SIRT3 Inhibited The Formation Of Calcium Oxalate-induced Kidney Stones Through Regulating NRF2/HO-1 Signaling Pathway

Backgroud and Objectives:Kidney stone is a common and frequently-occurring disease in the urinary system,which is caused by the abnormal accumulation of crystal substances in the kidney.Calcium oxalate(Ca Ox)stones are the most prevalent type.The injury and apoptosis of renal tubular epithelial cells caused by Ca Ox crystals are the key steps in the formation of Ca Ox stones.Mitochondrial oxidative stress mediated by reactive oxygen species(ROS)plays an important role in promoting injury and apoptosis of renal tubular epithelial cells caused by Ca Ox crystals.SIRT3 plays a critical role in removing ROS and reducing oxidative damage and mitochondrial dysfunction.In addition,SIRT3 plays a renal-protective role in some renal diseases.However,the role and underlying mechanism of SIRT3 in Ca Ox stones remain unclear.Studies have shown that SIRT3 protects nerve cells by up-regulating nuclear factor erythroid-2-related actor 2/heme oxygenase-1(NRF2/HO-1)pathway,which plays an important role in attenuating oxidative stress in kidney disease.Therefore,the purpose of this study was to investigate the role of SIRT3 in Ca Ox kideny stones and to explore whether its regulation of NRF2/HO-1 signaling pathway involves in this process.Methods:1.To examine apoptosis and expression of SIRT3,NRF2,HO-1 in renal tissues from patients with Ca Ox stones,we performed the following experiments:Renal tissues from patients with Ca Ox kidney stones and adjacent non-tumorousrenal tissues from patients who underwent renal resection due to renal tumors were collected.SIRT3 expression was detected by immunohistochemistry and western blot.TUNEL staining was performed to examine apoptosis in renal tissues.The protein levels of NRF2,HO-1,Bax,and SIRT3 in renal tissues were examined using western blot.2.To examine in vitro whether SIRT3 inhibits Ca Ox crystal-cell adhesion and cell apoptosis in HK-2 cells by activating NRF2/HO-1 pathway,we performed the following experiments:The human proximal tubular cell line HK-2 cells were treated with the following treatments:(1)HK-2 cells were treated with calcium oxalate monohydrate(COM,100 ?g/m L in DMSO)and human isolated nano-sized calcium oxalate(Ca Ox,100 ?g/m L in DMSO)crystals from kidney stones after comminution.(2)HK-2 cells were transfected with pc DNA3.1-SIRT3 or pc DNA3.1 vector,and then stimulated with COM(100 ?g/m L in DMSO).(3)HK-2 cells were co-transfected with pc DNA3.1-SIRT3 or pc DNA3.1 vector,and si-NRF2 or si-Ctrl,followed by stimulation with COM(100 ?g/m L in DMSO).Detection index: After the above cells were treated for 72 hours,the adhesion of crystals on the HK-2 cell surface was observed under the light microscope.Cell apoptosis was detected by annexin V-FITC/PI flow cytometry.The protein levels of SIRT3,NRF2,HO-1 and Bax in cells were detected using western blot.3.To examine in vivo whether SIRT3 overexpression alleviates the glyoxylate-induced damage,crystals deposition and apoptosis in mice,we performed the following experiments:Mice were randomly divided into four groups: Control(given normal saline),Model(given 100 mg/kg glyoxylate by daily intraabdominal injection),LV-Ctrl(given control lentivirus via tail vein),and LV-SIRT3(given SIRT3 overexpression lentivirus via tail vein)group.One week later,the kidneys of mice were isolated for subsequent experiments.The hematoxylin-eosin(HE)staining method was used to observe changes of renal pathology.Von-kossa staining to observe the adhesion of calcium oxalate crystals in the lumen of the renal tubules.SIRT3 expression in renal tissue was detected by immunohistochemistry.TUNEL staining was performed to evaluate apoptosis in renal tissues.The protein levels of NRF2,HO-1,Bax,and SIRT3 in renal tissues were detected using western blot.Results:1.Human kidney with stones showed enhanced apoptosis,downregulated SIRT3 and upregulated NRF2/HO-1 expression Immunohistochemistry analysis revealed that SIRT3 expression in human renal tissues with stones was significantly downregulated compared with the normal control group.The data of western blot further consolidated the results of immunohistochemistry analysis.Furthermore,human renal tissues with stones showed greatly enhanced number of TUNEL-positive cells compared with the control group.In agreement with this,the pro-apoptotic gene Bax in human renal tissues with stones was markedly upregulated as compared with the control group,indicating the enhanced apoptosis in human kidneys with stones.Moreover,protein expression of NRF2 and HO-1 in renal tissues with stones was significantly higher than that in the control renal tissues.2.SIRT3 inhibits Ca Ox crystal-cell adhesion and cell apoptosis in HK-2 cells by activating NRF2/HO-1 pathway Compared with DMSO group(vehicle control),Ca Ox crystal-cell adhesion and cell apoptosis were enhanced,NRF2 and HO-1 protein levels were increased,whereas SIRT3 protein level was decreased in the COM group and Ca Ox group.Overexpression of SIRT3 inhibited Ca Ox crystal-cell adhesion and cell apoptosisCa Ox crystal-cell adhesion and cell apoptosis,and activated NRF2/HO-1signaling pathway.More importantly,NRF2 knockdown effectively impaired the inhibitory effect of SIRT3 on Ca Ox crystal-cell adhesion and cell apoptosis under COM exposure.3.SIRT3 overexpression alleviated the glyoxylate-induced damage,crystals deposition and apoptosis in mice Compared with the control group,significant renal injury(HE staining),increased crystal deposition(von-kossal staining),TUNEL-positive cells(TUNEL staining)and pro-apoptotic Bax protein level(western blot)were observed in the kidney tissues from glyoxylate-induced stone mice.These results suggest that the mouse model of kidney stone has been successfully constructed.Compared with LV-Ctrl,LV-SIRT3 significantly increased renal SIRT3 expression and alleviated the glyoxylate administration-induced damage,crystals deposition and apoptosis in the kidney tissues from model mice.Furthermore,Lv-SIRT3 up-regulated the protein levels of NRF2 and HO-1 in renal tissues from the model mice.These results suggest that the protective effect of SIRT3 on renal stone injury in vivo may be related to the up-regulation of NRF2/HO-1 signaling pathway.Conclusion:1.Human kidney with stones showed enhanced apoptosis,downregulated SIRT3 and upregulated NRF2/HO-1 expression;2.SIRT3 inhibited Ca Ox crystal-cell adhesion and cell apoptosis in HK-2 cells by activating NRF2/HO-1 pathway;3.SIRT3 overexpression alleviated the glyoxylate-induced damage,crystals deposition and apoptosis in mice.