NAC,catalase),autophagy modifiers(rapamycin and 3-methyladenine)and small interfering RNA technology(knockdown of the autophagy key gene BECN1)were added to 4.0 m M CaOx crystal solution working with HK-2 cells 24h;Western blot,confocal microscopy,autophagic single-stranded plasmid(GFP-LC3)and Ad-m RFP-GFP-LC3 transfection techniques were used to elucidate the mechanism of ROS-activated autophagy mediated by calcium oxalate crystals and its regulation of autophagy.By modulating the level of autophagy in the calcium oxalate crystal-renal tubular epithelial cell intervention system,and cell apoptosis was detected by DAPI staining,cell viability was detected by CCK-8,and the expression level of LDH in cell supernatant was detected by the kit,and to investigate the role of autophagy in the damage of renal tubular epithelial cells induced by calcium oxalate crystals.Results: 1.Compared with the control group,the number of GFP-LC3 positive spots increased with increasing concentration of calcium oxalate crystals in a concentration-dependent manner under the confocal microscope(P < 0.01).Western blot results further showed that with the increase of CaOx crystal concentration,the expression levels of autophagic key proteins LC3-II and BECN1 gradually increased,peaked at 4.0 m M,and the expression level of p62 protein reached the lowest(P < 0.05).At the same time,with the prolonged intervention time of calcium oxalate crystals,the expression of LC3-II and BECN1 protein gradually increased,peaked at 24 h,and the expression of p62 protein reached the lowest(P < 0.05).Under transmission electron microscopy,we found that the number of autophagosomes and autolysosomes increased significantly after HK-2 cells were stimulated with 4.0 m M CaOx crystals for 24 h,compared with the control group(P < 0.01).2.Compared with the control group,after intervention of HK-2 cells with CaOx crystals for 24 h,the intracellular ROS fluorescence intensity gradually increased with the increase of stimulating concentration,and showed a concentration-dependent increase(P < 0.05).CaOx crystals could induce the number of GFP-LC3 positive spots and the expression of autophagic key protein LC3-II and BECN1 increased in HK-2 cells.ROS inhibitors NAC and catalase could down-regulate the number of GFP-LC3 positive spots and the expression of LC3-II and BECN1 proteins,while NAC and catalase itself did not affect the formation of autophagic vacuoles(P < 0.01).3.After transfection with Ad-m RFP-GFP-LC3,HK-2 cells were co-cultured with 4.0 mmol/L CaOx crystals for 24 h.Compared with the control group,the number of green,yellow,and red fluorescent spots increased significantly under confocal microscope,indicating that CaOx crystals can enhance intracellular autophagic flux after intervention with HK-2 cells.GFP dots were green fluorescence,m RFP dots were red fluorescence,and the yellow and red fluorescence points in the overlay graph represented autophagosomes and autolysosomes,respectively.Compared with the control group,the number of autophagosomes and autolysosomes increased in the rapamycin group,while the 3-methyladenine group further decreased.Compared with the CaOx crystals intervention group,the number of autophagosomes and autolysoso mes in the CaOx crystals + Rapa treatment group increased significantly,while the CaOx crystals + 3-MA treatment group significantly decreased(P < 0.01).It shows that rapamycin can enhance the autophagic flow,while 3-methyladenine can inhibit the autophagic flux.Western blot results confirmed that: compared with CaOx crystal intervention group,CaOx crystal + Rapa treatment group significantly increased the expression of LC3-II,while CaOx crystal + 3-MA treatment group showed significantly decreased the expression of LC3-II protein(P < 0.01).4.Compared with the control group,the apoptosis rate increased after 4.0mmol/L CaOx crystal intervention in HK-2 cells for 24h(P < 0.001);the activity of HK-2 cells decreased(P < 0.001);The level of lactate dehydro genase from the supernatant of cells increased(P < 0.001).Compared with CaOx crystals intervention group,the apoptosis rate in CaOx crystal + Rapa treatment group significantly increased(P < 0.01);the activity of HK-2 cells significantly decreased(P < 0.05).The expression level of lactate dehydroge nase in the supernatant of the cells significantly increased(P < 0.01),while in the CaOx crystal + 3-MA treatment group,the leve of HK-2 cell injury was alleviated.5.Using small interfering RNA technology to knockdown the autophagy key gene BECN1 pretreatment,adding 4.0m M CaOx crystal solution to intervene renal tubular epithelial cells for 24 h.Western blot showed that,compared with the CaOx + Control siRNA intervention group,the expression of LC3-II and BECN1 was significantly decreased in CaOx+BECN1 siRNA group(P < 0.001).Compared with Control siRNA group,the apoptosis rate was increased in CaOx+Control siRNA intervention group(P < 0.001);the activity of HK-2 cell was decreased(P < 0.01);the expression level of lactate dehydrogenase in cell supernatant was increased(P < 0.001);Compared with CaOx+Control siRNA intervention group,the apoptosis rate of CaOx+ BECN1 siRNA group was decreased(P < 0.01);the activity of HK-2 cell was increased(P < 0.05);the expression level of lactate dehydrogenase in the supernatant of the cells significantly decreased(P < 0.001);However,BECN1 siRNA itself did not affect cell activity.Conclusion:1.The optimal concentrations and time points for autophagy production in renal tubular epithelial cells induced by calcium oxalate crystals were clarified.2.Calcium oxalate crystals can induce the production of ROS in renal tubular epithelial cells,which in turn mediate the activation of autophagy through ROS.3.By using autophagy