| Podocytes,highly differentiated glomerular epithelial cells,are essential for the maintenance of glomerular filtration barrier.Podocyte injury is considered to be an important factor in the development of glomerular diseases,including diabetic kidney disease(DKD),focal segmental glomerulosclerosis(FSGS),membranous nephropathy(MN)and minimal change disease(MCD).Although some progress has been made in the understanding of pathophysiological mechanisms of podocyte injury,there is still a lack of podocyte-specific and effective therapies.Current studies have suggested that lipid dysfunction in podocytes is a major pathogenesis of glomerular disease and has become a research hotspot in this field.JAML is a new identified member of the junctional adhesion molecule(JAM)family which are type I transmembrane proteins differentially expressed at the junctions of endothelial cells,epithelial cells,and on various leukocytes.JAML has been implicated in mediating the transmigration of leukocytes,the release of inflammatory factors and growth factors of ??T cells,and play an important role in immune regulation,inflammatory response and damage repair.Our studies discovered a previously unknown function of JAML for the regulation of lipid metabolism and found that JAML contributes to podocyte injury in DKD.Importantly,we observed that the level of JAML expression was significantly increased in renal biopsies from patients with diabetic kidney disease and other glomerular diseases,which was correlated with lipid accumulation and glomerular filtration rate individually.We further found that podocyte-specific JAML knockout mice significantly ameliorated podocyte injury and proteinuria under diabetic conditions and adriamycin nephropathy.Mechanistically,JAML regulated podocyte lipid metabolism through Sirtl/AMPK-mediated SREBP1 signaling.Collectively,our studies suggested that JAML may represent an attractive therapeutic target for glomerular diseases.Objective1.Detect the expression patterns of JAML in glomerular diseases such as diabetic kidney disease.2.Explore the role of JAML in podocytes injury and lipid metabolism disorder under diabetic kidney disease,and provide a new target for the prevention and treatment of diabetic kidney disease.3.Clarify the mechanisms of JAML in podocytes lipid metabolism disorder,provide some theoretical basis for elucidating the mechanism of podocyte injury in glomerular diseases.Methods1 The expression patterns of JAML in diabetic kidney disease1.1 The expression levels of JAML in serum and renal biopsy samples of proteinuria patientsThe levels of JAML in serum of patients with glomerular diseases and normal subject were detected by ELISA.We firstly confirmed the levels of JAML in renal biopsies from DKD,FSGS,MN and MCD by immunohistochemistry(IHC)staining.Then,the correlations between JAML and estimated GFR(glomerular filtration rate),proteinuria or serum creatinine in all subjects were also analyzed.1.2 Detected the expression patterns of JAML in the kidney from diabetic mice The levels of JAML in serum of diabetic mice was detected by ELISA.In the renal cortex,the expression of JAML was detected by RT-qPCR,Western blot and immunohistochemistry.Moreover,the changes of JAML in podocytes were analyzed by double immunofluorescence staining for JAML and podocytes(synaptopodin)in samples and observed by a confocal microscope.1.3 In vitro,the expression levels of JAML were detected in podocytes with different stimulus treatmentConsidering that hyperglycemia and hyperlipidemia are the most prominent risk factors in diabetes,we applied high glucose(HG),palmitic acid(PA),cholesterol,advanced glycation end-products(AGEs),oxidized LDL(oxLDL)and serum obtained from diabetic patients to stimulate podocytes.Western blotting was used to confirm the changes of JAML protein levels in glomerular podocytes,tubular epithelial cells,glomerular endothelial cells and mesangial cells stimulated by high glucose(HG)and palmitic acid(PA).1.4 The expression patterns of JAML in ADR miceIn addition,we also constructed other podocyte-related disease mouse models,such as focal segmental glomerulosclerosis(FSGS)induced by adriamycin(ADR).ADR(12 mg/kg)was injected into the tail vein of mice,we detected the expression patterns