Mitochondrial Dysfunction In The Early Glomerular Podocyte Damage In The Role Of Research

Glomerular epithelial cells, podocytes, are highly specialized cells that serve as acrucial component of the glomerular filtration barrier. Podocytes are composed of acell body, major processes and foot processes. The neighboring foot processes derivedfrom different cell bodies are connected by a continuous membrane-like structurecalled the slit diaphragm and form an interdigitating pattern adhering to theglomerular basement membrane (GBM). It is becoming increasingly clear thatpodocyte injury leads to proteinuria and occurs in many glomerular diseases thatfinally progress to chronic kidney disease.Recently, a growing number of studies have shown that mitochondrialdysfunction (MtD) is associated with many types of kidney disease. MtD represents amalfunction in biochemical processes, resulting from either its own mitochondrialDNA (mtDNA) or from nuclear DNA. Children with mitochondrial cytopathies havebeen reported to develop focal segmental glomerulosclerosis (FSGS), andabnormalities of podocyte mitochondria have been detected by transmission electronmicroscopy (TEM) in renal biopsy specimens. A rat model of FSGS also found thatpuromycin aminonucleoside nephrosis (PAN), the expression of mtDNA and itsencoded proteins were reduced in podocytes.Peroxisome proliferator activated receptor-γ coactivator1α (PGC-1α), atranscriptional co-activator of PPAR-γ and other nuclear hormone receptors, is amajor regulator of oxidative metabolism and mitochondrial biogenesis. PGC-1α bindsto and coactivates the transcriptional function of nuclear respiratory factor-1(NRF-1),which binds specifically to the mitochondrial transcription factor A(TFAM) promoter,a direct regulator of mtDNA replication. The overexpression of PGC-1α results in arobust increase in mitochondrial number, cellular respiration, and intracellular ATPconcentration in a variety of cell types. In addition, the repression of PGC-1α expression by a mutant histone deacetylase5(HDAC5) resulted in loss of andmorphological changes to mitochondria and in downregulation of mitochondrialenzymes.Recently SIRT1(silent mating type information regulation2homolog1) hasbeen shown to function together with PGC-1α to regulate mitochondrial biogenesis.SIRT1is one of seven mammalian homologs of Sir2that catalyzes NAD+-dependentprotein deacetylation. Several reports have shown that SIRT1is not just a histonedeacetylase but that SIRT1can directly interact and regulate the activity of severaltranscription factors, such as p53and FOXO, that are involved either in setting athreshold for apoptosis or in prompting cell repair. Several lines of evidence indicatethat SIRT1-mediated regulation of PGC-1α activity may play a major role in themetabolic adaptation to energy metabolism in different tissues. An activator of SIRT1,resveratrol (RSV), was recently reported to promote mitochondrial biogenesis and toprotect cells from metabolic disease through SIRT1-dependent deacetylation ofPGC-1α.Part I Mitochondrial dysfunction mediates aldosterone-induced podocyte injuryObjective: To explore the effect of MtD inAldo-induced podocyte injury.Methods: To evaluate the time and dose effect of Aldo on mitochondrial functionand podocyte, podocytes were treated with Aldo at the concentration of25、50、100nmol/L and at different time points of2、4、8、12、24h, respectively. The expressionsof podocyte main slit diaphragm proteins nephrin and podocin were examined byreal-time PCR and western blotting. Mitochondrial morphology was observed byelectromicrograph morphometry. Cellular reactive oxygen species (ROS) productionwas determined by DCFDA fluorescence, and mitochondrial ROS levels weredetermined by MitoSOX staining. The ATP content was measured by using a luciferase-based luminescence assay kit and the mitochondrial membrane potentialwas examined by JC-1staining. Mitochondrial DNA (mtDNA) copy numbers weredetermined by real-time PCR. Podocyte apoptosis was assessed using Hoechst33258staining and annexin V/flow cytometry detection. Caspase-3,-8and-9activities inpodocytes were measured using caspase activity assay kits. In vivo, C57BL/6J micewere implanted subcutaneously with14-day-realease pellets containing Aldo.Aldo-infused mice were sacrificed at day1,3,5,7,14, respectively. Podocyteapoptosis was labelled by TUNEL assay. The podocyte ultramicrostructure wasinvestigated by transmission electron microscopy. The proteinuria was evaluated byurine protein/urine creatinine. The expression of nephrin was determined by westernblotting. The urine MDA was measured by using a Lipid Peroxidation MDA AssayKit. The ATP content was measured by using a luciferase-based luminescence assaykit and the mitochondria DNA (mtDNA) was measured by real-time PCR. Toinveastigate the effect of mineralocorticoid receptor (MR) and glucocorticoid receptor(GR) on Aldo-induced MtD and podocyte injury, podocytes were pretreatedeplerenone (EPL) or RU-486for30min and then incubated with Aldo for indicatedtime. Podocyte injury and mitochondrial function were detected.Results:(1) Aldo induced the production of ROS and the reduction ofmitochondrial membrane potential (MMP), ATP levels and mtDNAcopy number in adose-dependent manner. Mitochondrial morphology was observed by TEM. Swollenmitochondria with disorganized and fragmented cristae were seen in podocytestreated with Aldo for24h. Aldo induced podocyte apoptosis in a dose-dependentmanner. Aldo dose-dependently activated caspase-3and caspase-9and inhibited theglomerular slit diaphragm proteins nephrin and podocin expression. All these resultssuggested that Aldo induced MtD and podocyte injury in a dose-dependent manner.