| BACKGROUNDDiabetic nephropathy (DN) is an important and common complication of both type1and type2diabetes leading to end-stage renal disease (ESRD). Although current strategies slow disease progression, approximately one third of patients with diabetes develop end-stage renal disease 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.Glomerular filtration barrier is composed of glomerular endothelial cells (GeCs), the glomerular basement membrane (GBM), and podocytes. Podocytes are terminally differentiated cells reside on the outer surface of the glomerular basement membrane (GBM) and play a key role in maintaining the structure and function of the glomerular filtration barrier. Accumulating evidence suggests that glomerular podocytes play a pivotal role in the pathogenesis of diabetic kidney disease. Some studies in type1and type2diabetes have demonstrated that podocyte depletion and loss represented one of the earliest mechanisms in the pathogenesis of DN. High glucose (HG) induced podocyte apoptosis in vitro and in vivo, and podocyte apoptosis contributed to podocyte loss and reduced podocyte number. It has been suggested that high glucose increased reactive oxygen species (ROS) may be a trigger mechanism in podocyte apoptosis and loss in DN. However, the concert mechanisms that mediate hyperglycaemia-induced podocyte apoptosis is still far from being fully understood. Due to this reason, there are no current interventions for DN specifically preventing podocyte apoptosis.The nuclear factor of activated T cells (NFAT), which are the substrate for calcineurin(CaN), represent a family of Ca2+dependent transcription factors. Five isoforms, NFAT1, NFAT2, NFAT3, NFAT4and NFAT5, have been identified. NFAT is expressed in most immune system cells and plays a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. Although originally thought to be largely restricted to cells of the immune system, abundant evidence now indicates that NFAT family members are expressed in nonimmune cells with some family members expressed ubiquitously. NFAT is of critical importance in regulating the survival, proliferation, and function of multiple cell types. NFAT has been shown to regulate heart valve development, skeletal muscle and smooth muscle cell differentiation, and vascular development. NFAT has also been implicated in the pathogenesis of cardiac and skeletal muscle hypertrophy.The activities of NFAT proteins are tightly regulated by the Ca2+/calmodulin-dependent CaN, which can be inhibited by cyclosporine A (CsA). CaN controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells. In the nucleus, the NFATc proteins either alone or in combination with other nuclear partners form NFAT transcription complexes to control the transcription of target genes. In unstimulated cells, NFAT transcription factors are located in the cytoplasm and are highly phosphorylated. Dephosphorylation of NFAT isoforms by CN causes their translocation to the nucleus and stimulation of gene transcription.Recent data suggests that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle, pancreatic β-cells. A body of evidence has accumulated to suggest that NFAT was implicated in several types of cell apoptosis. Meanwhile, recent studies have also demonstrated that the activation of NFAT was involved in podocyte injury and glomerulosclerosis. But it is unknown whether hyperglycemia activates NFAT2in cultured mouse podocyte and whether this leads to podocyte apoptosis. The aim of the present study is to investigate if HG activated NFAT2and its function in hyperglycaemia-induced podocyte apoptosis and increased the expression of pro-apoptotic factor-Bax and changed the intracellular Ca2+concentration and study preliminarily the mechanism of glomerular podocyte injury in diabetic nephropathy meanwhile.METHODSPart one Culturing, identification and dividing subgroup of podocyte1. Mouse podocytes cultureConditionally immortalized mouse podocyte friendly present as a gift by Professor Danesh of Baylor Medical College. Podocyte should be cultured on the plates or dishes coated with collagen I. Mother podocyte should be incubated at33℃in5%CO2, with10%FBS RPMI1640(containing20-100U·mL-1INF-gamma) to pass, and those podocytes were grown under "growth permissive" condition.Then podocytes were cultured at37℃in5%CO2,with5%FBS DMEM (without INF-gamma)for differentiation. For10-14days, the cells became differentiated and acquired a quiescent phenotype, being ready to experiment.2. Identification and morphology observation of differentiated podocyteWe photographed the growing podocyte cultured in RPMI1640with INF-gamma at33℃and the differentiated podocyte cultured in DMEM without INF-gamma at37℃for10-14days, observing the variance of podocytes morphology in different situation.Cells Which expressed synaptopodin