The Role And Mechanisms Of Nrf2 Activator Dimethyl Fumarate In Inhibiting The Formation Of Oxalate-induced Renal Stones

Part ? The effect of DMF on calcium salt deposition and osteogenic transformation in renal tissue of hyperoxalate rat model?Objective? To investigate the effect of dimethyl fumarate on calcium salt deposition and osteogenic transforming protein expression in renal tissue of hyperoxalate rats induced by glyoxylic acid monohydrate.?Methods? Forty male SD rats were randomly divided into 4 group: control group,glyoxylic acid monohydrate modeling group(GAM group),therapeutic group(GAM+DMF group)and solvent control group(GAM+MC group),with 10 rats in each group.Rats in the control group were given free drinking purified water,and those in the glyoxylic acid monohydrate modeling group were given intraperitoneal injection of glyoxylic acid monohydrate to induce the establishment of hyperoxalate renal stone model.The therapeutic group was given DMF gavage on the basis of modeling group;In the solvent control group,MC was given by gavage on the basis of modeling group.The body weight of the rats was measured every 3 days,and 24-hours urine,serum samples and kidneys samples of the rats in each group were collected after 10 days of continuous treatment,and the serum creatinine value,the content of calcium and oxalic acid in the urine were measured.The expression levels of OPN and BMP2 in kidney tissue of rats were detected by immunohistochemistry and western blot.HE staining was used to analyze the histology of rat kidney tissue sections.The von kossa staining and polarized optical microscopy were used to analyze the crystal deposition.?Results? The body weights in the control group were significantly heavier than those in the other three groups.There was no significantly difference in 24-hours urine volume between the groups.There was no significant difference in urinary calcium excretion between the groups.The urinary oxalate excretion levels in GAM group,GAM+DMF group and GAM+MC group were significantly higher than those in the control group.Serum creatinine levels in the GAM group and GAM+MC group were significantly higher than those in the control group,and DMF treatment significantly reduced serum creatinine levels.HE staining showed that the renal tissues of GAM group and GAM+MC group had obvious histological damage,and these changes were dramatically ameliorated by DMF administration.In GAM group and GAM+MC group,there was significantly calcium salt deposition at the junction of renal medulla and cortex,and DMF significantly reduced the calcium salt deposition level in renal tissue.Immunohistochemistry and western blot indicated that the expression levels of OPN and BMP2 in renal tissue of GAM group and GAM+MC group were significantly higher than those of the control group.DMF significantly reversed these effects.?Conclusion? Hyperoxaluria can induce renal tissue injury,calcium salt deposition,and osteogenic transformation,while DMF can alleviate the above effects.It is expected to be used to prevent the formation of hyperoxalate-induced kidney stonesPart ? Study on the mechanism of DMF in alleviating hyperoxalate-induced renal tubular epithelial cell injury?Objective?To explore the role and mechanism of DMF in oxidative stress injury,inflammatory response and apoptosis of renal tubular epithelial cells induced by hyperoxalate.?Methods?NRK-52 E cells were treated with oxalate,and CCK-8 method was used to study the effect of DMF on the activity of NRK-52 E cells exposed to oxalate.DHE fluorescence staining was used to determine the level of reactive oxygen species(ROS)in renal tissue of the rat model of hyperoxalate kidney stones and the DMF treatment model.Cell membrane injury toxicity was determined by using lactic dehydrogenase(LDH)kit.Malondialdehyde(MDA)assay was used to detect cellular lipid peroxidation injury.Western blot and immunohistochemistry were used to detect the expressions of inflammatory cytokines(IL-6 and MCP-1)and apoptosis-related proteins(caspase-3).TUNEL assay was used to detect the level of renal tissue apoptosis.?Results?Our results showed that DMF could reduce the inhibition of NRK-52 E cell activity by oxalate.DMF treatment reduced MDA production and LDH release induced by oxalate.DMF can significantly reduce ROS level in renal tissue induced by hyperoxalate.DMF can reduce the release level of inflammatory cytokines in renal tissue induced hyperoxalate.DMF can alleviate renal cell apoptosis induced by hyperoxalate.?Conclusion?DMF can reduce oxidative stress,inflammatory reaction and apoptosis of renal tubular epithelial cells induced by oxalate.Part ? DMF alleviates hyperoxalate-induced renal tubular epithelial cell injury by activating Nrf2/Keap1 pathway?Objective?To explore the role and mechanism of Nrf2/Keap1 pathway in oxidative stress and inflammatory response of renal tubular epithelial cells induced by hyperoxalate.?Methods?NRK-52 E cells were treated with oxalate and small interfering RNA(si RNA)kit was used for gene knockout experiments.The expressions of Nrf2 and Keap1 m RNA were detected by q PCR.Cell ATP level was detected with ATP kit.Mitochondrial membrane potential was detected by jc-1 fluorescent probe.The expressions of Nrf2,Keap1,NADPH oxidase subunits(p22,Nox2,Nox4),inflammatory related proteins(MCP-1,IL-6),osteogenic related proteins(OPN,BMP2)and apoptosis-related protein caspase-3 were detected by immunohistochemistry and western blot.The distribution of Nrf2 in cells was analyzed by immunofluorescence.Cell apoptosis was analyzed by flow cytometry,and the intracellular and mitochondrial ROS levels were analyzed by flow cytometry.?Results?Immunohistochemistry and western blot showed that the expression of Nrf2 was down-regulated and the expression of Keap1 was up-regulated in renal tissue of hyperoxalate kidney stone rats,and DMF treatment could reverse this effect.Immunofluorescence experiments showed that,under the induction of potassium oxalate,the fluorescence intensity of Nrf2 antibody in the nucleus of NRK-52 E cells decreased,and DMF could promote Nrf2 to enter the nucleus.Western blot analysis showed that DMF inhibited the expression of Keap1 in potassium oxalate-induced NRK-52 E cytoplasm and up-regulated the expression of nuclear Nrf2.Western blot analysis showed that DMF could reduce the expressions of oxalate-induced NRK-52 E cells in inflammation(MCP-1,IL-6),apoptosis(caspase-3)and osteogenic(OPN,BMP2)related proteins.Flow cytometry showed that DMF could reduce the apoptosis level of NRK-52 E cells exposed to potassium oxalate.DMF alleviated the damage of mitochondrial membrane potential in NRK-52 E cells induced by potassium oxalate.DMF enhanced the ATP level of NRK-52 E cells inhibited by potassium oxalate.Western blot analysis showed that DMF significantly reduced the up-regulation of protein expressions of Nox4 and p22 in NRK-52 E cells induced by potassium oxalate compared with the control group,and the expression of Nox2 remained unchanged in this process.Flow cytometry showed that DMF pretreatment significantly reduced intracellular and mitochondrial ROS levels compared with oxalate-induced cells.These effects were partially blocked by Nrf2 si RNA transfection,and enhanced by Keap1 si RNA transfection?Conclusion?These results suggest that DMF inhibits the increase of NADPH oxidase subunit Nox4 and p22 hyperoxalate-induced by upregulating the level of Nrf2 in renal tubular nucleus,thereby improving the intracellular and mitochondrial oxidative stress,correcting the abnormalities of mitochondrial metabolism,and inhibiting cellular inflammatory reaction,osteogenic differentiation and apoptosis.