The Role And Mechanism Of Septin7 Activating Ca²⁺/CaN/NFAT₂ Signal Pathway In Podocyte Injury Of Diabetic Nephropathy

BACKGROUNDDiabetic nephropathy（diabetic nephropathy,DN）is one of the common microvascular complications of DM,all reported incidence between 20-50%;In addition,it is an important cause of death in patients with diabetes,and also contributed to a major cause of end-stage renal disease（ESRD）.The pathogenesis of DN is complex.Although several interventions have been shown to slow the progression of diabetic nephropathy currently,such as tight glucose and blood pressure control and blockade of the renin-angiotensin system,none of the available treatments could cure or prevent DN,and about one-third of patients eventually develop ESRD,requiring renal replacement therapy.As a result,much effort has been devoted to understanding the mechanisms that promote glomerular damage in diabetic kidney disease with the hope of identifying new therapeutic strategies.Previous studies have shown that podocyte apoptosis is the major cause of podocyte depletion,which plays a pivotal role in the pathogenesis of DN.However,the concert mechanism of podocyte apoptosis in DN has not been fully understood and there is still no effect intervention to prevent and treat podocyte apoptosis in patients with DN.Therefore,it is important to explore the key molecules and pathogenesis of podocyte apoptosis in patients with DN.Septins are a novel family of GTP-binding proteins with ubiquitous and tissue-specific expression that influence the function and localization of cell surface proteins.Septins are originally identified in yeast,and also expressed in most eukaryotic cells,except for plants.They assemble into hetero-oligomers and filamentous polymers.In mammalian cells,septins serve as molecular scaffolds that associate with actin microfilaments,microtubules,and other elements of the cytoskeletal network.More and more studies identified important role of septins in the morphogenesis and physiological functions,such as vesicle fusion,cytokinesis,cell polarity and apoptosis.The alterations of septin expression is associated with pathogenesis of numerous disease states,such as cancer,neurodegenerative and kidney disease.Recent studies revealed that septin7 may participate in the regulation of glucose transport in podocytes.Meanwhile,it has been demonstrated that overexpression of septin7 induce human glioma cell apoptosis.In additional,Septins induced human cervical cancer passage cell injury by activating Cal+/CaN/NFAT signaling pathway.In our previous study,we had demonstrated that high glucose induced cultured podocytes apoptosis through increasing intercellular Ca²⁺ concentration,which subsequently activated the Ca²⁺/CaN/NFAT₂ signal pathway.Studies has shown that the intracellular Ca²⁺ level is tightly regulated by multiple pumps,channels,and binding proteins.TRPC6（Transient receptor potential channel 6）is the main channel of Ca²⁺ in podocytes.Previous studies have also found that increased expression or activation of TRPC6 in podocyte lead to continuous increasing of Ca²⁺ influx,which subsequently activated Ca²⁺/CaN/NFAT₂ signaling pathway and induced cell damage,even glomerular sclerosis.Therefore,to explore the mechanism of podocyte apoptosis with the connection of Septin7,TRPC6 and Ca²⁺/CaN/NFAT₂ signal pathway,may present a potential target for preventing podocyte apoptosis in DN.In this study,we evaluated the potential role of Septin7 in podocyte apoptosis in patients with type 2 diabetic nephropathy and db/db mice.Meanwhile,we will explored the mechanism of septin7 regulating TRPC6 and activating Ca²⁺/CaN/NFAT₂ signal pathway in high glucose induced cultured podocytes apoptosis.METHODSPart one Septin7 and TRPC6 expression in glomerular podocytes of patients with type 2 DN.control group:normal adjacent renal tissue of renal tumor patients after surgical resection which excluded kidney tissue infiltration by cancer cells;DN group:kidney tissue of patients with type 2 DN;Kidney tissue specimens were stained with periodic acid Schiff（PAS）for light microscopic examination.The glomerular podocyte injury was checked by electron microscopy.The expression of Septin7,TRPC6 and synaptopodin were observed by Immunofluorescence..Part two The role of Septin7 activating the Ca²⁺/CaN/NFAT₂ signaling pathway in db/db mice with DN.The body weight,diet,glucose and urine albumin/creatinine ratio were measured in twenty C57BLKS/J db/db mice with 7-week-old and five wild-type BKS mice with the same genetic background.After spontaneous type 2 diabetes mouse were successfully established,these db/db mice were divided into four groups:each group of five,namely:DM group,DN group,DN+11R-VIVIT group（11R-VIVIT:NFAT₂ inhibitor;Usage:lmg/kg,intraperitoneal injection,once every other day,for eight weeks）and DN+DMSO（solvent control）group;and five wild-type BKS mice named as the Control group.Mice were anaesthetized（10%chloral hydrate;0.25ml/50g i.p.）and blood samples were obtained from the retro-orbital venous plexus for determination of the plasma concentration of creatinine in control group and the DM group,The