of JAML by Western blot and immunohistochemistry after ADR injection for 5 and 10 weeks.Furthermore,ADR(0.15?g/ml)stimulated podocytes for 24h,Western blot and RT-qPCR were used to detect JAML levels.2 The role of JAML in podocytes under diabetic condition2.1 Conditional knockout mice in which JAML specifically ablated in podocytes by using Cre-LoxP recombination systemTo better study the role of JAML in podocytes,we used JAML(Amical)flox mice(constructed by Shanghai Southern Model Biotechnology Development Co.,Ltd.)to hybridize with podocyte-Cre mice(donated by the partner Dr Ningjun Li research group)to obtain podocyte-specific JAML knockout mice(Cre+/JAMLfl/fl),which was identified by tail genotyping,immunofluorescent analysis and the protein level of JAML in isolated glomeruli from Cre+/Jamlfl/fl mice2.2 The role of JAML in podocytes injury under diabetic kidney disease condition2.2.1 Mouse model podocyte-specific knockout of JAML ameliorates podocyte injury and proteinuriaThe body weight,kidney weight/body weight,blood glucose,triglyceride,cholesterol,HDL-C(high density liptein cholesterol),heart rate and blood pressure of STZ/HFD mice were measured.The urinary albumin/creatinine ratio,periodontal acid-schiff stain(PAS),transmission electron microscope(TEM)as well as immunofluorescence staining of the podocyte specific marker nephrin were used to detect glomeruli and podocyte injury.2.2.2 JAML gene silencing ameliorates podocyte damageThe relative mRNA level of interleukin-6(IL-6),intercellular cell adhesion molecule-1(ICAM-1),monocyte chemotactic protein 1(MCP-1)and tumor necrosis factor-?(TNF-?)in glomeruli of STZ/HFD mice were detected by RT-qPCR.In addition,JAML lentivirus transfection was used to silence JAML in podocytes.Proinflammation response was detected in podocytes under high glucose.Podocyte apoptosis was detected by flow cytometry.F-actin immunofluorescence staining and confocal imaging was used to observe the rearrangement of podocyte cytoskeleton.2.3 JAML knockout improves lipid disorders in podocytes of diabetic kidney disease2.3.1 Specific knockout of JAML improves glomerular lipid deposition in a mouse model of diabetic kidney diseaseNeutral lipid was assessed by Oil Red O staining and lipid droplets in podocytes were quantified according to the colocalization of adipophilin with synaptopodin by immunofluorescent staining.2.3.2 JAML silencing improves lipid deposition in podocytes induced by high glucoseThe lipid droplets in podocytes were stained with oil red O staining and Nile Red fluorescent antibody.By ultra-fast liquid chromatography tandem mass spectrometry(UFLC-MS),renal cortical lipidomics(including sphingolipids,phospholipids)were quantitative analyzed.2.3.3 Analysis of correlation between JAML and glomerular lipid deposition in renal biopsy samples of patients with proteinuria-related nephropathyIn addition,we detected the expression of lipid droplet marker Adipophilin in patients with glomerular diseases by immunohistochemistry,including diabetic kidney disease,focal segmental glomerulosclerosis,membranous nephropathy and minimal-change disease,and analyzed the correlation between the level of adipophilin and the expression of JAML in glomerulus.2.4 The role of JAML in adriamycin nephropathy2.4.1 JAML knockout alleviates kidney damage of adriamycin nephropathyThe urinary albumin/creatinine ratio in ADR mice was measured,and the morphological changes were detected by PAS staining and TEM.2.4.2 JAML silence alleviates the disorder of podocyte lipid metabolism in adriamycin nephropathyLipid accumulation in podocytes were assessed by Oil Red O staining under ADR treatment.3 The mechanism of JAML in the dysfunction of lipid metabolism in podocytes of diabetic kidney disease3.1 JAML regulates Sirtl expression in podocytesThe glomeruli were isolated to detect the protein level of sirtuin(Sirt1-7).In order to further study the regulatory effect of JAML on Sirtl,we performed double immunofluorescence staining to detect the co-localization of Sirtl and synaptopodin in the kidneys of STZ/HFD mice.In vitro,podocytes were treated high glucose(40 mmol/L)for 24 hours.Western blot was used to measure the protein level of SIRT1.Furthermore,the effect of JAML overexpression on SIRT1 protein level was also detected.3.2 JAML regulates the expression of phosphorylated AMPK through SirtlP-AMPK?