The Role And Mechanism Of R-spondin3 In Aerobic Exercise Ameliorating Sepsis-associated Acute Kidney Injury

Objectives:Sepsis is a clinical syndrome in which a dysregulated host responses to infection resulting in multiple organ dysfunction and a high mortality rate,and acute kidney injury（AKI）is one of the most common complications of sepsis.The kidney is rich in endothelial cells,which are the major target for pathogens and their toxins.However,endothelial glycocalyx plays a vital role in maintaining the integrity of the renal endothelial cell barrier and is the primary site of the attack by pathogens and toxins during sepsis.It is worth noting that aerobic exercise as a non-pharmacological therapy exerts a protective effect against sepsis-associated multiple organ dysfunction.In addition,moderate-intensity endurance training exerts a pronounced protective effect on endothelial glycocalyx integrity.However,whether exercise training attenuates sepsis-associated AKI（S-AKI）via preserving the endothelial glycocalyx and improving endothelial dysfunction remains unknown.RSPO3 is a secreted protein involved in the development and functional maintenance of vascular endothelial cells.Intriguingly,a recent study indicates that muscular RSPO3expression is significantly up-regulated in mice after wheel running.However,whether exercise training exerts protection against S-AKI via modulating RSPO3expression is still unclear.Therefore,this study will reveal the role and mechanism of RSPO3 in aerobic exercise ameliorating S-AKI.Methods（1）C57/BL6 mice were allocated to Control,lipopolysaccharide（LPS）,Exercise,and Exercise+LPS groups.The exercise regimen is constructed with 6 weeks of treadmill training;The AKI model was induced by intraperitoneal injection of lipopolysaccharide（LPS）.The pathological changes and function of kidney tissues were assessed,and endothelial barrier-related protein levels,renal endothelial permeability,and pro-inflammatory cytokine levels were measured.（2）Peripheral blood was collected from clinical S-AKI patients,and the changes of plasma RSPO3 protein concentration were detected by ELISA.And the correlation between plasma RSPO3 levels and biomarkers of renal function levels were analysed.（3）RT-PCR was used to detect the expression of RSPO3 in kidney tissues in order to explore the effect of aerobic exercise.And the correlation between RSPO3 m RNA expression and biomarkers of renal function levels were analysed.（4）C57/BL6 mice were allocated to Control,LPS,RSPO3,and RSPO3+LPS groups.RSPO3 was intraperitoneally injected 30 minutes before LPS treatment.Damage biomarkers were detected as above.（5）Inducible endothelial cell-specific RSPO3 knockout(RSPO3^EC-/-)and wild-type（WT）mice were assigned to Control,LPS,RSPO3^EC-/-,RSPO3^EC-/-+LPS groups.AKI was induced by intraperitoneal injection of LPS.Damage biomarkers were detected as above.（6）To investigate the potential mechanisms of aerobic exercise and RSPO3protecting against LPS-induced AKI,we analysed differentially expressed MMP genes in gene expression profiles of S-AKI models downloaded from the NCBI GEO database.Results:（1）Aerobic exercise training attenuates renal endothelial hyperpermeability,inflammation and AKI induced by systemic LPS administration.（2）RSPO3 protein concentration was decreased in plasma of S-AKI patients and was negatively correlated with renal function biomarkers.（3）Aerobic exercise training improves LPS-induced downregulation of RSPO3expression in renal tissues.（4）Supplementation of RSPO3 attenuates renal endothelial hyperpermeability,inflammation and AKI induced by systemic LPS administration.（5）Endothelial cell-specific RSPO3 deletion aggravates renal endothelial hyperpermeability,inflammation and AKI induced by systemic LPS administration.（6）Bioinformatics analysis showed that MMPs expression was upregulated during sepsis-associated AKI.RSPO3 and aerobic exercise inhibited LPS-induced upregulation of MMPs and attenuates glycocalyx loss.Conclusion:Aerobic exercise inhibits MMPs-mediated endothelial glycoclyx damage and disruption of tight and adhesion junctions by upregulating the expression of RSPO3 in renal tissue,and thus attenuates renal endothelial hyperpermeability and renal damage during S-AKI.