(2)Aldo induced mitochondrial superoxide anion production in a time-dependent manner, which became noticeable at2h and was sustained until24h. Aldo induced decreasesin mtDNA copy number and ATP production after4–8h of treatment. Nephrinexpression was downregulated at12h in Aldo-treated podocytes. In Aldo-infusedmice, examination of kidney ultrastructure morphology showed foot-processeffacement after5days of Aldo treatment, and urinary protein excretion increasedafter5days of Aldo infusion in mice and continued to increase until day14.Consistent with the podocyte damage and proteinuria, the expressions of nephrindecreased after5days in Aldo-infused mice. The urine MDA showed a progressiverise that was noticeable after3days of Aldo infusion. Similary, the mtDNA copynumber and ATP production in the kidney cortex were decreased on day3. All theseresults suggested that Aldo induced MtD and podocyte injury in a time-dependentmanner in vivo and vitro, and MtD was the early event in Aldo-induced podocyteinjury.(3) Pretreating podocytes with the mineralocorticoid receptor (MR) antagonisteplerenone but not with the glucocorticoid receptor (GR) antagonist RU-486,abrogated Aldo-induced MtD. Similarly, eplerenone rather than RU-486notablyinhibited nephrin decrease induced by Aldo. These results strongly indicate that thestimulatory effect of aldosterone on MtD and podocyte injury is mediated via MRactivation.Conclusion: MtD was the early event in Aldo-induced podocyte injury, and MtDmight mediateAldo-induced podocyte injury. Objective: To explore the effect of SIRT1/PGC-1α axis in Aldo-induced MtD andpodocyte injury. Methods: To explore the effect of Aldo on PGC-1α and SIRT1expressions,podocytes were treated withAldo at the concentration of25、50、100nmol/L. PGC-1αexpression was determined by RT-PCR, immunofluorescence, real-time PCR andwestern blotting. SIRT1expression was determined by real-time PCR and westernblotting. The time course of PGC-1α acetylation treated by Aldo was measured byimmunoprecipitation (IP) and then by western blotting with an anti-acetylated lysineantibody. To examine the role of endogenous PGC-1α in mitochondrial function andpodocyte damage, podocytes were transfected with with PGC-1α shRNA. Thereduction of PGC-1α was measured by real-time PCR and western blotting. The ATPcontent was measured by using a luciferase-based luminescence assay kit and themitochondrial membrane potential was examined by JC-1staining. MitochondrialDNA (mtDNA) copy numbers were determined by real-time PCR. Podocyteapoptosis was assessed using Hoechst33258staining and annexin V/flow cytometrydetection. Caspase-3,-8and-9activities in podocytes were measured using caspaseactivity assay kits. The expression of nephrin was determined by western blotting. Toteste the role of PGC-1α overexpression on Aldo-induced MtD, Podocytes wereinfected with Ad-PGC-1α that expressed murine PGC-1α at multiplicities of infection(MOI)50. Overexpression of PGC-1α was confirmed by western blotting analysis.Cytochrome c release was tested by western blotting. Mitochondrial functions andpodocyte injury were determined as described above. To investigate the role of SIRT1in Aldo-induced MtD and podocyte injury, podocytes were infected with Ad-SIRT1vectors. Overexpression of SIRT1was confirmed by western blotting analysis.PGC-1α and TFAM expressions were determined by western blotting. The interactionbetween SIRT1and PGC-1α was determined by using a luciferase reporter systemand immunoprecipitation. To evaluate the hypothesis that SIRT1overexpressionprevents Aldo-induced MtD via PGC-1α, podocytes were co-infected with Ad-SIRT1 and PGC-1shRNA for24h and stimulated with Aldo for another24h.Mitochondrial functions and podocyte injury were determined as described above.Results:(1) Aldo treatment reduced both mRNA and protein levels of PGC-1α andSIRT1in a dose-dependent manner. MR antagonist eplerenone perfectly inhibited thedecreases of SIRT1and PGC-1α protein levels, suggesting involvement ofaldosterone/MR cascade.(2) Downregulation of PGC-1α by shRNA reduced MMP,ATP levels, and mtDNA copy number. Also, PGC-1α shRNA increased thepercentage of cells undergoing apoptosis by65%, and increased levels of caspase-3and caspase-9activity. Moreover, nephrin and podocin protein concentrationsdecreased by35%and25%, respectively, following knockdown of PGC-1α.Therefore, suppression of endogenous PGC-1α expression induced MtD andpodocyte injury.