inducer(rapamycin),inhibitor(3-methyladenine)and small interfering RNA technology(knockdown of autophagy key gene BECN1)could effectively regulated the levels of autophagy induced by calcium oxalate crystals in the renal tubular epithelial cells.4.Inhibition of autophagy can effectively attenuated calcium oxalate crystals induced renal tubular epithelial cells injury.Part 3 The role and pathogenesis of autophagy in rat model of calcium oxalate nephrolithiasisObjective: To establish a rat model of calcium oxalate kidney stones,and to investigate the role of ROS mediated autophagy in the formation of calcium oxalate nephrolithiasis.Methods: The rat model of calcium oxalate nephrolithiasis were established by administration of 0.75% ethylene glycol in their drinking water.32 healthy male Sprague-Dawley rats were randomly divided into four groups(eight rats/group): normal control group,stone model group,stone model + rapamycin group,and stone model + chloroquine group.After 4 weeks of treatment,the expression of autophagic key proteins LC3-II,BECN1 and p62 in renal specimens were detected by Western blot.The number of autophagic vacuoles were observed by transmission electron microscopy.The expression of autophagic key proteins LC3-II was detected by immunohisto-chemistry.32 healthy male SD rats were randomly divided into four groups(eight rats/group): normal control group,NAC treatment group,stone model group,stone model + NAC group.After 4 weeks of treatment,the expression levels of T-SOD and GSH-PX in urine of each group for 24 hours were measured using a kit.Western blot and immunohistochemistry were used to detect the expression of autophagic key protein LC3-II in each group.The number of autophagic vacuoles in each group of kidneys were observed by electron microscopy.Metabolic cages were used to collect 24 hours of urine from the rats.The expression level of NGAL and Kim-1 in the urine of each group were detected by ELISA kits.Rat blood was collected and the serum creatinine and urea nitrogen levels were measured by automatic biochemical analyzer.Von Kossa silver nitrate method was used to detect the deposition of crystals and the pathological changes of renal tissues.Results: 1.Compared with the normal control group,Western blot analysis showed that the expression of autophagy key proteins LC3-II and BECN1 increased in the stone model group,while the expression level of p62 protein decreased(P < 0.05).Immunohistochemical results showed that the expression level of autophagy key proteins LC3-II was increased.The number of autophagic vacuoles were significantly increased by transmission electron microscopy(P < 0.01).Compared with the stone model group,the number of autophagic vacuoles and the expression of autophagy key proteins LC3-II,BECN1 and p62 in the stone model + chloroquine group increased significantly(P < 0.05).Compared with the stone model group,the number of autophagic vacuoles and the expression levels of key autophagy proteins LC3-II and BECN1 in the stone model+rapamycin group increased significantly,while the expression level of p62 protein significantly decreased(P < 0.05).2.Compared with the stone model group,the number of autophagic vacuoles and the expression of autophagic key protein LC3-II in the NAC + stone model group decreased(P < 0.01).Compared with the normal group,the expression of T-SOD and GSH-PX in the stone model group decreased(P < 0.01).Compared with the stone model group,the activity of T-SOD and GSH-PX in the rapamycin + stone model group decreased,while the activity of GSH-PX and T-SOD increased in the NAC treated group and the chloroquine treated group(P < 0.05).3.Compared with the normal control group,serum creatinine and urea nitrogen levels in rat blood increased in the stone model group(P < 0.01);NGAL and Kim-1 expression levels in rat urine increased(P < 0.01);the number of TUNEL-positive cells in the renal tissue increased(P < 0.001);the deposition of calcium oxalate crystals in the kidney,tubular lumen and glomerular injury increased;the normal structure of mitochondria disappeared,swollen and damaged under transmission electron microscope.In addition,the size and the damage degree of the kidney was increased.Compared with the stone model group,the expression levels of serum creatinine and urea nitrogen in the rapamycin + stone model group increased significantly(P < 0.05);the expression levels of NGAL and Kim-1 increased significantly(P < 0.01);the number of TUNEL-positive cells in kidney tissue was significantly increased(P < 0.01);the deposition of CaOx crystals in the kidneys,tubular lumen and glomerular injury were significantly increased;mitochondria were significantly swelled and damaged by transmission electron microscopy;the size and the damage degree of the kidney was increased significantly.However,in the chloroquine + stone model group,ethylene glycol induced calcium oxalate crystals deposition and kidney injury were significantly alleviated.Conclusion: 1.Administration of 0.75% ethylene glycol could successfully induced the rat model of calcium oxalate nephrolithiasis.2.Ethylene glycol can induce the production of ROS in the kidney of rats,and then it can mediate the activation of autophagy through ROS.3.By using autophagy inducer(rapamycin)and inhibitor(chloroquine)could effectively regulated the levels of autophagy induced by ethylene glycol in a rat model of calcium oxalate nephrolithiasis.4.ROS-mediated autophagy could promote ethylene glycol-induced calcium oxalate kidney stone formation in rats,while inhibition of autophagy could effectively attenuated ethylene glycol induced crystals deposition and renal injury,thus reducing the formation rate of kidney stones. |
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