The Protection Effect And Mechanism Of MFG-E8 In Sepsis Induced Acute Kidney Injury

Background:Sepsis is a systemic inflammatory response syndrome(SIRS)caused by various infectious,which is one of the most common diseases in the intensive care unit(ICU).Sepsis often causes the patient to be in a state of shock and multiple organ failure Kidney is one of the target organs for damage in sepsis.Sepsis is closely related to kidney injury.About 30%-50%of patients with sepsis in the ICU have acute kidney injury(AKI).More than half of patients with new onset of acute kidney injury are also due to sepsis.Despite the continuous development of early diagnosis and treatment techniques for sepsis-induced acute kidney injury(SAKI)in recent years,there are still no effective means to prevent and treat SAKI.SAKI will further increase the mortality rate of patients,and domestic and foreign studies have shown that the mortality rate is maintained at 50-70%.Acute kidney injury causes a huge economic burden.The average cost of AKI in China is 5071 USD,while in the USA patients with acute kidney injury have an additional hospitalization cost of 7933 USD per patient,requiring an additional 4077 USD per patient for dialysis patientsThe high mortality of SAKI is closely related to the unclear pathogenesis.The pathogenesis of SAKI is complex,and a series of pathological processes are involved in the the state of SAKI,including renal hemodynamic disorders,the death of endothelial and epithelial cells,thrombosis of the renal tubules,and excessive immune and inflammatory reactions.Previously,the main pathogenesis of SAKI was considered as renal tubular epithelial cell necrosis caused by shock and renal hypoperfusion.However,the current study found that the degree of renal tubular necrosis was not parallel with the degree of renal function damage.Renal tissue biopsy in patients with acute kidney injury did not reveal significant tubular necrosis,suggesting that other types of cell death are involved in the development of acute kidney injury.In addition to ischemia,renal cell apoptosis triggered by factors such as exogenous toxins and endogenous cytokines may be involved in the pathophysiological process of acute kidney injury.It is speculated that renal cell apoptosis may play an important role in SAKIThe pathogenesis of SAKI are complex.A series of cytokines are involved in the pathogenesis of SAKI.At present,the commonly used indicators for the diagnosis of SAKI are serum creatinine and urine output.Bsides,some new biomarkers including NGAL,KIM-1,IL-18,and cystatin c have also been used in the early diagnosis of SAKIMilk fat globule-EGF factor 8(MFG-E8)is a lipophilic glycoprotein isolated from mammalian breast tissue and is a major component of mammalian milk fat globule membrane.MFG-E8 is an important bridge molecule in the process of apoptotic cell clearance,which can promote the phagocytosis of apoptotic cells by macrophages Evidence suggests that MFG-E8 plays an important role in promoting angiogenesis,maintaining intestinal epithelial cell integrity,and regulating the inflammatory response As the important role in apoptotic cell clearance,MFG-E8 are involved in the occurrence and development of lung injury,liver injury,intestinal inflammatory disease,Alzheimer's disease,dementia,cerebral ischemia-reperfusion injury,and other diseasesMFG-E8 also plays an important role in the pathogenesis of sepsis.After 20 hours in sepsis model,the expression MFG-E8 was decreased(decreased by 48%in spleen cell expression,decreased by 70%in hepatocyte,decreased by 45%in plasma).Similar results were observed in the LPS-induced sepsis model,where LPS treatment of macrophages decreased MFG-E8 protein expression.MFG-E8 mRNA expression was dose-dependently reduced in the spleen with LPS treatment.This indicates that the expression of MFG-E8 is decreased in sepsis.However,the research on the role of MFG-E8 in SAKI is limitedThis study focused on the role of MFG-E8 in SAKI and its mechanism,through the construction of cecal ligation and puncture(CLP)to perform SAKI model.Using immunohistochemistry,TUNEL,RT-PCR,western blot and other cell molecular biology techniques to observe renal injury markers,histopathology changes and the expression of MFG-E8 in SAKI.To determine whether MFG-E8 protects against SAKI and whether MFG-E8 is possible down-regulating the activation of NF-?B pathway,in order to achieve the purpose to find the mechanism of its renal function protection effects for SAKIPart ? Construction of a mouse model of SAKI and expression of MFG-E8Objective:To establish a model of acute kidney injury in mice sepsis,and to detect the expression of MFG-E8 in mice with SAKI by RT-PCR,western blot and other molecular biological methods for further interventionMaterials and method:Sixty-eight male C57BL/6 mice were randomly divided into 3 groups.Control group:16 mice.Sham group:20 mice did not receive procedure of cecal ligation and puncture.Cecal puncture ligation group(CLP