(3) Podocytes infected with empty vector and then treated with Aldohad swollen mitochondria with disorganized and fragmented cristae. In contrast,typical mitochondria with normal intact cristae and dense matrices were observed inPGC-1α-overexpressed podocytes treated with Aldo. PGC-1α overexpression blockedthe effects of Aldo on ROS production, MMP, ATP levele and mtDNAcopy numbers.Moreover, PGC-1α overexpression conferred protection against Aldo-inducedpodocyte apoptosis, blocked the release of cytochrome c, reduced the activation ofboth caspase-3and-9, and inhibited Aldo-induced reduction of nephrin and podocin.(4) SIRT1and PGC-1α interact physically and secondly that SIRT1regulatedPGC-1α at both the transcriptional and posttranslational levels. SIRT1overexpressionprevented the Aldo-induced reduction in MMP, ATP production, and mtDNA copynumber. Meanwhile, SIRT1overexpression decreased Aldo-induced apoptosis andrestored nephrin and podocin mRNA and protein levels after Aldo treatment.Knockdown of PGC-1α, using PGC-1α shRNA, abolished these protective effectsConclusion: SIRT1/PGC-1α axis might protect against Aldo-induced podocyte injury via inhibiting MtD. Objective: To explore the effect of RSV inAldo-induced MtD and podocyte injury.Methods: In vitro, podocytes were pretreated with RSV (10–50M) or RSV (50M)plus nicotinamide (NAM)(10mM) for30min followed by incubation with Aldo. Theexpressions of nephrin, podocin, PGC-1α and TFAM were measured by real timePCR and western blotting. The effect of RSV on Aldo-induced PGC-1α acetylationwas measured by immunoprecipitation (IP) and then by western blotting with ananti-acetylated lysine antibody. The ATP content was measured by using aluciferase-based luminescence assay kit and the mitochondrial membrane potentialwas examined by JC-1staining. Mitochondrial DNA (mtDNA) copy numbers weredetermined by real-time PCR. In vivo, C57BL/6J mice were implantedsubcutaneously with14-day-realease pellets containing Aldo. Histology of kidneywas tested by Periodic acid Schiff (PAS) staining.24h urinary albumin excretion wasdetermined by using enzyme linked immunosorbant assay (EIA) kits, and the footprocesses of podocytes were observed by transmission electron microscopy. Theexpressions of nephrin, podocin, PGC-1α, TFAM and SIRT1were measured by realtime PCR and western blotting. The effect of RSV on PGC-1α acetylation inAldo-infused mice was measured by immunoprecipitation (IP) and then by westernblotting with an anti-acetylated lysine antibody. Mitochondrial morphology inglomerular podocytes was observed by transmission electron microscopy. The MMPof isolated mitochondria from glomeruli were monitored using JC-1. ATP levels were determined in isolated glomeruli with a luciferase-based bioluminescence assay kit.Mitochondrial DNA (mtDNA) copy numbers were determined by real-time PCR.Complex I, II and IV enzyme activities of isolated mitochondria were measured usingmicroplate assay kits, and the enzyme activities of mitochondrial complex III wereassessed spectrophotometrically using the Complex III Assay Kit. Renal cortex MDAlevels were analyzed by using a Lipid Peroxidation MDA Assay Kit. Urinaryconcentration of F2-isoprostane was determined using EIA kits. Isolated glomeruliand mitochondrial ROS levels were measured by DCFDAstaining.Results:(1) RSV reduced Aldo-induced podocyte apoptosis in a dose-dependentmanner and restored nephrin and podocin mRNA and protein expression. In addition,the SIRT1targets, PGC-1α and TFAM, were upregulated following RSV treatment.Also, RSV treatment prevented the Aldo-induced reduction in MMP, ATP production,and mtDNA copy number. These effects were blocked by the SIRT1inhibitornicotinamide (NAM).(2) We sought in vivo support for the protective effect of RSVon mitochondrial function and podocytes by studying mice that had been treated withAldo for21days. Aldo treatment resulted in glomerular enlargement and increasedmesangial area, and RSV restored the normal structure of the glomeruli. Moreover,albuminuria was significantly reduced by RSV treatment in Aldo-infused mice. Inelectron micrographs, extensive fusion of foot processes was evident in Aldo-treatedmice. In contrast, treatment of the Aldo-infused mice with RSV maintained thenormal shape of the foot processes. RSV restored nephrin and podocin mRNA andprotein levels. Similarly, SIRT1, PGC-1α and TFAM were significantly induced uponRSV treatment, which also translated into the increase in proteins levels. Also, RSVtreatment rescued the MMP, ATP levels, and mtDNA copy number in Aldo-infusedmice. RSV protection against Aldo-induced MtD was further confirmed by measuringmitochondrial respiratory chain enzyme activities. RSV treatment restored the activities of complexes I, III and IV. Furthermore, RSV treatment in Aldo-infusedmice abolished the induction of ROS in glomeruli, which was assessed by urinaryF2-isoprostane levels, Malondialdehyde (MDA) levels and DCF staining.Conclusion: RSV might protect against Aldo-induced podocyte injury by regulatingmitochondrial function.