The Role And Mechanism Of Ang ?/AT1R-mediated Oxidative Stress In Renal Tubular Cells In Calcium Oxalate Stones Formation

Part? The role of NADPH oxidase-mediated oxidative stress induced by calcium oxalate on expression of stone related proteins in renal tubular cellsObjctive To explore the role and mechanism of NADPH oxidase and cellular oxidative stress on expression of stone related proteins in calcium oxalate-induced renal tubular cell.Methods NRK-52 E cells were treated with calcium oxalate crystals.The content of reactive oxygen species(ROS)was measured by flow cytometry using DCFH-DA.The contents of intracellular SOD,CAT,MDA and supernatant 8-OHd G were detected.Intensity of intracellular Ca2+ was detected by confocal microscopy.Expression of AT1 R was detected by real-time PCR.Expression of cellular AT1 R,NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65),and stone related proteins(OPN,CD44,and MCP-1)were detected by western blot.At the same time,cells were treated with NADPH oxidase inhibitor apocynin to observe its effects on the above changes.Hyperoxaluric rat model was established by intraperitoneal injection of glyoxylic acid.The serum Ang ? concentration was detected by Elisa detection.The expression of AT1 R in rat kidney was detected by western blot and immunohistochemical staining.Results Calcium oxalate increase ROS production in NRK-52 E cells,and activities of SOD and CAT were decreased,while contents of MDA and 8-OHd G were increased.Western blot analysis showed that expression of NADPH oxidase subunits(nox2 and nox4),NF-?B subunits(p50 and p65),and stone related proteins(OPN,CD44 and MCP-1)were significantly increased.Administration of apocynin reduced ROS levels,inhibited the expression of NADPH oxidase subunits(nox2 and nox4),NF-?B subunits(p50 and p65),and stone related proteins(OPN,CD44,and MCP-1).In addition,calcium oxalate increased intracellular Ca2+ concentration in NRK-52 E cells.Real-time PCR and western blot analysis showed that expression of AT1 R was increased,and serum Ang ? concentration in hyperoxaluric rats was also increased.Western blot and immunohistochemistry showed that expression of AT1 R in rat kidney was increased.Conclusions Calcium oxalate activated NADPH oxidase-mediated oxidative stress and ROS production in renal tubular cells,which further promoted the expression of NF-?B subunits(p50 and p65)and stone related proteins(OPN,CD44 and MCP-1).Calcium oxalate increased intracellular Ca2+ concentration and the expression of Ang ?/AT1 R,which may be involved in cellular oxidative stress.Part? Role and mechanism of Ang ?/AT1R-mediated endoplasmic reticulum calcium release in renal tubular cells oxidative stress and renal stone formationObjctive To explore the role and mechanism of Ang ?/AT1 R and endoplasmic reticulum calcium release in renal tubular cells oxidative stress and renal stone formation.Methods NRK-52 E cells were treated with calcium oxalate crystals.The intracellular IP3 content was detected by Elisa detection.The expression of IP3R(IP3R1,IP3R2,and IP3R3)was detected by western blot.IP3-R inhibitor 2-APB was used to inhibit endoplasmic reticulum calcium release,intensity of intracellular Ca2+ was detected by confocal microscopy,intracellular ROS was measured by flow cytometry using DCFH-DA,and expression of NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65)and stone related proteins(OPN,CD44 and MCP-1)were detected by western blot.si RNA or losartan was used to inhibit AT1 R expression,and then intracellular Ca2+ concentration,oxidative stress level,NF-?B pathway subunits(p50 and p65),and stone related proteins(OPN,CD44 and MCP-1)expression were examined.Hyperoxaluric rat model and losartan treatment model were established by intraperitoneal injection of glyoxylic acid.Expression of NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65)and stone related proteins(OPN,CD44 and MCP-1)were detected by western blot.Kidney expression of stone related proteins(OPN,CD44 and MCP-1)was detected by immunohistochemistry.Formation of calcium oxalate crystals in rat kidney was detected by Von Kossa StainingResults Expression of IP3 and IP3R(IP3R1 and IP3R2)were increased after treatment with calcium oxalate.2-APB inhibited the increase of intracellular Ca2+,intracellular ROS generation,expression of NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65)and stone related proteins(OPN,CD44 and MCP-1).AT1R-si RNA or losartan treatment inhibited ER calcium release and ROS production,decreased expression of NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65),and stone related proteins(OPN,CD44 and MCP-1).Losartan treatment inhibited the expression of NAPDH oxidase subunits(nox2 and nox4),NF-?B pathway subunits(p50 and p65),and stone related proteins(OPN,CD44,and MCP-1)in kidneys of hyperoxaluric rats.The formation of calcium oxalate crystals in rat kidney was also significantly reduced.Conclusions Calcium oxalate induces Ang ?/AT1 R in renal tubular cells,and activates NADPH oxidase and oxidative stress through IP3/IP3R-mediated calcium release,increases activity of NF-?B pathway and expression of stone related proteins,and promotes adhesion of calcium oxalate crystals and formation of kidney stones.