Protective Role Of Endothelial Calpain Knockout To Lipopolysaccharide Induced Acute Kidney Injury

Background:Sepsis is a clinical syndrome that has defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.However,no precise and effective treatment has been found.As a result,sepsis remains one of the leading causes of death in critically ill patients.Acute kidney injury(AKI)is one of the major components of multi-organ dysfunction syndrome.AKI caused by sepsis accounts for nearly half of all AKI events,which is associated with worse outcomes.Vascular endothelial cells play a crucial role in maintaining homeostasis and integrity of the endothelial barrier in kidney.During the process of sepsis,endothelial cells expose to variety of stimuli,which makes endothelium undergoing structural changes and resulting in the increased permeability of endothelium.Endothelial dysfunction leads to the leakage of large endogenous molecules and leukocyte transmigration to the renal interstitium,which damages to renal tubular cells.Even though endothelial dysfunction has been found playing a central role in sepsis-associated AKI(SA-AKI),the targeted preventive and therapeutic measures are still lacking for its unclear pathological mechanisms.Calpains are calcium-dependent cysteine proteases,which are significant in a wide range of cellular calcium-regulated functions.Calpain activation has been shown to induce hepatic inducible nitric oxide synthase(iNOS)during lipopolysaccharide(LPS)stimulation and be correlated with an increase in reactive oxygen species(ROS)production peroxynitrite formation.Endothelial cells are sensitive to vasoactive substances such as ROS and Nitric oxide(NO),thereby regulating the microcirculation and glomerular filtration.But what doubts us is that whether endothelial calpain activity is associated with the SA-AKI and whether endothelial calpain-targeted treatment can be a therapeutic method.Methods:1?We established sepsis animal models on the white type(WT)mice,the endothelial specific Capn4 knockout(TEK/Capn4-/-)mice,the over-expression of calpastatin(Tg-CAST)mice and the lymphoage Capn4 specific knock-out(LYZ/Capn4-/-)mice and in vitro cell models using pulmonary microvascular endothelial cells(PMECs).2?Renal function,cellular apoptosis,renal inducible nitric oxide synthase(iNOS)and endothelial NOS(eNOS)expression,plasma and renal levels of NO and reactive oxygen species(ROS),mitogen-activated protein kinase(MAPK)family members(p38,ERK1/2,JNK1/2)phosphorylation were examined.3?A calpain inhibitor,calpastatin overexpression adenoviruses and inhibitors of MAPKs were used and caspase-3,iNOS,eNOS,NO and ROS expression and MAPK family members phosphorylation were examined.Results:1?significant renal dysfunction was induced by LPS stimulation,and part of recovery was seen in TEK/Capn4-/-and Tg-CAST mice but not in LYZ/Capn4-/-mice.2?Endothelial Capn4 knockout decreased the LPS-induced increments in renal iNOS expression,NO and ROS levels in the plasma and kidney,caspase-3 activity and apoptosis,but did not obviously affect renal eNOS expression.3?LPS increased both calpain and caspase-3 activity and the expression of only iNOS without eNOS in PMECs,accompanied by increasing phosphorylation of p38 and JNK.4?Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression,NO/ROS production and cellular apoptosis induced by LPS.Conclusion:These results suggested that endothelial calpain knockout may play a protective role in LPS-induced AKI by inhibiting p38 phosphorylation,thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis.