The Role Of Junctional Adhesion Molecule-Like Protein In Acute Kidney Injury

Objective and SignificanceAcute kidney injury(AKI)is a recognized critical clinical disease with high mortality rate.Nephrotoxic drugs,kidney hypoperfusion,sepsis and infection are easy to cause acute kidney injury.Immune inflammatory response plays a very important role in acute kidney injury.When renal tubular epithelial cells are stimulated by antigen,they will show immunocompetence just like inflammatory cells.On the one hand they produce more inflammatory mediators by autocrine or paracrine,on the other hand they will express major histocompatibility complex II(MHC II)class molecules,pattern recognition receptors(PRRs),etc.Peripheral microvascular endothelial cell permeability increased,the expression of adhesion molecules,and promote the adhesion and migration of inflammatory cells.In addition,a variety of immune inflammatory cells in the damaged area of infiltration,and further activation of the release of a large number of inflammatory mediators,leading to inflammatory reaction amplification and further damage of renal tubular epithelial cells.The three major effector cells in the kidney tissue interact with each other and participate in the local innate immunity and adaptive immune dysfunction of the kidneys,which leads to the persistent inflammation of the kidneys and the main mechanism of acute renal injury.The role of immune inflammatory response in acute kidney injury is very important.In recent years,the role of junctional adhesion molecule-like protein(JAML,amical)in immune cell activation and inflammatory response has been paid more and more attention.JAML is a novel connective adhesion molecule that belongs to the secreted type I transmembrane glycoprotein.Its structural features make it not only mediate intercellular interactions,but also bind to intracellular proteins to mediate downstream signaling pathways.So far,whether JAML plays a key role in acute kidney injury has not been reported.Therefore,it is essential to study the role of JAML in AKI.This topic will explore how JAML participate in the immune regulation of the kidney area.In order to clarify the immune mechanism of the kidney,to provide some reference,we hope that it will help with finding a new immunotherapy target.Methods1.The expression of JAML in mice model of renal ischemia/reperfusion injury1.1 Establishment of mice model of renal ischemia/reperfusion in vivo and detection of JAMLThe acute renal ischemia/reperfusion model was constructed by wild type(WT)C57BL/6.Western blotting(WB),real-time quantitative reverse transcription polymerase chain reaction(Real-time RT-PCR)and immunohistochemistry(IHC)were used to detect the expression of JAML in renal ischemia-reperfusion tissue.In addition,intraperitoneal injection of cisplatin was used to construct acute renal injury model,and the expression of JAML was detected by IHC.1.2 Co-localization of JAML with segment-specific tubular markersImmunofluorescence(IF)was used to co-target JAML with different parts of renal tubular marker protein.1.3 Establishing in vitro model and detection of JAMLCultured human kidney proximal tubular epithelial cells(HK-2),then simulated renal Ischemia reperfusion models in three ways:oxygen glucose deprivation(OGD);administration of anti-mite AD/2-deoxyglucose(AA-2DG);administration of cobalt chloride(CoCl2).WB was used to detect the protein level of JAML in HK-2 cells under three conditions.Besides,After reoxygenation of HK-2 cells which was treated with hypoxia,then use the supernatant to treated with macrophages.WB was used to detect the protein level of JAML in macrophages.2.The role of JAML in mice model of renal ischemia/reperfusion2.1 Assessment of renal function in WT and JAML-/-miceSeram creatinine was measured by an Agilent 1100 HPLC system.Blood urea nitrogen was measured by Cobas 8000 modular analyzer.2.2 Comparison of morphological injury between wild type and JAML-/-miceHematoxylin-eosin staining(HE)was used to stain the paraffin sections of wild type and JAML-/-mice,and the morphological damage was scored.Apoptosis renal cells were detected in wild type and JAML-/-mice by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL).2.3 Detection of inflammation related indexes in renal ischemia-reperfusion model of miceReal-time RT-PCR was used to detect inflammation factors in wild type and JAML-/-mice.Immunohistochemistry(IHC)was used to detect macrophage marker protein CD68,neutrophil marker protein Ly6B.3.Effects of JAML on downstream molecule Clec4e in renal ischemia reperfusion injuryDifferential genes in wild type and JAML-/-mice were analyzed by gene chip technique,and the molecular thermal map of the pattern recognition receptor family was drawn.Real-time RT-PCR was used to further validate the selected genes.RT-PCR was used to detect the expression of Clec4e in macrophages and HK-2 cells.Furthermore,the changes of Clec4e were observed by AA/2-DG stimulation on HK-2 cells.Results1.The expression of JAML in mice model of renal ischemia/reperfusion1.1 JAML was significantly increased in the kidney from mice model of renal ischemia/reperfusionReal-time RT-PCR and WB were used to detect JAML expression in mice kidney.The results showed that the mRNA level and protein level of JAML were significantly higher than those in sham operation group.These results were also confirmed in IHC.1.2 JAML was mainly expressed in the renal cortex and medullary proximal tubulesIF detection found that JAML was mainly expressed in the renal proximal tubules,it was also expressed in outer medulla distal convoluted tubules,the expression in the medullary collecting duct is weaker.1.3 The protein level of JAML was significantly increased under in vitro modelHK-2 cells were treated with three methods OGD,AA-2DG,CoCl2.The protein level of JAML was significantly increased under these three simulated conditions.The protein of JAML in macrophages was also increased after macrophage culture with HK-2 cells supernatant treated with hypoxia.2.JAML promotes injury in renal ischemia-reperfusion modelThe levels of creatinine and urea nitrogen in serum of JAML-/-mice were lower than those in wild type mice.The levels of pro-inflammatory mediators in renal homogenate were decreased and the numbers of neutrophils and macrophages decreased,the apoptotic cells were also decreased,indicating that JAML deficiency reduces renal ischemia-reperfusion injury.3.JAML regulates Clec4e involved in in vitro ischemia reperfusion renal injuryExpression of Clec4e and its family members was significantly up-regulated after ischemia-reperfusion injury by gene chip technique,and JAML-/-could significantly restore the expression of these genes induced by injury.RT-PCR showed that Clec4e had a low expression level in HK-2 cells compared with macrophages,and that of Clec4e was induced by AA/2-DG stimulation,indicating that Clec4e was involved in hypoxia/2 cell damage.Conclusions and Innovation1.Expression of JAML in renal ischemia-reperfusion injury was observed for the first time.The expression of JAML in renal ischemia-reperfusion injury was significantly increased.2.It was the first time that JAML promoted the development of renal ischemia-reperfusion injury,the down-regulation of JAML could inhibit the development of renal ischemia-reperfusion injury and alleviate the inflammatory response.3.The mechanism of JAML promoting the occurrence and development of renal ischemia-reperfusion injury was preliminarily discussed,and it was proved that it was positive regulation of downstream model recognition receptor Clec4e.In summary,on the one hand this subject prevent acute kidney injury to provide potential drug targets,on the other hand,JAML will be possible as acute renal injury occurrence and development of an important indicator.