(T172)/AMPK? protein level in mouse glomeruli were detected by Western blot to verify the regulatory effect of JAML on AMPK(Adenosine 5'-monophosphate(AMP)-activated protein kinase)activity.In vitro,JAML and Sirtl were silenced simultaneously in podocytes followed by high glucose(40 mmol/L)treatment for 24 hours.Western blot was used to detect the protein level of p-AMPK?.3.3 JAML-Sirtl signaling pathway regulates the expression of SREBP1 and downstream target genes3.3.1 Gene sequence to detect JAML downstream signaling pathwayBy RNA sequencing for global gene expression analysis,we observed the significant changes of genes that important for regulating cellular lipid homeostasis in HG-treated podocytes compared with their controls.3.3.2 JAML regulates SREBP1 and its downstream signaling pathwaysFurthermore,the results of microarray were verified by RT-qPCR.At the same time,we also detected the mRNA and protein level of SREBP1(sterol-regulatory element binding proteins 1)and SREBP2 in the glomerulus from diabetic kidney disease mice.Compared with the control group,the expression of mSREBP1,and downstream fatty acids,cholesterol synthesis protein FASN(fatty acid synthase),ACC1(acetyl-CoA carboxylase 1),SCD1(stearoyl-CoA desaturase 1)and SQLE(squalene epoxidase)were detected in podocytes.3.4 JAML-Sirtl-AMPK signaling pathway regulates SREBP1 expressionIn order to clarify the regulatory effect of JAML on SREBP1 expression though Sirtl and AMPK,we silenced JAML or AMPK to obseve the expression of mSREBP1 in JAML deficiency podocytes under high glucose stimulation.3.5 JAML regulates the expression of SREBP1 through the Sirtl signaling pathway in podocytes with adriamycin treatmentIn vitro,podocytes were treated by ADR(0.15 ?g/ml)for 24 hours after gene silencing of JAML.Western blot was used to detect the protein levels of SIRT1,phosphorylated AMPKa and mSREBP1.Results1 The expression patterns of JAML in diabetic kidney disease1.1 The expression levels of JAML in serum and renal biopsy samples of proteinuria patientsSerum levels of JAML were much higher in patients with DKD than healthy control subjects.The levels of JAML in glomeruli were significantly increased in subjects with DKD and positively correlated with different DKD classes by IHC staining.Notably,upregulation of JAML was further observed in renal biopsies from patients with other different forms of glomerular diseases such as FSGS,MN and MCD.The levels of JAML were positively correlated with serum creatinine,negatively correlated with eGFR in all subjects,but no correlation was found between JAML and proteinuria.1.2 Detected the expression patterns of JAML in the kidney from diabetic mice In the serum,the levels of JAML were markedly increased in the kidney from db/db mice,by Western blot and IHC analyses.Immunofluorescent results showed a significant increase of podocyte JAML expression in the kidney from db/db diabetic mice.Similarly,we also observed the elevation of JAML in serum and in the kidney from streptozocin plus high fat diet(STZ/HFD)-induced diabetic mice.1.3 In vitro,the expression levels of JAML were increased in podocytes with different stimulus treatmentWe applied high glucose,palmitic acid,cholesterol,advanced glycation end-products,and oxLDL.All of these stimuli that are closely associated with the etiology of DKD,significantly induced JAML expression in podocytes.Similar results were also found in podocytes treated with serum obtained from diabetic patients.However,other renal parenchymal cells including GECs,RMCs and HK-2 exhibited no obvious changes in JAML expression in response to hyperglycemia or hyperlipidemia,indicating that the upregulation of JAML may selectively contribute to podocyte injury under diabetic conditions.1.4 The expression patterns of JAML in ADR miceJAML expression was upregulated in the renal glomeruli of mice after ADR injection for 10 weeks.Consistently,in vitro,ADR increased the JAML expression in podocytes.2 The role of JAML in podocytes under diabetic condition2.1 Conditional knockout mice in which JAML specifically ablated in podocytes were generated by using Cre-LoxP recombination systemTo elucidate