were differentiated and quiescent phenotype podocytes.3. The groups of differentiated podocytes were treated as follows(1) The expression of NFAT2in the podocyte nucleus with different concentration of glucose①Normal glucose group:the podocytes were cultured with glucose (5.3mM) for2h;②High glucose group:the podocytes were cultured with glucose (10mM,20mM,30mM and40mM respectively) for2h;(2) The expression of NFAT2in the podocyte nucleus with different time①The podocytes were cultured with glucose (5.3mM) for0.5h,1h,2h and4h respectively;②The podocytes were cultured with glucose (20mM) for0.5h,1h,2hand4h respectively;(3) The expression of NFAT2in the podocyte nucleus with different treatments①The podocytes were cultured with glucose (5.3mM) for2h;②The podocytes were cultured with glucose (20mM) for2h;③The podocytes were cultured with glucose (5.3mM) and Mannitol (14.7mM) for2h;④The podocytes were cultured with glucose (20mM) and11R-vivit (100nM) for2h:⑤The podocytes were cultured with glucose (20mM)and CsA (500nM)for2h:⑥The podocytes were cultured with glucose (20mM) and0.1%DMSO for2h；⑦The podocytes were cultured with glucose (5.3mM) and Ionomycin (500nM) for2h.(4)The apoptosis of podocyte with different treatments①The podocytes were cultured with glucose (5.3mM) for12h；②The podocytes were cuItured with glucose (5.3mM) for24h；③The podocytes were cultured with glucose (5.3mM) for48h；④The podocytes were cultured with glucose(20mM)for12h；⑤The podocytes were cultured with glucose (20mM) for24h；⑥The podocytes were culttred with glucose (20mM) for48h；⑦The podocytes were cultured with glucose (20mM) and11R-vivit (100nM) for48h:⑧The podocytes were cultured w-th glucose (5.3mM) and Mannitol (14.7mM) for48h:(5)The expression of pro-apoptotic factor Bax in podocyte with different treatments①The podocytes were cultured with glucose (5.3mM) for24h；②The podocytes were cultured with glucose(20mM)f.or24h；③The podocytes were cultured with glucose (5.3mM) and Mannitol (14.7mM) for24h:④The podocytes were cultured with glucose (20mM) and11R-vivit (100nM) for24h:⑤The podocytes were cultured with glucose (5.3mM) for48h；；⑥The podocytes were cultured with glucose (20mM) for48h； ⑦The podocytes were cultured with glucose (5.3mM) and Mannitol (14.7mM) for48h⑧The podocytes were cultured with glucose (20mM) and11R-vivit (100nM) for48h;(6) The change of the intracellular Ca2+concentration in podocytes with different treatments①The podocytes were cultured with glucose (5.3mM) for5min;②The podocytes were cultured with glucose (20mM) for5min;③The podocytes were cultured with glucose (5.3mM) and Mannitol (14.7mM) for5min;④The podocytes were cultured with glucose (5.3mM) and lonomycin (500nM) for5min;Part two The activation of NFAT2, apoptosis, the expression of Bax and the change of Ca2+concentration in podocytesTo evaluate the activation of NFAT2by Immunofluorescence and Western blot respectively; To evaluate the apoptosis of podocyte by Flow cytometry; To evaluate the expression Bax by quantitate-PCR and Western blot. To observe dynamicly the level of Ca2+concentration in podocytes using fluorescent dye by confocal.Part three Statistical AnalysesAll measurement data were expressed as mean±SD. Statistical analyses were conducted with SPSS13.0for Windows, comparing continuous variables of groups used One-way ANOVA,LSD method was used as multiple comparison for homoscedasticity, and Dunnett’s T3method was used as multiple comparison for heterogeneity of variance.Significance was difined as P<0.05.RESULTSPart one The activation of NFAT2in concentration-dependent with glucose in podocytesThe rateio of NFAT2/Histone3in podocytes with Glucose5.3mM, Glucose10mM, Glucose20mM, Glucose30mM and Glucose40mM respectively for2h by Western blot were0.999±0.001;1.652±0.188;2.405±0.281;1.850±0.397and2.000±0.381. Compared to the Glucose5.3mM group, the high glucose series groups significantly increased NFAT2nuclear accumulation (P5.3mM group,10mM group=0.020; P5.3mM group,20mM group<0.001; P5.3mM group,30mM group=0.005;, P5.3mM group,40mM group=0.002). With the same incubation time, the NFAT2nuclear accumulation was in a concentration-dependent. It would reach the peak when the cultured podocyte exposed to the glucose (20mM) for2h.Part two The activation of NFAT2in time-dependent with high glucose in podocytesThe rateio of NFAT2/Histone3in podocytes with Glucose5.3mM (NG) for0.5h,1h,2h and4h respectively by Western blot were1.000±0.000;1.080±0.321,1.229±0.385and1.112±0.458. Compared it among them, none increased NFAT2nuclear accumulation (P NG0.5h group, NG1h group=0.792; P ng0.5h group NG2h group=0.455; P NG0.5h group NG4h group=0.713; P NG1h group,NG2h group=0.626; P NG1h group,NG4h group=0.916; P NG2h group, NG4h group=0.701); The rateio of NFAT2/Histone3in podocytes with Glucose20mM (HG) for0.5h,1h,2h and4h