Effects Of MG53 And Gastrin On Relieving Acute Kidney Injury

Acute kidney injury(AKI)has become a public health problem worldwide.It has a n acute onset,high morbidity,no specific treatments,and has a high risk of death,while it can also progress to chronic kidney disease with poor prognosis,which brings us a heavy medical burden.In recent years,owing to the wide application of cardiovascular interventional diagnosis and treatment as well as extensive use of the iodine contrast agents,and the occurrence of trauma,shock and the surgery of kidney transplantation,contrast-induced AKI(CI-AKI)and ischemia/reperfusion(I/R)-induced AKI have become two common types of AKI.The body is an organic whole,we privously found that other organs such as skeletal muscle and gastrointestinal tract have a certain “dialogue” effect on kidney.When AKI occurs,what are the effects of skeletal muscle and gastrointestinal tract on damaged kidneys? Therefore,around this idea,we performed the following two major parts of the studies:(1)The effect of muscle factor Mitsugumin 53(MG53)on CI-AKI and the underlying mechanisms;(2)The role of gastrointestinal hormone gastrin on I/R-induced AKI and the underlying mechanisms.Part I The effect of MG53 on contrast-induced acute kidney injury and the underlying mechanismsBackgroundMG53 is a newly discovered membrane repair protein secreted by skeletal muscle.Through its membrane repair function,MG53 could alleviate the damage of various organs;CI-AKI is a common renal complication caused by the use of iodine contrast media(CM).It has been reported the toxic effect of iodine contrast agent on renal tubular cells may be caused by its direct damage to the cell membrane.Therefore,we hypothesized that MG53 could alleviate CI-AKI via membrane repair.ObjectiveThis study was designed to investigate the effect of MG53 on CI-AKI and the underlying mechanisms,and to provide possible new strategies for the clinical treatment and prevention of CI-AKI.Methods(1)The rat CI-AKI model was established,which was verified by detecting the levels of the serum creatinine(SCr)and the blood urea nitrogen(BUN).Besides,the changes of MG53 protein content in the plasma and the kidney of CI-AKI rats were detected by the Western Blot(WB).The alteration of the localization of MG53 in the kidneys of CI-AKI rats was accessed by immunohistochemistry(IHC),and whether exogenous fluorescent labeled recombinant human MG53(rh MG53)could accumulate in the kidneys of CI-AKI rats was examined by laser confocal microscopy.(2)Whether MG53 has the alleviating effects on CI-AKI was confirmed by detecting the levels of SCr and BUN,as well as the hematoxylin-eosin(H&E)staining of the rat kidney sections.(3)The effect of CI-AKI on rat renal tubular cell apoptosis and the role of rh MG53 pretreatment were explored by the TUNEL staining.(4)The in vitro cell model of iopromide-induced renal proximal tubular(RPT)cell injury was established,and the TUNEL staining was performed to verify the effect of iopromide on RPT cell apoptosis and to study whether rh MG53 pretreatment has a protective effect on it.(5)The cell counting kit-8(CCK-8)was used to detect the effect of rh MG53 on RPT cell injury induced by iopromide.(6)The lactate dehydrogenase(LDH)assay and FM1-43 fluorescent staining were performed to detect the effect of iopromide on RPT cell membrane and the effect of rh MG53 on it.(7)The immunofluorescence staining was used to detect the localization and distribution of MG53 in RPT cells.(8)The q PCR and WB were performed to detect the changes of MG53 m RNA and protein levels,respectively.(9)The immunofluorescence staining was used to check the co-localization of MG53 and Annexin V,a sensitive and specific probe to mark phosphatidylserine(PS)on the outer membrane of apoptotic cells.Results(1)The rat CI-AKI model was successfully established,and the MG53 protein in the plasma was decreased while that in the kidney was increased in CI-AKI rats,suggesting that MG53 was recruited from plasma to kidney.MG53 was expressed in rat renal tubular cells while gathered to the lumen side of tubules after CI-AKI,and the fluorescence-labeled exogenous rh MG53 accumulated in the kidney of CI-AKI rats.(2)The levels of SCr and BUN,as well as the H&E staining showed that rh MG53 pretreatment alleviated the renal function deterioration and renal pathological damage of CI-AKI.(3)The renal tubular cell apoptosis was significantly increased in CI-AKI rats,which was relieved after pretreatment with rh MG53.(4)Iopromide caused apoptosis of cultured RPT cells,which was alleviated by rh MG53.(5)Iopromide caused RPT cell injury in concentration-and time-dependent manners,which was ameliorated by rh MG53.