The Role And Machanism Of DGKE On Podocyte Injury In Experimental Glomerular Diseases

Glomerular diseases are a group of renal diseases characterized by bilateral glomerulus damage,which are common causes of chronic renal failure.Although the etiologies,clinical classifications,pathological types,treatments and prognosises may be different,the emergence of large amounts of proteinuria is the common remarkable cilincal features of glomerular diseases.Podocytes are specialized cells with a complex cellular morphology that reside in the kidney glomerulus and cover the outer surface of the filtering capillaries.Podocyte and glomerular endothelial cells,together with glomerular basement membrane(GBM)form the glomerular filtration barrier,which acts as the important maintenance of glomerular homeostasis.Podocyte injury is now considered as a hallmark of glomerular filtration barrier disruption,which may induce the emergence of proteinuria and result in the exacerbation of kidney disease.Therefore,searching for the key molecular that is associated with podcyte injury in glomerular diseases,and trying to find possible effective intervention methods are of great significance.Diacylglycerol kinases(DGKs)are important regulators of inositol phospholipids signal system,which regulate cellular activities and functions through phosphorylating diacylglycerol(DAG)to phosphatidic acid(PA)under physiological state.Considering of that both the substrate(DAG)and the product(PA)are important signaling molecules in the body,DGKs have important biological functions.Currently,ten DGK family members are identified in mammals,which belong to 5 subclasses.Except for the common catalytic domain and C1 domain,the molecular structures,tissue distributions as well as biological functions are different from each other.Among them,diacylglycerol kinase E(DGKE)is the only member of the III subclass,which possesses the simplest structure and the arachidic acid-diacylglycerol(AA-DAG)substrate specificity compared with the other family members.These characteristics suggest that DGKE may play a special role under physiological and pathological conditions.Recent studies found that recessive mutations in DGKE were related to some kidney diseases,such as atypical hemolytic uremic syndrome(aHUS)and glomerular microangiopathy,which suggested us that DGKE had become an important target for exploring the pathophysiological mechanism of proteinuria related kidney diseases.But current studies focus mainly on the gene variants in clinic,the related systematic experimental research works are very limited.ObjectiveIn this study,we will use podocyte specific knockin mice and cultured human podocytes to investigate the role of DGKE on podocyte injury in experimental glomerular diseasess and furtherly explore the molecular biological mechanisms.1.To detect the expression of DGKE on podocyte injury in glomerular diseases.2.To confirm the role of DGKE on podocyte injury in glomerular diseases.3.To explore the machanism of DGKE on podocyte injury in glomerular diseases.Methods and Results1.The expression of DGKE on podocyte injury in glomerular diseasesWild type C57BL/6J mice aged 8-12 weeks were randomly divided into normal control group and glomerular disease model group after a week of adaptive feeding under laboratory conditions.After anaesthesia,glomerular disease animal models were established by intravenous injection of nephrotoxic drugs puromycin aminonucleoside(PAN,360mg/kg)or adriamycin(ADR,15mg/kg)respectively.A successful glomerular disease model was confirmed by morphology and electron microscopy detection.Real-time PCR method was used to measure the mRNA level and Western blot(WB)and immunohistochemistry(IHC)methods were used for the detection of protein levels of DGKE in the kidney cortex from different groups.Double immunofluorescent labeling method was used for detecting the cellular location of DGKE.The results showed that compared with control group,the expression of DGKE in the renal cortex was significantly decreased in glomerular disease model animals.The immunofluorescent results identified the podocyte location of DGKE in glomerulus.Human podocytes(HPC)were cultured and treated by PAN and ADR respectively,similar results were obtained in vitro studies as in vivo studies.2.The role of DGKE on podocyte injury in glomerular diseasesDGKEflox/flox mice were established and crossbred with Podocin-Cre mice to obtain conditional podocyte DGKE knockin later generations(gene type:Podocin-Cre/DGKEflox/flox),the animals with gene type as Podocin-Cre-/DGKEflox/flox were used as controls.Experimental glomerular disease models were established by intravenous injection of PAN or ADR.Kidney paraffin slides staining with PAS and transmission electron microscope(TEM)was employed to observe the morphology of glomerulus and the ultrastructure of podocytes.The results of PAS staining showed that podocyte conditional knockin of DGKE significantly alleviated glomerular mesangial matrix proliferation and glomerulosclerosis.The ultrastructure of podocytes indicated a lighter injury of podocyte charactered by improvement of foot process fusion and effacement.Labelled by immunofluorescent method,it was found that compared with control group,podocyte conditional knockin of DGKE partly recovered expression of podocyte structural proteins Nephrin and Podocin induced by PAN and ADR respectively.Similar results were obtained in vitro studies.These results suggest that DGKE plays a protective role on podocyte injury in glomerular diseases.3.The mechanism by which DGKE in the protection of podocyte injuryWe found that compared with normal control group,the protein and mRNA levels of TRPC6 and concentration of intracellular calcium in model group were significantly increased,while podocyte conditional knockin of DGKE markedly suppressed the increased expression of TRPC6 and concentration of intracellular calcium by real-time PCR,Western blot and flow cytometry methods on podocyte injury in experimental glomerular diseases.Our current studies indicate that the role of DGKE on podocyte injury in glomerular diseases may be associated with the downregulation of TRPC6 and concentration of intracellular calcium,but the exact mechanism is need further research.Considering the kinase activity of DGKE,we also detected the phosphorylated proteins in kidney cortex from podocyte conditional DGKE knockin mice.The protein mass spectrometry analysis results showed a DGKE protein associated serine phosphorylation of coil-coil domain containing protein 37(CCDC37)in podocyte conditional DGKE knockin mice.We furtherly confirmed that DGKE and CCDC37 interact with each other by using immunoprecipitation technology(Co-IP)method.Since the biological function of CCDC37 remains unclear now,whether the protective effect of DGKE on podocyte injury is related to its phosphorylatory regulating CCDC37 or not also needs further study.Conclusion and innovationIn summary,this study firstly addressed a protective role of DGKE on podocyte injury in experimental glomerular diseases by using conditional knockin animals together with cultured human podocytes,and to some extent,preliminarily investigated the possible mechanisms.This study will suggest a new pathogenic molecular contributing to podocyte injury in glomerular diseases as well as other kidney diseases,which may provide effective treatment for proteinuric kidney diseases.