respectively by Western blot were:1.253±0.332,1.656±0.464,2.080±0.319and1.601±0.431. Compared to the HG0.5h group, HG1h group didn’t increase NFAT2nuclear accumulation (P HG0.5h group, HG1h group=0.197).But HG2h group significantly increased NFAT2nuclear accumulation (P HG0.5h group HG2h group=0.014). Compared to the HG2h group,HG4h group didn’t increase NFAT2nuclear accumulation (P HG2h group, HG4h group=0.128).As a result:the NFAT2nuclear accumulation was in a time-dependent. It would reach the peak when the cultured podocyte exposed to the glucose (20mM) for2h.Part three1.The activation of NFAT2by Western blot in podocytes with different treatmentsThe rateio of NFAT2/Histone3in podocytes by Western blot:Glucose20mM2h (HG) group (2.039±0.373), was similar to Glucose5.3mM+Ionomycin500nM2h group (2.353±0.506) and P HG group, Ionomycin group=0.220. Compared to the HG2h group, Glucose20mM+11R-vivit100nM2h group(1.223±0.188) and Glucose20mM+CsA500nM2h group (1.208±0.207) significantly reduced NFAT2nuclear accumulation (P11R-vivit group, HG group=0.005; P CsA group, HG group=0.004),but were similar to Glucose5.3mM2h (NG) group (1.000±0.000) and P11R-vivit group, NG group=0.378; P CsA group, NG group=0.409. The Glucose5.3mM+Mannitol14.7mM (osmotic control) group(1.173±0.194), was similar to the Glucose5.3mM2h group and P osmotic control group, NG group=0.491. As a result:high glucose could increase NFAT2nuclear accumulation but didn’t depend on increasing osmotic pressure in podocytes. And this could be suppressed by CsA (inhibition of CaN) or11R-vivit.2.The expression of NFAT2by confocal in podocyte with different treatments.NFAT2stained in red, nuclei stained blue, two colors overlap in purple..As a result:NFAT2were expressed most in the cytoplasm but just a small amount in the nucleus in NG group, osmotic control group,11R-vivit group and CsA group. While NFAT2were highly expressed not only in the cytoplasm but also in the nucleus in HG group, DMSO group and Ionomycin group.these were similar to the result of Western blot.Part four The apoptosis of podocyte with different treatments.The apoptosis of podocyte was detected by Flow cytometry Using Annexin V/PI kit. The apoptotic cells are Annexin V (+)/PI (-). When the podocytes were cultured with glucose (5.3mM)(NG) for12h,24h,48h, the apoptosis rates were: (10.00±2.04)%.,(11.39±1.62)%and (12.99±2.22)%. And with glucose (20mM)(HG) for12h,24h,48h, the apoptosis rates were:(11.79±3.24)%,(15.08±3.35)%and (22.72±6.68)%. But with Glucose5.3mM+Mannitol14.7mM (osmolality control) for48, the apoptosis rate was (13.17±5.36)%and similar to the podocytes cultured with Glucose5.3mM for48h.(P osmolaiity control48h group, NG48h group=0.174). When the podocytes were cultured with Glucose20mM+11R-vivit100nM for48h, the podocyte apoptosis rate was (13.62±3.82)%and significantly reduced than the podocytes cultured with Glucose20mM for48h (P11R-vivit48hgroup, HG48h group <0.001). As a result:high glucose would induce podocyte apoptosis in a time-dependent, but it didn’t depend on the increased osmotic pressure, and it should be reduced by inhibiting excessive activation of NFAT2.Part five High glucose increase the expression of BaxBax is one of pro-apoptotic factors in cells. By real-time quantitative PCR and Western blot,compared to the podocytes cultured with Glucose5.3mM (NG) for48h, the podocytes cultured with Glucose20mM (HG) for48h significantly increased the expression of Bax factor mRNA and protein (P HG48h group NG48h group<0.01, P HG48h group, NG48h group<0.01).But the podocytes cultured with Glucose5.3mM+Mannitol14.7mM for48h is similar. Compared to the podocytes cultured with Glucose20mM for48h, the podocytes cultured with Glucose20mM+11R-vivit100nM for48h significantly reduced the expression of Bax factor mRNA and protein(P HG48h group,11R-vivit group<0.01, P HG48h group,11R-vivit group<0.01) and similar to the podocytes cultured with Glucose5.3mM for48h. The result is consistent with apoptosis.Part six High glucose increased intracellular Ca2+concentration in podocytesThe change of intracellular Ca2+concentration marked by Fluo-3/AM was Observed by confocal. The results show that:compared to the podocytes cultured with Glucose5.3mM, the podocytes cultured with Glucose20mM, Glucose5.3mM +Ionomycin500nM significantly increased intracellular Ca2+concentration.But the podocytes cultured with Glucose5.3mM+Mannitol14.7mM was similar. As a result:high glucose (20mM) increased intracellular Ca2+concentration in podocyte but didn’t depend on the increasing osmotic pressure.CONCLUSIONSHigh glucose may be mediated podocyte apoptosis through CaN/NFAT/Bax signaling pathways. And blocking the CaN/NFAT2/Bax signaling pathways could reduce high glucose-induced podocyte apoptosis. |
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