group):32 mice.Follow the method of Daniel Rittirsch for cecal ligation and puncture.The ligation position is on half the length of the cecum.Two holes were punctured with 21G needle.The levels of serum creatinine and blood urea nitrogen were measured at the time of Oh,6h,12h and 24h after CLP.Urine were collected for measurement of NGAL,KIM-1.The expression of MFG-E8 was detected by RT-PCR and western blot at the time of Oh,6h,12h and 24h in the CLP group.The remaining 6 mice in each group were observed for the changes in biological behavior and 7-day survival analysis.The expression of MFG-E8 in renal tissues was determined by RT-PCR and western blot.Survival analysis was performed for the remaining 6 mice in each group.Statistical analysis of the measurement data was expressed as mean ± standard deviation(X ± S).Comparison between groups was performed using one-way analysis of variance,Kaplan-Meier method was used to calculate cumulative survival rate and plot survival curves,p<0.05 was considered to be statistically significant.Results:1.Comparison of renal function in each groupCompared with the control group,there was no significant difference in serum creatinine between the sham operation groups at same time points(p>0.05).Serum creatinine increased progressively after procedure in the CLP group.Serum creatinine increased at 6 hours after surgery,and the difference was statistically significant(p<0.01).Serum creatinine in the CLP group was significantly different from Oh after ligation and perforation(p<0.01).Creatinine was more than 1.5 times higher than baseline.The mice in the CLP group could be diagnosed as acute kidney injury.There was no significant difference in serum urea nitrogen between the sham operation group and the control group at each time point(p>0.05).The serum urea nitrogen level of the mice increased at 6 h after CLP and increased continuously at 12 h and 24 h.The difference was statistically significant compared with the sham operation group(p<0.01).2.Comparison of urine NGAL and KIM-1There was no significant difference in urinary NAGL and KIM-1 levels between the sham group and the control group(p>0.05).Urinary NAGL and KIM-1 were elevated at 6 hours after operation in the CLP group,which was statistically significant compared with the sham operation group(p<0.01).3.Comparison of renal pathology and pathological scores in miceNo obvious abnormalities were found in the renal histopathology of the sham group and the control group.The kidney tissue structure is normal and clear.Kidney injury in the CLP group was marked by renal tubular lesions.It is characterized by tubular epithelial cell swelling and vacuolar degeneration.Renal pathological scores were significantly increased in the CLP group.4.Comparison of biological behavior and survival rate of mice in each groupSham group of mice recovered faster from anesthesia than that in the CLP group.The mice in the sham group were flexible and free to eat.The mice in the CLP group had delayed recovery and showed vertical hair,chills,shortness of breath,and burnout.The mice in the sham group had no death for 4 days and 7 days,and the survival rate was 100%.The 4-day and 7-day survival rates of the mice in the CLP group were 66.6%and 33.3%respectively5.MFG-E8 expressionThere was no significant difference in the expression of MFG-E8 mRNA and protein between the sham group and the control group.Compared with the control group,the expression of MFG-E8 mRNA and protein in the CLP group decreased significantly at 24 hours after surgery(p<0.01)Conclusion:1.The serum creatinine and BUN levels in the CLP group were significantly increased,the levels of urinary NGAL and KIM-1 were significantly increased.The pathological changes of renal tissues were consistent with the acute renal injury of sepsis,and the renal pathological score was significantly increased.24 h after surgery can be used as the time point for the subsequent study2.SAKI model can be stably replicated by controlling the surgical procedure,selecting appropriate C57BL/6 mice3.The expression of MFG-E8 was decreased in SAKI,which established a theoretical basis for further intervention of exogenous rmMFG-E8Part ? Study on the protective effect of recombinant mouse MFG-E8 on renal function and inflammation in mice with SAKIObjects:To construct a model of SAKI and study the effect of exogenous MFG-E8 on renal function in mice with SAKI and its effect on inflammatory markersMaterials and Methods:Male C57BL/6 mice(128)were randomly divided into 8 groups.Control group:16 mice,control group.Sham group:16 mice did not receive cecal ligation and perforation.CLP group:16 mice underwent cecal ligation and puncture.CLP+PBS group:16 mice receive cecal ligation and perforation with intraperitoneal injection PBS 0.1ml.CLP+rmMFG-E8(5?g/kg)group:16 mice with cecal ligation and perforation,intraperitoneal injection of 5?g/kg rmMFG-E8 CLP+rmMFG-E8(10?g/kg)group:16 mice with cecal ligation and puncture,10 ?g/kg of rmMFG-E8 