| Part I Expression of Nox4 in renal papilla of patients with calcium oxalate stone and its mechanism Objective: To explore the expression of nicotinamide adenine dinucleotide phosphate oxidase subunit 4(Nox4)in renal papilla of patients with calcium oxalate stone and its potential mechanism.Methods: Randall plaque chip was searched by GEO database.According to the sample description of GSE73680,the samples were divided into stone-free control group and calcium oxalate stone group.There were 6 samples in the control group and 24 samples in the calcium oxalate stone group.GEO2 R was used to analyze the differential expression of Nox4 and osteogenic-associated protein genes in renal papilla of normal subjects and patients with calcium oxalate stone.The renal stone rat model of hypercalciuria was established by using 1,25(OH)2D3.The renal tubular epithelial cells(NRK-52E)were treated with high calcium(1.0mg/ml)for 24 hours.The expression of Nox4 was detected by immunofluorescence(IF),and the expression of Nox4,Nox2,p22,mitogen activated protein kinase(MAPK),bone morphogenetic protein 2(BMP2)and osteopontin(OPN)was detected by western blot.The activities of oxidative stress markers malondialdehyde(MDA)and superoxide dismutase(SOD)were determined by chemiluminescence method.Results: GEO2 R analysis showed that the expression of Nox4,OPN,osteocalcin(OCN)and osteoprotegerin(OPG)were increased in calcium oxalate stone group,while the expression of matrix Gla protein(MGP)was decreased.The results of immunofluorescence showed that the average fluorescence absorbance of Nox4 in NRK-52 E cells treated with high calcium was significantly higher than that in the control group.Western blot showed that the expression of Nox4,p22,MAPK,BMP2 and OPN in NRK-52 E cells treated with high calcium was significantly increased compared with the untreated normal control group,but the expression of Nox2 had no significant change.The expression of p22 in the kidney of hypercalciuria model rats was increased compared with the control group,but the expression of Nox2 protein had no significant change.In addition,after NRK-52 E cells were treated with high calcium,the content of MDA was increased and the activity of SOD was decreased.Conclusion: The expression of Nox4 gene in renal papilla tissue of patients with calcium oxalate stone is significantly increased.Oxidative stress,activation of MAPK signal pathway and osteogenic transformation in renal tubular epithelial cells may be the specific mechanisms of Nox4 in regulating calcium oxalate stone formation.Part II Effects of high calcium activated Nox4/ROS-mediated oxidative stress on injury and apoptosis of renal tubular epithelial cells.Objective: To explore the effect of high calcium activated Nox4/ROS-mediated oxidative stress on injury and apoptosis of renal tubular epithelial cells and its potential mechanism.Methods: The renal stone rat model of hypercalciuria was established by using 1,25(OH)2D3.Adeno-associated virus 2(AAV2)was injected under the renal capsule to block the expression of Nox4 in the rat kidney,and GKT137831,a selective Nox1/4 inhibitor,was used to inhibit the activity of Nox4 in the rat kidney.PKC inhibitor GF109203 X was used to inhibit the activity of PKC in NRK-52 E cells.NRK-52 E was treated with high calcium(1.0mg/ml)for 24 hours.The expression of Nox4 was silenced by CRISPR/Cas9 technique,and the lentivirus transfection technique was used to up-regulate the expression of Nox4.The effect of high calcium on cell viability was detected by CCK-8.The 24-hour urine volume of rats was collected and the concentration of urinary calcium was determined.The intracellular calcium and reactive oxygen species(ROS)levels and apoptosis in NRK-52 E cells were detected by flow cytometry.Western blot was used to detect the expression of p-PKC/PKC,Nox4 and Keap1/Nrf2 in cell and rat kidney.The histological morphology of rat kidney was detected by hematoxylin-eosin(HE)staining.TUNEL was used to detect the apoptosis in rat kidney.The morphological changes of NRK-52 E cells treated with high calcium were observed by transmission electron microscope(TEM).DHE fluorescence probe showed the production of ROS in renal tissue.The activity and content of redox substances(SOD,CAT,MDA and 8-OHd G)in cells and kidneys were measured by chemiluminescence.Results: The viability of NRK-52 E cells significantly decreased after high calcium treatment in a concentration / time dependent manner.The 24-hour urinary calcium level and urinary calcium concentration of SD rats induced by 1,25(OH)2D3 were significantly higher than those of the control group,but the 24-hour urine volume had no significant change.The level of cellular Ca2+ in NRK-52 E cells increased under high calcium