Protective Effects And Mechanisms Of Remote Ischemic Preconditioning On Renal Tissue Injury After Percutaneous Nephrolithotomy

Objective:The protective effect of remote ischemic preconditioning(RIPC)on renal injury due to various causes has been reported,but whether RIPC can alleviate renal injury caused by percutaneous nephrolithotomy(PCNL)remains unclear,and its mechanism needs to be further explored.To clarify the renal injury after PCNL and evaluate the protective effect of RIPC on renal injury after PCNL.Animal and cellular models are constructed to clarify that extracellular vesicles(EVs)mediated the protective effect of RIPC on renal tissue injury.We used 4D Label-free quantitative proteomics to detect the changes in protein expression in plasma-derived EVs from healthy volunteers before and after RIPC and analyzed the results by bioinformatics to explore the potential protective mechanism of RIPC against kidney injury.Method:1.According to the inclusion and exclusion criteria,we enrolled patients with nephrolithiasis who underwent PCNL in our center from June 2020 to October 2021.We divided the patients into RIPC and control groups following the principle of randomized controlled experiments and collected the peripheral blood and urine samples from both groups before the operation,8 hours,and 24 hours after the operation;We checked the levels of inflammatory indicators and kidney injury markers,observing whether RIPC has protective effects on kidney injury after RIRS.2.We established the rat RIPC model by tying the hind limbs of the rat intermittently with a tourniquet;We detected the blood flow of the hind limbs by laser Doppler color ultrasound;We ligated the left ureter of the rat and injected LPS derived from Escherichia coli into the renal pelvis from the ureter to induce the U-L kidney injury model.To observe whether RIPC has a protective effect on kidney damage in the U-L model,we divided the rats into three groups: the Sham group,U-L group and RIPC+U-L group.The rats were sacrificed 24 hours after modeling,and their kidney and blood samples were collected for detection.We isolated and identified the EVs in the plasma of rats before and after RIPC.We randomly divided the rats into the Sham group,U-L group,RIPC-EVs+U-L group,and Nor-EVs+U-L group.Before establishing the U-L model,we injected EVs into the rats through the caudal vein(protein quantification 200 μg).The rats were sacrificed 24 hours after modeling,and we observed the kidney injury and systemic inflammatory response in the four groups.3.We gave different final concentrations(0,1,10,20,50,100ug/ml)of LPS,intervened in HK-2 cells for 24 hours,and evaluated cell viability by trypan blue staining.We used flow cytometry to detect cell apoptosis and screened the optimal concentration of LPS intervention.The EVs in the plasma of healthy volunteers before and after RIPC were isolated and identified.The EVs were labeled with PKH26 and added to HK-2 cells for coculture.The uptake of EVs by HK-2 cells was observed by fluorescence microscopy,and the co-culture of the uptake of EVs by HK-2 cells at different time points was detected by flow cytometry.We added the EVs from the plasma of healthy volunteers before and after RIPC to HK-2 cells and used LPS to intervene after co-cultivation for 12 hours.We divided the experiment into the Sham group,LPS group,LPS+RIPC-EVs,and LPS+Nor-EVs.We used flow cytometry and Western Blot(WB)to detect apoptosis,ELISA was used to detect the level of inflammatory factors in the supernatant of cells,and EDU was used to detect proliferation.4.Through 4D Label-free proteomics,the differential proteins significantly increased in EVs after RIPC were screened,and the bioinformatics analysis of the differential proteins was performed.WB verified the expression of the tenascin C(TNC),fibronectin(FN1),and recombinant human carbonic anhydrase-1(CA1)in plasma-derived EVs before and after RIPC.Result:1.Patients who received PCNL significantly increased postoperative inflammation indicators and kidney injury markers(NGAL,Kim-1,DKK3).In contrast,there were no significant changes in serum creatinine and blood urea nitrogen;Compared with the control group,the expressions of postoperative kidney injury markers(NGAL,Kim-1/creatinine,DKK3/creatinine)in the RIPC group were significantly decreased.2.We successfully established the U-L model and verified that extremity RIPC could attenuate the kidney injury caused by U-L.We successfully extracted plasma-derived EVs before and after RIRS.Plasma-derived EVs after injection of RIPC in the caudal vein can notably improve the degree of U-L model kidney injury in the form of decline in serum creatinine,kidney tissue injury score,inflammation,and apoptotic cells.3.50 μg/ml was selected as the optimal concentration of LPS for intervention in HK-2 cells,when HK-2 cell viability was significantly reduced and apoptosis level was increased;Compared with the LPS group,HK-2 cells in the LPS+Nor-EVs group had no significant changes in apoptosis,inflammation,and proliferation,while the HK-2 cells in the LPS+RIPC-EVs group significantly decreased in apoptosis and inflammation,and the ability of proliferation was significantly improved.4.In this study,37 differential proteins that were significantly elevated in human plasmaderived EVs after RIPC were screened;Bioinformatics analysis of the differential proteins suggests that these proteins are involved in ECM receptor interaction and PI3K-Akt and other pathways;WB results showed that the expressions of TNC,FN1,and CA1 in plasmaderived EVs were significantly increased after RIPC.Conclusion:RIPC can significantly decrease the expression of kidney injury markers,revealing that RIPC may play a potential role in renal protective effect.Plasma EVs may mediate the protective effect of RIPC against intrapelvic hypertension and LPS-induced renal injury,and specific proteins significantly elevated within EVs may be critical for their protective effect.