was intraperitoneally injected.CLP+rmMFG-E8(20 ?g/kg)group:16 mice were treated with cecal ligation and perforation with 20 ?g/kg rmMFG-E8CLP+rmMFG-E8(40?g/kg)group:16 mice underwent cecal ligation and perforation 40?g/kg rmMFG-E8.According to the results of the previous study,the end point of modeling was selected as 24 h after CLP.10 mice in each group were taken,blood and kidney samples were taken for detection of serum creatinine,blood urea nitrogen Urinary NGAL and KIM-1 were detected.Kidney tissue was stained with PAS stain and pathological scores of kidney injury were performed.The remaining 6 mice in each group were subjected to survival analysis using the Kaplan-Meier method and log-rank test.p<0.05 was considered to be statistically significantResults:1.Compared with the control group,the renal pathological score of the mice with SAKI was significantly increased(p<0.01).Compared with the CLP group,the renal pathological scores of the CLP+rmMFG-E8 group were significant decreased(p<0.01)2.Compared with the sham group,the levels of IL-1,IL-6,and TNF-a in renal tissue of CLP group were significantly higher.The expression of IL-1,IL-6,and TNF-a in renal tissue of group were significantly reduced in CLP+rmMFG-E8 groups3.Compared with the sham group,the expression of IL-1,IL-6 and TNF-a mRNA in the renal tissue of the sepsis group was significantly higher.The expression of IL-1,IL-6 and TNF-a mRNA in renal tissue of MFG-E8 group was significantly decreased in CLP+rmMFG-E8 groups(p<0.01)4.Compared with the sepsis group,CLP+rmMFG-E8 could decrease the serum creatinine and BUN levels in the CLP group(p<0.01)5.Compared with the sepsis group,CLP+rmMFG-E8 could decrease the expression of NGAL and KIM-1 in SAKI group compared with the control group6.All the mice of the sham group and control group were survived.The survival rate of CLP mice was 33.33%.The survival rate of 5 ?/kg rmMFG-E8 mice was 33.33%;The survival rate of 10 ?g/kg rmMFG-E8 mice was 40%.The survival rate of 20 ?g/kg rmMFG-E8 mice was 66.7%.The survival rate of 40 ?g/kg rmMFG-E8 mice was 66.7%Conclusion:1.Exogenous MFG-E8 can improve the pathological score of acute kidney injury in sepsis and reduce the level of serum and urine kidney injury markers2.Exogenous MFG-E8 can down-regulate IL-1,IL-6,TNF-a and IL-1,IL-6,TNF-a mRNA levels in mouse kidney tissue,and reduce acute sepsis acute kidney injury3.Exogenous MFG-E8 can improve mortality in mice4.The dose of 20 ?g/kg and 40 ?g/kg rmMFG-E8 has similar protection effects on kidney injury.Further studies can be performed with rmMFG-E8 at a dose of 20 ?g/kgPart ? Mechanism of recombinant mouse MFG-E8 on renal protection in mice with SAKIObjective:To investigate the effect of rmMFG-E8 on renal cell apoptosis in SAKI.To investigate the effect of rmMFG-E8 on NF-?B signal transduction pathway in SAKIMaterials and methods:Same as the previous studyResults:1.The expression of the cleaved caspase-3 protein was detected by western blot Western blot analysis showed that cleaved caspase-3 expression in the CLP group was significantly higher than that in control group(p<0.05).The rmMFG-E8 intervention group decreased cleaved caspase-3 levels in kidney tissue2.Western blot was used to detect the expression of Bax protein in mice kidney Western blot analysis showed that the expression of Bax in CLP group was significantly higher than that in the control group(p<0.05),and the level of Bax in renal tissue was decreased by rmMFG-E8(p<0.05)3.Western blot was used to detect the expression of Bcl-2 protein in mouse kidney Western blot analysis showed that the expression of Bcl-2 in CLP group was significantly lower than that in the sham group(p<0.05),and the level of Bcl-2 in renal tissue was increased by rmMFG-E8(p<0.05)4.Compared with the control group and the sham group,the expression of phosphorylated p65 and I?B? in CLP group was significantly increased,and p65 in the nucleus was also significant increased,which indicating that NF-?B transduction passway was in the activated state.Compared with the CLP group,phosphorylated p65 and IKBa were decreased in the rmMFG-E8 group,and p65 decreased in the nucleus These results suggest that rmMFG-E8 deactivated of the NF-?B pathway,which alleviated acute kidney injury in sepsis.Conclusions:1.In SAKI the expression of cleaved caspase-3 and Bax protein in the kidney tissue were increased with more apoptotic kidney cell.Recombinant mouse lipodermal epidermal growth factor-8 can reduce the cleaved caspase-3 and Bax protein levels,reduce renal cell apoptosis,and reduce the degree of acute kidney injury in sepsis.2.NF-?B p65 was highly phosphorylated in the kidney tissues of septic mice,and the NF-?B signaling pathway was activated.Recombinant mouse milk globulin epidermal growth factor-8 can inhibit the activation of NF-?B,thereby reducing the degree of acute kidney injury in sepsis.It is suggested that MFG-E8 can ameliorate acute kidney injury in sepsis through NF-?B signal transduction pathway.