| 【Background】 Kidney stone is a common and frequently occurring disease in our country.A recent cross-sectional epidemiological study across the country showed that the prevalence of kidney stones in Chinese adults was 5.8%.Combined with China’s huge population,there are about 61 million 200 thousand adults suffering from kidney stones.The study also shows that the stones in China are mainly concentrated in Southern China and southwest China,which is related to the average temperature in the South higher than in the north.Kidney stones are also a global disease,a number of foreign studies show that about 5-15% of the population will suffer from kidney stones.In all patients with kidney stones,about 80% of the kidney stones are calcium stones,calcium oxalate stones are the most common types of kidney stones,accounting for about 70%-80%.In recent years,minimally invasive surgery in the treatment of urinary stones has been a great success,kidney stones patients also benefit.However,the pathogenesis of calcium oxalate stone includes many aspects,such as heredity,environment,diet,disease and metabolism.The recurrence rate is still high,most of the patients in life because of recurrent kidney stones need to accept multiple treatment,seriously affecting the quality of life of patients and their families,but also bring huge economic burden to individuals and society.After treatment,the recurrence rate is still high,because of recurrent kidney stones,most of the patients need to accept multiple treatment in life,seriously affecting the quality of life of patients and their families,but also bringing huge economic burden to individuals and society.Therefore,it is of great scientific and clinical value to further reveal the exact pathogenesis of calcium oxalate stone and to find new effective targets for intervention.【Purpose】 To study the effect of autophagy on the formation of calcium oxalate crystals in rat kidney and its possible mechanism.【Methods】 40 healthy male SD rats were randomly divided into four groups(n = 10): control group,model group,chloroquine intervention group and rapamycin intervention group.The model of calcium oxalate stone was established by freely drinking water with 1% ethylene glycol and 1% ammonium chloride for 28 days.Chloroquine intervention group was given intraperitoneal injection of chloroquine solution(40mg/kg/day),rapamycin intervention group received intraperitoneal injection of rapamycin(0.5mg/kg/day).28 days after molding,24-hour urine samples and kidney sections were collected and the risk factors of stone forming in 24 h urine were detected.The levels of autophagy in rats were detected by immunohistochemistry and transmission electron microscopy,and the morphology of mitochondria was observed by transmission electron microscope.We examined crystal formation by using hematoxylin-eosin(HE)staining and polarized light optical microscopy.The expressions of oxidative stress injury related molecular markers were detected by immunohistochemistry.【Results】 1.Analysis of 24 hours urine risk components in rats for each groupCompared with the control group,the excretion of urinary oxalate was significantly higher in the model group(P<0.05),and the excretion of urinary citrate was significantly lower(P<0.05).Compared with the control group,the urine p H value,urine potassium,urinary chloride,urinary sodium,urinary uric acid and urinary citrate excretion were significantly lower in the intervention group(P<0.05).Compared with the control group,the urinary chloride,urinary potassium,urinary sodium,urinary phosphorus,urinary uric acid and urinary citrate excretion were significantly lower in the rapamycin group(P<0.05).Compared with the model group,the urine p H value,urine potassium,urinary chloride,urinary sodium,urinary phosphorus and urinary oxalate excretion in rats of chloroquine intervention group were significantly lower(P<0.05).Compared with the control group,the urinary chloride,urinary potassium,urinary sodium,urinary phosphorus,urinary uric acid and urinary citrate excretion were significantly lower in the rapamycin group(P<0.05).Compared with the model group,the urine p H value,urine potassium,urinary chloride,urinary sodium,urinary phosphorus and urinary oxalate excretion in rats of chloroquine intervention group were significantly lower(P<0.05).2.The formation of calcium oxalate crystals in kidney of rats in each groupIn the control group,there was no calcium oxalate crystals in the kidneys of rats,but a large number of calcium oxalate crystals were found in the renal tubules of model group.Compared to the model group(32.4±5.1/HP),chloroquine intervention group(4±0.9/HP)with renal calcium oxalate crystals decreased significantly(P<0.05),and rapamycin group(102.4±8.5/HP)with calcium oxalate crystals was significantly increased(P<0.05).3.The autophagy and the lysosome were observed by transmission electron microscopeIn control group,there was no autophagy in renal cells.In the model group,there was a typical globular monolayer membrane autophagic lysosome in rat kidney cells.In the chloroquine intervention group,the typical globular monolayer membrane of the kidney cells was found to be autophagic lysosome.In the rapamycin intervention group,double layer membrane autophagic vacuoles were found in the renal cells of rats.4.Immunohistochemistry was used to detect the level of autophagy markers in kidney of rats in each groupCompared with the control group,the expression of LC3 in the model group was significantly higher than that in the control group,while the P62 of the autophagy marker was significantly decreased.Compared with the model group,the expression of LC3 and P62 were significantly increased in the rats treated with chloroquine.Compared with the model group,the expression of LC3 was significantly increased in the intervention group,while the expression of P62 was significantly decreased.5 The morphology of mitochondria in renal cells of rats were observed by transmission electron microscopeIn the control group,the mitochondria in the cells of kidney of rats in the control group were found to be intact.In the model group,the mitochondria in the kidney of the model group were damaged,which showed the swelling of mitochondria,the structure of the double membrane of the mitochondria was fuzzy,the number of cristae of mitochondria was decreased,and the arrangement was disordered.The mitochondria structure in the renal cells of rats in the chloroquine intervention group was clear,and the mitochondria were similar to those in the control group.The mitochondria in the renal cells of the intervention group were obviously edema,the structure of the double membrane of the mitochondria was fuzzy,the number of cristae of mitochondria was decreased,and the arrangement was disordered.6.Immunohistochemistry was used to detect the expression level of oxidative stress markers in ratsCompared with the control group,model group,renal superoxide dismutase(SOD)expression was significantly reduced,and monocyte chemotactic protein 1(MCP-1)and 8-hydroxy deoxyguanosine(8-OHd G)expression was significantly increased.Compared with the model group,the expression of SOD was significantly increased in the rats of chloroquine intervention group,while the expression of MCP-1 and 8-OHd G were significantly decreased.Compared with the control group,the expression of SOD in the kidney of rapamycin group was significantly decreased,while the expression of MCP-1 and 8-OHd G were significantly higher.【Conclusions】 For the first time,this study demonstrated that autophagy plays an important role in the formation of calcium oxalate crystals in rats.The inhibitor of autophagy chloroquine can significantly inhibit the formation of calcium oxalate crystals induced by ethylene glycol in rats by decreasing the level of urinary oxalate in rats,inhibiting the level of autophagy in rat kidney and the degree of injury,but the autophagy inducer rapamycin can induce the formation of more calcium oxalate crystals in the kidney by inducing autophagy in renal tissue and improving the degree of renal injury.Our study confirmed that autophagy is involved in the regulation of calcium oxalate stone formation,which may be the mechanism of autophagy regulating the high oxalate induced oxidative stress injury of renal tubular epithelial cells,mitochondrial damage and function,ROS crystal formation,adhesion and formation of calcium oxalate calculus. |
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