kidney samples were frozen and kept in liquid nitrogen until use.In the remaining three groups（DN group,DN+11R-VIVIT group and DN+DMSO group）,Fasting blood glucose was measured in tail-vein blood using one touch ultra glucometer and and Test Strip（Lifescan,Milpitas,CA）after 6 h of fasting weekly.Body weight was obtained at one week intervals.For urine collection,individual mice were caged once weeks in a metabolic cage for 24 h.After 8 weeks of treatment,mice were anaesthetized（10%chloral hydrate;0.25ml/50g i.p.）and blood samples were obtained from the retro-orbital venous plexus for determination of the plasma concentration of creatinine.The samples were frozen and kept in liquid nitrogen until use.Urinary albumin/creatinine ratio and serum creatinine were determined by ELISA.The glomerular podocyte injury was checked by electron microscopy.The number of podocytes per glomerulus was determined based on the WT1 immunohistochemistry.Septin7,TRPC6 and nucleal NFAT₂ expression were detected by Immunofluorescence.Septin7,TRPC6 expression were also analyzed by western blot.Part three1.Mouse podocytes culture and identification1.1 Mouse podocytes culture:The conditionally immortalized mouse podocyte cell line（MPC）was kindly provided by Dr.Jochen Reiser（Rush University Medical Center,Chicago,USA）.Cells were grown to 80%confluence in type-Ⅰ collagen（Shengyou Biotechnology,China）-coated flasks at 33℃ in RPMI-1640（Gibco BRL,Gaithersburg,USA）supplemented with 10%fetal bovine serum（FBS,Gibco BRL,USA）and 50 U ml-1 γ-interferon（IFN-γ,ProSpec,Israel）.To induce differentiation,podocytes were reseeded and cultured at 37℃ in 50 cm2 culture dish coated with type-I collagen in the absence of IFN-y for 10 to 14 days.1.2 Identification and morphology observation of differentiated podocyte:The cultured podocyte in RPMI 1640 with INF-gamma at 33℃ and the differentiated podocyte in DMEM without INF-gamma at 37℃ for 10-14 days were checked by contrast phase microscope.The variance of podocytes morphology in both conditions was observed.The skeleton protein synaptopodin is expressed in differentiated podocytes.However,it is not expressed in undifferentiated cells.2.The differentiated podocytes were treated as follows2.1 Septin7 expression in podocyte（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 3,6,12,24,48 hours respectively;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 3,6,12,24,48 hours respectively.2.2 the rate of podocyte apoptosis and pro-apoptotic factor Bax expression（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 48 hours;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 48 hours;（3）MA group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose and 24.7 mmol/1 Mannital for 48 hours;（4）HG+ Septin7-SiRNA group:the podocytes were interfered with Septin7-SiRNA for 48 hours and then cultured in DMEM medium with 30 mmol/1 D-glucose for 48 hours;（5）HG+ Septin7-SiNC:the podocytes were interfered with Septin7-SiNC for 48 hours and then cultured in DMEM medium with 30 mmol/1 D-glucose for 48 hours;（6）HG+SAR7334 group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/1 SAR7334 for 48 hours;（7）HG+11R-VIVIT group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/11R-VIVIT for 48 hours;（8）NG+ Septin7-cDNA group:the podocytes were transfected with Septin7-cDNA for 48 hours and then cultured in DMEM medium with 5.3 mmol/1 D-glucose for 48 hours;（9）NG+ Septin7-virus empty shell group:the podocytes were transfected with Septin7-cDNA-virus empty shell for 48 hours and then cultured in DMEM medium with 5.3 mmol/1 D-glucose for 48 hours;（10）NG+ Septin7-cDNA+SAR7334 group:the podocytes were transfected with Septin7-cDNA for 48 hours and then cultured in DMEM medium with 5.3 mmol/1 D-glucose with 100 nmol/1 SAR7334 for 48 hours;（11）NG+ Septin7-cDNA+11R-VIVIT group:the podocytes were transfected with Septin7-cDNA for 48 hours and then cultured in DMEM medium with 5.3 mmol/1 D-glucose with 100 nmol/1 11R-VIVIT for 48 hours.2.3 the nuclear NFAT₂ expression in podocyte（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 24 hours;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（3）HG+ Septin7-SiNC group:the podocytes were interfered with Septin7-SiNC for 48 hours and then cultured in DMEM medium with 30mmol/1 D-glucose for 24 hours;（4）HG+ Septin7-SiRNA group:the podocytes were interfered with Septin7-SiRNA for 48 hours and then cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（5）HG+SAR7334 group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/1 SAR7334 for 24 hours;（6）HG+11R-VIVIT group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100nmol/1 11R-ⅥⅥT for 24 hours.2.4 Intracellular calcium concentration（1）NG group:the podocytes were intervened in DMEM medium with 5.3 mmol/1 D-glucose for 5 minutes;（2）HG group:the podocytes were intervened in DMEM medium with 30 mmol/1 D-glucose for 5 minutes;（3）HG+ Septin7-SiRNA group:the podocytes were interfered with Septin7-SiRNA for 48 hours and then intervened in DMEM medium with 30 mmol/1 D-glucose for 5 minutes;（4）HG+SAR7334 group:the podocytes were pretreatment with 100nmol/1 SAR7334 for 48 hours,and then intervened in