Study On The Mechanism Of Mitochondrial Division In Podocyte Injury

The prevalence of the global chronic kidney disease(CKD)has been continued to keep growing.In 2012,an epidemiology investigation showed 10.8% CKD rate in Chinese.CKD has become an important public problem which threatens human health.An increasing number of studies have found that the injury of podocyte plays a predominant role in the pathogenesis and progression of CKD.As a result,how to prevent and to treat podocyte damage is the key to block the occurrence and development of CKD.The process of mitochondrial fission and fusion is referred to as mitochondrial dynamics,of which mitochondrial fission was shown to be pivotal in renal tubular damage models.However,it's not fully clear the role of mitochondrial fission in podocyte.The molecular mechanism governing mitochondrial dynamics was initially explored in budding yeasts.The key proteins of mitochondrial fission are Dnm1,Mdv1 and Fis1 in yeast as well as dynamin related protein 1(Drp1)and mitochondrial fission protein 1(Fis1)in mammals.Drp1 is a GTPase of the dynamin superfamily.In normal physiological conditions,Drp1 is mainly cytosolic;Fisl is anchored on the outer membrane of mitochondria.When activated,Drp1 translocates to mitochondria by the chemotaxis of Fis1,then localize to the fission sites where they form spirals surrounding the mitochondria,fission is finally activated when the intermolecular distance or angle of Drp1 change through GTP hydrolysis.After mitochondrial fission,Drp1 returns to the cytosol.As a result,it cycles between the cytosol and mitochondria.In 2008,Ann Cassidy-Stone and others discovered a new mitochondrial division inhibitor(Mdivi-1),which inhibits mitochondrial fission by suppressing Drp1 accumulation in mitochondrial outer membrane.Balance disorders in mitochondrial dynamics are involved in apoptosis,autophagy,aging and so on.Study confirmed mitochondrial fission was the key step to cause mitochondrial dysfunction(Mt D).However,One report showed that Mt D mediated aldosterone-induced podocyte injury and abrogating Mt D protected against podocyte damage induced by Aldo.From these,we suppose mitochondrial fission mediates Aldo-induced podocyte injury,which may be inhibited by Mdivi-1.Drp1 is an initiator of mitochondrial fission,various upstream signal pathways have been associated with the regulation of Drp1 gene and protein.Actively controlling the upstream signals for Drp1 may prevent mitochondrial fission induced by Drp1.Recently the role of the tumor suppressor gene p53 has been attracted much attention on mitochondrial dynamics.As a tumor suppressor gene,transcriptional regulation factor p53 can lead to cell apoptosis and retardation of cell growth.Increased p53 expression was confirmed in some models of podocyte injury.One investigation reported that p53 was upregulated;p53 transcriptionally activated Drp1 and finally induced apoptosis in neonatal rat cardiomyocytes injury upon H2O2 treatment.We further guess that increased p53 expression induced by Aldo targets Drp1 and controls the consequent podocyte impairment through mitochondrial fission.Part I Objective: To investigate the effect and underlying mechanism of mitochondrial fission and Mdivi-1 in Aldo-induced podocyte injury.Methods:C57BL/6J mice were randomly divided into four groups: Sham group?Mdivi-1 group?Aldo group and Aldo +Mdivi-1 group.Aldo-infused mice were implanted subcutaneously with 14-day-release pellets containing Aldo.Mdivi-1 was given by peritoneal injection.At day 14,mice were sacrificed.Western blot and laser scanning confocal microscopy were used to detect the transient expression for Drp1;the mitochondrial apoptotic signaling pathways and podocyte injury were measured.Results:Compared with sham group,Aldo decreased expression of nephrin and podocin and increased Drp1 expression.Cytochrome C was released from mitochondria and Caspase-3 and Caspase-9 were activated after exposure to Aldo.Administration of Mdivi-1 reduced Drp1 expression and Caspase-3,9 activities.In addition,Mdivi-1 dramatically ameliorated podocyte impairment in Aldo treatment mice.Conclusion:Aldosterone induces mitochondrial fission and podocyte injury,Mdivi-1 may alleviate podocyte injury by mitochondrial apoptotic signaling pathway.Part II p53 may be involved in mitochondrial fission and cell injury in Aldo-treated podocyte through its downstream target Drp1 Objective: To explore the effect of p53 in Drp1 mediated mitochondrial fission and podocyte injury induced by Aldo.Methods:In vitro,podocytes were treated with Aldo at the concentration of 0?25?50?100 mmol/L respectively to evaluate the dose effect of Aldo.The expressions of Drp1 and p53 were examined by RT-PCR and western blot.Then podocytes were transfected with Drp1 sh RNA and p53 sh RNA.The reduction of Drp1 and p53 was measured by RT-PCR and western blot.Then glomerular slit diaphragm proteins nephrin and podocin expressions were investigated by western blot;mitochondrial fission was measured by Mito-Tracker Red;podocyte apoptosis was labelled by TUNEL assay;cytochrome C release was evaluated by immunofluorescent microscopy;mitochondrial permeability transition pore(MPTP)and the mitochondrial membrane potential were examined by MPTP assay kit and JC-1 staining respectively.Results:(1)Aldo induced increased Drp1 and p53 expressions in a dose-dependent manner;(2)down regulation of Drp1 by sh RNA increased nephrin and podocin expressions in Aldo-treated podocytes.Also,Drp1 sh RNA decreased mitochondrial fission and the open of MPTP as well as the decrease of the mitochondrial membrane potential compared with the single Aldo intervention group;(3)Down regulation of p53 by sh RNA led to a decline in Drp1 protein level.Consistent with the results of decreased Drp1 expression,inhibiting p53 expression suppressed mitochondrial fission,mitochondrial and podocyte injury induced by Aldo.Conclusion:Aldo induced increased expression of Drp1 and p53,Aldo induced podocyte injury may be mediated via p53/Drp1/mitochondrial signaling pathway.