The Study Of Sodium Hydrosulfide On Alleviating Renal Ischemia-reperfusion Injury By Regulating Mitochondrial Fusion

Objective:To investigate the role and regulatory mechanism of sodium thiosulfate（Na HS）in renal ischemia-reperfusion injury（IRI）,and provide a potential therapeutic strategy for the prevention and treatment of IRI during renal transplantation.Methods:Animal level:18 SPF-rated adult male Wistar rats were selected and randomly numbered into three groups:sham-operated group（Sham group）,ischemia-reperfusion group（IR group）and Na HS-pretreated group（IR+Na HS group）.The right kidney of the rats was surgically removed,and the left renal pedicle was clamped with a non-invasive arterial clip for 45 minutes.Blood and renal tissues were retrieved for24 hours of reperfusion to establish a rat renal ischemia-reperfusion injury model.Serum creatinine（Scr）and urea nitrogen（BUN）levels were measured by a fully automated biochemical assay to assess changes in renal function.Western blot was performed to detect the expression of MFN2,Parkin and P62 proteins in the kidney tissues.MFN2 m RNA expression was detected by RT-PCR.Cell level:Human renal tubular epithelial（HK-2）cells in a good growth condition and free of contamination were selected and randomly divided into four groups:control group（Control group）,hypoxic reoxygenation group（HR group）,50u M Na HS pretreatment group（HR+50u M Na HS group）,and 100u M Na HS pretreatment group（HR+100u M Na HS group）.HK-2 cells were incubated in a triple gas incubator（37℃,1%O₂,5%CO₂）for 12 hours,followed by DMEM/F12 medium,normal cell culture incubator（37℃,21%O₂,5%CO₂）for 2 hours to establish the HR model.DCFH-DA reactive oxygen fluorescence probe was used to detect the change of reactive oxygen species（ROS）content in HK-2 cells,and select the optimal concentration for Na HS action for subsequent experiments according to the fluorescence staining results.Use JC-1 as a fluorescent probe,and observe the changes in mitochondrial membrane potential（MMP）of HK-2cells under laser confocal microscopy.Mito-Tracker Red CMXRos label Mitochondria specifically.Changes in mitochondrial morphology of HK-2 cells were observed under laser confocal microscopy.The expression of MFN2 protein in HK-2 cells was detected by immunoblotting.MFN2 and LC3 m RNA expression was detected by RT-PCR.Results:（1）Compared with the Sham group,the serum Scr and BUN concentrations in the IR and Na HS pretreatment groups were significantly higher（P<0.05）;compared with the IR group,the serum Scr and BUN levels in the Na HS pretreatment group were significantly lower（P<0.05）.（2）HE and PAS staining results:compared with the IR group,the degree of kidney tissue damage was significantly reduced in the Na HS pretreatment group;the tubular necrosis score showed that the score in the IR group was significantly higher than that in the Sham group（P<0.05）,while the score in the Na HS pretreatment group was significantly lower than that in the IR group（P<0.05）.（3）Immunohistochemical results:the expression of P62 was significantly higher in the IR group compared with the Sham group;the expression of P62 was significantly increased in the IR+Na HS pretreatment group compared with the IR group.（4）Western blot and RT-PCR results:compared with the Sham group,the expression of MFN2 was significantly lower in the IR group（P<0.05）;compared with the IR group,the expression of MFN2 was significantly higher in the IR+Na HS pretreatment group（P<0.05）.The expression of Parkin was significantly higher in the IR group compared with the Sham group（P<0.05）;the expression of Parkin was significantly lower in the IR+Na HS pretreatment group compared with the IR group（P<0.05）.The expression of P62 was significantly increased in the IR group compared with the Sham group（P<0.05）;the expression of P62 was significantly increased in the IR+Na HS pretreatment group compared with the IR group（P<0.05）.Compared with the Control group,the expression of LC3 was significantly increased in the HR group（P<0.05）;compared with the HR group,the expression of LC3 was significantly decreased in the HR+Na HS pretreatment group（P<0.05）.（5）Compared with the Control group,the ROS content was significantly increased in the HR group（P<0.05）;compared with the HR group,the content of ROS in HK-2 cells was reduced in the HR+50u M Na HS pretreatment group and significantly decreased in the HR+100u M Na HS pretreatment group（P<0.05）,which indicated that 100u M was the optimal concentration for the effect of Na HS（6）Compared with the Control group,MMP was significantly lower in the HR group（P<0.05）;Compared with the HR group,MMP was significantly higher in the HR+100u M Na HS pretreatment group（P<0.05）.（7）Mitochondria in the Control group showed normal strip or linear shape;mitochondria in the HR group of HK-2 cells were significantly altered in morphology,showing dotted or fragmented shape;mitochondria in the HR+100u M Na HS pretreatment group restored their normal morphology.Conclusion:（1）Hydrogen sulfide pretreatment attenuates renal ischemia-reperfusion injury.（2）Hydrogen sulfide may protect against ischemic renal injury by regulating mitochondrial fusion.