the role of JAML in podocytes in vivo,we generated conditional knockout mice in which Jaml is specifically ablated in podocytes by using Cre-LoxP recombination system.Podocin-Cre mice were crossed with Jamlfl/fl mice to generate Podocin-Cre Jamlfl/fl mice(Cre+/Jamlfl/fl mice),which was identified by tail genotyping,immunofluorescent analysis in podocytes and a significant reduction in the protein level of JAML in isolated glomeruli from Cre+/Jamlfl/fl mice.All mice were viable and fertile.2.2 The role of JAML in podocytes injury under diabetic kidney disease condition2.2.1 Mouse model podocyte-specific knockout of JAML ameliorates podocyte injury and proteinuriaBy using conditional podocytes knockout mice,we generated STZ/HFD-induced DKD model.STZ/HFD-induced diabetic mice had hyperglycemia and lower body weight compared with their non-diabetic counterparts,no difference in blood pressure among these groups.Diabetic Cre+/Jamlfl/fl mice exhibited a significant decrease in urinary albumin excretion as compared with diabetic Cre+/Jaml+/+mice.Morphological examinations showed the glomerular and podocyte injuries as evidenced by glomerular basement membrane(GBM)thickening,podocyte foot process broadening and effacement from diabetic Cre+/Jaml+/+ mice,all of which were alleviated in diabetic Cre+/Jamlfl/fl mice.The loss of key podocyte differentiation markers including Nephrin and Podocin was partially recovered in diabetic Cre+/Jamlfl/fl mice.Moreover,db/db diabetic mice(20-week old)had hyperglycemia and higher body weight compared with their control,no difference in blood pressure among these groups.Up to 8-week after birth,these mice were indistinguishable from control littermates as analyzed by renal histology,albuminuria,and glomerular ultrastructure.In a 12-week follow-up,compared with their controls,db/db//Cre+/Jaml+/+ mice developed albuminuria,mesangial matrix expansion and podocyte injury,which were alleviated either in db/db//Cre+/Jamlfl/+or db/db//Cre+/Jamlfl/fl mice.2.2.2 JAML gene silencing ameliorates podocyte damageCre+/Jamlfl/fl diabetic mice alleviated inflammatory responses in isolated glomeruli.Consistently,in vitro,except that gene silencing of JAML reduced the levels of apoptosis and proinflammatory mediators,and ameliorated actin cytoskeleton derangement in podocytes with HG treatment.2.3 JAML knockout improves lipid disorders in podocytes of diabetic kidney disease2.3.1 Specific knockout of JAML improves glomerular lipid deposition in a mouse model of diabetic kidney diseaseWe found that the podocyte-specific Jaml knockout mice in db/db diabetic model(db/db//Cre+/Jamlfl/fl),even heterozygous genotype(db/db//Cre+/Jamlfl/+)had less lipid droplets and neutral lipids deposition in glomeruli assessed by Oil Red O staining and in podocytes according to the colocalization of Adipophilin,a lipid droplet-specific marker,with Synaptopodin by immunofluorescent staining.The reduced lipid accumulation was also found in STZ/HFD-induced Cre+/Jamlfl/fl diabetic mice.We also found that JAML deficiency reduced lipid accumulation in podocytes under HG condition by using Oil Red O staining and Nile Red staining.2.3.2 JAML silencing improves lipid deposition in podocytes induced by high glucoseWe further used LC-MS based lipidomics analysis to quantify the different lipid species in podocytes under HG condition.Gene silencing of JAML reduced lipid accumulation such as the cholesteryl esters(CE),free fatty acids(FFA),ceramides,phosphatidylethanolamines(PE),phosphatidylcholines(PC)and sphingomyelins(SM)in HG-treated podocytes.2.3.3 JAML was associated with glomerular lipid deposition in renal biopsy specimens of patients with proteinuria associated nephropathyyFurthermore,given that DKD is associated with the presence of lipid deposits which were also described in other contexts including FSGS,MN and MCD,we further assessed the amount of lipid droplets in glomeruli from these patients.It was found that lipid deposits were accumulated in glomeruli and the degree of lipid accumulation was positively correlated with the level of JAML2.4 The role of JAML in adriamycin nephropathy2.4.1 JAML knockout alleviates kidney damage of adriamycin nephropathy Compared with the ADR-treated Cre+/Jaml+/+mice,ADR-treated Cre+/Jamlfl/fl