DMEM medium with 30 mmol/1 D-glucose for 5 minutes;（5）HG+Ionomycin group:the podocytes were intervened in DMEM medium with 5.3 mmol/1 D-glucose and 500 nmmol/1 Ionomycin for 5 minutes;2.5 TRPC6 expression in podocyte（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 24 hours;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（3）HG+ Septin7-SiRNA group:the podocytes were interfered with Septin7-SiRNA for 48 hours and then cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（4）HG+ Septin7-SiNC group:the podocytes were transfected with Septin7-SiNC for 48 hours and then cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（5）HG+11R-VIVIT group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/1 11R-VIVIT for 24 hours2.6 Septin7 expression in podocyte（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 24 hours;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours;（3）HG+SAR7334 group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/1 SAR7334 for 24 hours;（4）HG+11R-VIVIT group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose with 100 nmol/1 11R-VIVIT for 24 hours.2.7 combination of Septin7 and TRPC6（1）NG group:the podocytes were cultured in DMEM medium with 5.3 mmol/1 D-glucose for 24 hours;（2）HG group:the podocytes were cultured in DMEM medium with 30 mmol/1 D-glucose for 24 hours.3.detection of podocyte apoptosisAccording to the detection process Annexin V/PI kit from roche,the rates of cell apoptosis under different interventions were detected by flow cytometry.Annexin V（+）/PI（-）was defined as apoptotic cells.4.detection of total podocyte proteins Bax,TRPC6,Septin7,nuclear protein NFAT₂,and membrane proteins TRPC6 and Septin7Total protein Bax,TRPC6,Septin7,nuclear protein NFAT₂,membrane protein TRPC6 and Septin7 were detected by western blot.And nuclear protein NFAT₂ was also observed by immunofluorescence assay.5.Intracellular calcium concentration the level of Ca²⁺ concentration in podocytes was observed dynamicly using fluorescent dye by confocal.6.Septin7 and TRPC6 co-immunoprecipitation（co-ip）Total proteins from cells were extracted using IP lysate and the protein concentration was quantified according to the detection kit of nanjing keji BCAprotein concentration.Co-immunoprecipitation experiments were performed using the Pierce Co-Immunoprecipitation Kit as per the manufacturer’s instructions.Briefly,300ig of protein lysates were pre-cleared using a control agarose resin to minimize non specific binding.These lysates were then applied to columns containing 5ig immobilized Septin7 or TRPC6 antibody covalently linked to an amine-active resin and incubated overnight at 4℃.Equal volumes of the lysates were also applied to columns containing control resin and processed the same as the antibody coupling resin for negative controls.The co-immunoprecipitate was then eluted and analyzed by SDS-PAGE along with the input controls.Part four Statistical analysisAll values are expressed as mean±SEM.Statistical analysis was performed using the statistical package SPSS for Windows Ver.17.0.Multiple comparisons among the groups were conducted by one-way analysis of variance with LSD test for homogeneity of variance,or Dunnett’s T3-test for heterogeneity of variance.Differences between two groups were analyzed with a two-tailed test of significance.Significance level test for a=0.05,P-values less than 0.05 was considered significant.RESULTS1.The glomerular podocytes septin 7 expression was markedly increased in patients with DN compared with those without DN.We also found reduction of synaptopodin expression in patients with DN which probably means podocytes depletion.2.The protein expression of Septin7 and TRPC6 were markedly increased in glomerular podocytes in both db/db mice with DN and without DN（DM group）.Additionally,glomerular synaptopodin expression was diminished in db/db mice with DN compared with db/db mice without DN and BKS mice.And when inhibiting NFAT₂ activity,it can’t reduced Septin7 and TRPC6 expression in db/db mice.3.（1）Septin7 expression was increasing induced by high glucose.（2）Septin7 and TRPC6 mediated high glucose-induced apoptosis and Bax expression in cultured podocytes.（3）Septin7 and TRPC6 mediated high glucose-induced the activation of Ca²⁺/CaN/NFAT₂ signaling pathway in cultured podocytes.（3）Septin7 and TRPC6 mediated high glucose-induced Ca²⁺ influx in cultured podocytes.（4）Septin7 mediated high glucose-induced TRPC6 expression on the cell membrane in cultured podocytes,but TRPC6 had no similar effect on Septin7.Meanwhile,effectively inhibiting the activition of NFAT₂ failed to reduce the expression of Septin7 or TRPC6 induced by high glucose.（5）High glucose promoted the combination of Septin7 and TRPC6.CONCLUSIONSIn this study,we first found that:Under the stimulation of DM,such as high glucose,Septin7 was highly expressed in podocytes,promoted TRPC6 translocation to cell membrane,increased Ca²⁺ influx,activated Ca²⁺/CaN/NFAT₂ signaling pathway,increased the expression of pro-apoptotic factor Bax,induced podocyte apoptosis,and then induced albuminuria and impaired renal function,which lead to the occurrence and progress of DN.