(6)Iopromide caused RPT cell membrane damage,while it was relieved by rh MG53 in a concentration-dependent manner.(7)MG53 was distributed in the RPT cell membrane and cytoplasm,while it was accumulated in the cell membrane after iopromide treatment.(8)MG53 m RNA and protein did not change significantly after iopromide treatment,while large quantities of rh MG53 proteins was uptake by RPT cells.(9)The immunofluorescence staining revealed the co-localization of MG53 and Annexin V,suggesting MG53 exerts its membrane repair effect by combining with phosphatidylserine.ConclusionMuscle factor MG53 protects against CI-AKI through cell membrane repair and reducing cell apoptosis,and rh MG53 might be a potential effective means to treat or prevent CI-AKI.Part II The role of gastrin on ischemia/reperfusion-induced acute kidney injury and the underlying mechanismsBackgroundIschemia/reperfusion(I/R)is another major cause of acute kidney injury(AKI).There is a close regulatory connection between the gastrointestinal tract and kidney,“gastro-renal axis” is a novel concept proposed recently.Gastrin,a gastrointestinal hormone,has been reported to participate in the regulation of renal function like sodium excretion,thereby further participating in the blood pressure regulation.However,whether gastrin also has an effect on renal I/R injury remains unclear.Therefore,we hypothesized that gastrin might play a protective role in renal I/R injury.ObjectiveThis study was designed to explore the effect of gastrin on renal I/R injury and its underlying mechanisms,and to provide potential new ideas for clinical prevention and treatment of renal I/R injury.Methods(1)The mouse model of renal I/R injury was established,the expression and localization of gastrin receptor,also named cholecystokinin B receptor(CCKBR),were tested by q PCR,WB and IHC,respectively.(2)Whether gastrin pretreatment alleviates renal I/R injury was evaluated by SCr level,BUN level,H&E staining,periodic acid-Schiff(PAS)staining and the expression of kidney injury molecule-1(KIM-1).(3)Whether gastrin has a protective effect on I/R-induced tubular cell apoptosis was tested by TUNEL staining and caspase-3 enzyme activity assay,and the effect of gastrin on Akt phosphorylation was detected by WB.(4)The human kidney-2(HK-2)cell model of hypoxia/reoxygenation(H/R)injury was established in vitro,and the effect of gastrin on the viability of HK-2 cells was detected by CCK-8 and LDH assay.(5)The alteration in the expression of CCKBR in HK-2 cells after H/R treatment in vitro was assayed by WB.(6)The effect of gastrin on the apoptosis of H/R-treated HK-2 cells in vitro was analyzed by TUNEL staining and caspase-3 enzyme activity assay.(7)The effect of gastrin on the oxidative stress in H/R-treated HK-2 cells in vitro was tested by dihydroethidium(DHE)staining,superoxide dismutase(SOD)assay and malondialdehyde(MDA)assay.(8)The effect of gastrin on the phosphorylation of PI3 K,Akt and Bad in H/R-treated HK-2 cells was examined by WB.(9)Whether wortmannin,a specific PI3 K inhibitor,and Akt inhibitor VIII could block the gastrin-mediated protective effect of HK-2 cells was detected by CCK-8 assay.Results(1)The m RNA and protein levels of CCKBR were significantly up-regulated in I/R-injuried mouse kidneys,and CCKBR was distributed in the tubular cell membrane and cytoplasm.(2)The pre-administration of gastrin alleviated the increment of SCr and BUN caused by I/R,ameliorated the pathological damage of renal tubules,and reduced the elevation of KIM-1 expression.(3)The I/R injury induced renal tubular cell apoptosis,which was alleviated by gastrin pretreatment,and gastrin pretreatment also promoted the phosphorylation of Akt.(4)Gastrin had no significant effect on HK-2 cell viability under normoxia,while it alleviated the HK-2 cell damage caused by H/R in a concentration-dependent manner,and the protective effect could be blocked by CI-988,a specific CCKBR antagonist.(5)The CCKBR expression was significantly up-regulated in HK-2 cells after H/R treatment.(6)Gastrin attanuated H/R-induced apoptosis and ROS increment in HK-2 cells.(7)Gastrin increased the phosphorylation of PI3 K,Akt and Bad in H/R-treated HK-2 cells.(8)Wortmannin,the specific PI3 K inhibitor,and Akt inhibitor VIII blocked the protective effect of gastrin on H/R-treated HK-2 cell viability.ConclusionGastrointestinal hormone gastrin could ameliorate the I/R-induced renal tubular cell apoptosis through the PI3K/Akt/Bad pathway,thereby alleviating I/R-induced AKI.Gastrin might become a potential effective drug for the clinical prevention or treatment of AKI.