treatment,and it was not affected by the regulation of Nox4 expression.High calcium can activate the expression of p-PKC and Nox4 in NRK-52 E cells,while PKC inhibitor can inhibit the expression of Nox4,suggesting that high calcium may activate the expression of Nox4 through PKC.In addition,high calcium can increase the production of ROS/H2O2 in rat kidney and NRK-52 E cells,and regulating the expression and activity of Nox4 can change this phenomenon.The level of oxidative stress in rat kidney and NRK-52 E cells regulated by high calcium was dependent on the expression of Nox4.Renal injury and apoptosis were significantly increased in rats with hypercalciuria,and inhibition of the expression or activity of Nox4 could significantly improve these lesions.Injury and apoptosis occurred in NRK-52 E cells treated with high calcium,and the regulation of Nox4 expression will aggravate or alleviate the above changes.High calcium activated the keap1 expression and decreased the nrf2 expression in NRK-52 cells and rat kidney.Conclusion: On the one hand,high calcium can activate the expression of Nox4 in renal tubular epithelial cells through PKC,and Nox4-derived ROS/H2O2 can lead to oxidative stress;On the other hand,high calcium can induce changes in intracellular Keap1/Nrf2 pathway and inhibit the expression of antioxidant enzymes.The imbalance of redox homeostasis in renal tubular epithelial cells mediates cell injury and apoptosis,which leads to the formation of calcium oxalate stones.Part III The role of osteoblast-like transformation of renal tubular epithelial cells mediated by high calcium activation Nox4/MAPK pathway in the formation of Randall calcium plaque and calcium oxalate stone.Objective: To explore the role of osteoblast-like transformation of renal tubular epithelial cells mediated by high calcium activation Nox4/MAPK pathway in the formation of Randall calcium plaque and calcium oxalate stone.Methods: The renal stone rat model of hypercalciuria was established by using 1,25(OH)2D3.Adeno-associated virus 2(AAV2)was injected under the renal capsule to block the expression of Nox4 in the rat kidney,and GKT137831,a selective Nox1/4 inhibitor,was used to inhibit the activity of Nox4 in the rat kidney.NRK-52 E was treated with high calcium(1.0mg/ml)for 24 hours.The expression of Nox4 was silenced by CRISPR/Cas9 technique,and the lentivirus transfection technique was used to up-regulate the expression of Nox4.P38 inhibitor SB203580,JNK inhibitor SP600125 and ERK inhibitor PD98059 were used to inhibit the activity of MAPK(p38/JNK/ERK)in NRK-52 E.Calcium deposition in rat kidney and NRK-52 E cells was detected by Von Kossa staining and alizarin red staining respectively.The expression of Nox4 in NRK52 E cells was detected by immunofluorescence.Western blot was used to detect the expression of Nox4,P38/p-P38,ERK/p-ERK,JNK/p-JNK,BMP2 and OPN in rat kidney and NRK-52 E cells.The expressions of Nox4,BMP2 and OPN in rat kidney were detected by immunohistochemical staining.Real-time quantitative PCR was used to detect the expression of Nox4,BMP2 and OPN m RNA in the cells.Results: Calcium deposition was found in hypercalciuria kidney tissue and NRK-52 E cells induced by high calcium.It was decreased by inhibiting the Nox4 expression or activity,while calcium deposition was significantly increased by up-regulating the Nox4 expression.High calcium activated the expression of Nox4 and MAPK signal pathway protein in rat kidney and NRK-52 E cells,inhibiting Nox4 expression or activity significantly decreased high calcium-induced MAPK phosphorylated protein expression,while up-regulating Nox4 expression significantly enhanced high calcium-induced MAPK phosphorylated protein expression.In addition,Nox4 is involved in regulating the expression of osteogenic proteins BMP2 and OPN in renal tubular epithelial cells induced by high calcium.ERK and JNK inhibitors significantly decreased the expression of BMP2 and OPN in NRK-52 E cells induced by high calcium,suggesting that high calcium mediates the expression of osteogenic related proteins in NRK-52 E cells by activating Nox4/MAPK pathway.In addition,MAPK inhibitors significantly attenuated the increase of Nox4 in NRK-52 E cells induced by high calcium,indicating that MAPK can regulate the expression of Nox4 through a positive feedback loop in NRK-52 E cells exposed to high calcium.Conclusion: Nox4 induced by high calcium mediates the abnormal activation of osteoblast-related proteins BMP2 and OPN through ERK/JNK MAPK signal pathway,and induces renal tubular epithelial cells to transdifferentiate into osteoblast-like cells,resulting in the formation of Randall calcium plaque and calcium oxalate stones. |
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