mice exhibited significant decreased in urinary albumin excretion and glomerulosclerosis.2.4.2 JAML silence alleviates the disorder of podocyte lipid metabolism in adriamycin nephropathy Furthermore,JAML deficiency attenuated ADR-induced lipid accumulation in podocyte.3 The mechanism of JAML in the dysfunction of lipid metabolism in podocytes of diabetic kidney disease3.1 JAML regulates Sirtl expression in podocytesConsidering that sirtuins function as a master switch tomaintain lipid and glucose homeostasis by regulating important metabolic regulators.We then measured the levels of sirtuin members(Sirtl-7)in isolated glomeruli from STZ/HFD-induced diabetic Cre+/Jamlfl/fl mice and their controls.Among them,the levels of Sirtl,Sirt3,Sirt6 and Sirt7 were significantly reduced in diabetic Cre+/Jaml+/+mice and Sirtl was markedly restored in diabetic Cre+/Jamlfl/fl mice,indicating that JAML may regulate Sirtl expression.Simultaneously,we also found Sirt7 was partially recovered by JAML deficiency.Then,overexpression of JAML significantly reduced Sirtl expression,indicating that JAML is essential for the regulation Sirtl in podocytes with HG treatment.3.2 JAML regulates the expression of phosphorylated AMPK through SirtlSince AMPK is a key metabolic integrator of Sirtl-mediated signaling and share the similarities on the regulation of cellular and mitochondrial metabolism with Sirtl,we then measured AMPK activation and found that JAML reduced AMPK activation by measurement the level of phosphorylated AMPK(p-AMPK)in the kidney.In vitro,gene silencing of JAML recovered HG-reduced Sirtl expression and AMPK activation.3.3 JAML-Sirtl signaling pathway regulates the expression of SREBP1 and downstream target genes3.3.1 Gene sequence to detect JAML downstream signaling pathwayBy Agilent Whole Human Genome Oligo Microarray for global gene expression analysis,we observed the significant changes of genes important in regulating cellular lipid homeostasis in HG-treated podocytes compared with their controls.Among them,JAML deficiency significantly reduced the levels of transcription factor SREBP1 and its target genes that govern cholesterol and fatty acid biosynthesis such as FASN,ACC1,SCD1 and SQLE in podocytes with HG treatment.3.3.2 JAML regulates SREBP1 and its downstream signaling pathwaysThe decrease in the level of SREBP1 was also found in glomeruli isolated from STZ/HFD-induced Cre+/Jamlfl/fl diabetic mice compared with Cre+/Jaml+/+diabetic mice.3.4 JAML-Sirtl-AMPK signaling pathway regulates SREBP1 expressionIn order to clarify the regulatory effect of JAML,Sirt1,AMPK on SREBP1.We silenced both JAML and Sirt1,and verified the expression of mSREBP1 under high glucose stimulation.In addition,we also silenced JAML and AMPK,and verified that after high glucose stimulated podocytes and silenced JAML and AMPK,mSREBP1 protein expression changed.Finally,we provided evidence that Sirt1 and AMPK took center roles in JAML-mediated SREBP1 expression by either gene silencing of Sirt1 or AMPK.3.5 JAML regulates the expression of SREBP1 through the Sirt1 signaling pathway in podocytes with adriamycin treatmentJAML deficiency,recovered the decrease of Sirt1 level and AMPK activation,as well as significantly attenuated the increase of SREBP1 in podocytes.Conclusion and innovation1.Our studies for the first time demonstrate that JAML is significantly increased in the serum and renal biopsies from patients with glomerular diseases.Notably,correlation analysis showed that JAML protein level was positively correlated with serum creatinine and negatively correlated with glomerular filtration rate.2.In mice,podocyte-specific conditional knockout of JAML significantly ameliorated the proteinuria and glomerular sclerosis in diabetic kidney diseases,and silence of JAML inhibits high glucose-inducesd lipid metabolic disorder in podocytes.3.Mechanistically,we found that JAML regulated podocyte lipid metabolism through SIRT1/AMPK-mediated SREBP1 and its target genes associated with cholesterol and fatty acid biosynthesis.4.JAML was also increased in FSGS.JAML contributed to podocyte injury under ADR condition,indicating that JAML may have the same function in both glomerular diseases. |
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