Mitochondrial Dysfunction Mediates Podocyte Damage And Targeted Therapy

Proteinuria is an important clinical manifestation of various glomerular diseases. It is not only a marker of renal diseases, but also a major deteriorating factor for the disease progression, including to end-stage renal disease. Accumulating evidence has shown that proteinuria with glomerular injury is mainly due to the abnormalities of the glomerular filtration barrier with increased glomerular permeability, leading to the abnormal excretion of proteins to urine. The glomerular filtration barrier is composed of 5 layers: endothelial surface layer, the capillary endothelial cells and fenestra, the glomerular basement membrane, podocytes and subpodocyte space. Podocytes line the outer surface of the basement membrane and serve as the final defense against urine protein loss in normal glomerulus. More Recent findings have provided compelling evidence that podocyte injury is intimately related to proteinuria, and to prevent podocyte injury is most important for proteinuria and progression of renal disease, so it may be the target for therapy.Podocytes,also called visceral epithelial cells, are terminally differentiated cells, the main roles of podocytes include involvement in the synthesis of glomerular basement membrane, regulating the glomerular selective permeability. It has been known that podocyte injury involve in many renal disease, such as minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), IgA nephropathy, diabetic nephropathy and HIV-associated nephropathy. But the mechanism of podocyte injury is still being debated.Recently, more and more attentions have been paid on the mitochondrial dysfunction in renal disease. In 2000, Doleris et al. first reported that the lesion of focal segmental glomerulosclerosis occurred in four mitochondrial cytopathies patients, and progressed to end-stage renal failure. Then several studies suggested that mutations of mitochondrial DNA and abnormalities of mitochondrial ultramicrostructure may be associated with podocyte injury and focal segmental glomerulosclerosis. But until 2006, Hagiwara et al. first reported mitochondrial dysfunction in focal segmental glomerulosclerosis, they found that in puromycin aminonucleoside nephrosis, the mitochondrial oxidative phosphorylation and mtDNA copy numbers was downregulated, mtDNA mutation and podocyte apoptosis occurred. All these studies indicated that mitochondrial dysfunction may play an important role in podocyte injury. So with the more understanding of mitochondrial function in podocyte, it will provide new insight for podocyte injury and also provide a possible target for therapy.qObjective: To explore the effect of mitochondrial dysfunction in aldosterone-induced podocyte injury.Methods: C57BL/6J mice were implanted subcutaneously with 14-day-realease pellets containing Aldo. And all the mice were divided into two groups: Sham group, Aldo group. Mice podocytes were cultured in vitro, and divided into control group and Aldo treated group. Moreover, the podocytes were transfected transiently with pcDNA3-h TFAM vector, and were divided into control group, TFAM overexpression group, Aldo treated group and TFAM overexpression combined with Aldo group. The expression of Nephrin, P-cadherin, ZO-1, Fibronectin and Desmin were examined by real-time PCR and western blot. ROS production was determined by DCFDA fluorescence. Mitochondria ultramicrostructure was displayed by electromicrograph morphometry. The ATP content was measured with ATP lite-glo, a luciferase-based luminescence assay kit and the mitochondrial membrane potential was determined by JC-1 staining. Mitochondrial DNA (mtDNA) copy number was detected by real-time PCR. PGC-1αand TFAM expression was also detected by real-time PCR and western blot.Results: (1) Aldo decreased the expression of Nephrin, P-cadherin, ZO-1 and increased the expression of Fibronectin and Desmin on dose-dependent and time-dependent manner; (2) In vivo, Aldo induced the podocyte foot process detachment and effacement., and Aldo aldo induced the downregulation of Nephrin, P-cadherin, ZO-1 and upregulation of Fibronectin and Desmin; (3) After 12h treatment of 100 nmol/L Aldo, the mitochondria ultramicrostructure morphology displayed vacuolization and decreased distribution in podocytes, and moreover ROS was increased, while the mitochondrial membrane potential, the mtDNA and ATP synthesis were decreased as well as the expression of PGC1αand TFAM; (4) Overexpression of TFAM inhibited the downregulation of mitochondrial membrane potential and mtDNA, and prevent podocyte injury induced by Aldo. Conclusions: Aldo induced podocyte injury in vivo and in vitro, mitochondrial dysfunction mediates Aldo-induced podocyte injury.Objective: To explore the effect of rosiglitazone (Rosi) on podocyte injury and mitochondrial dysfunction induced by aldosterone. Methods: C57BL/6J mice were implanted subcutaneously with 14-day-realease pellets containing Aldo. And all the mice were divided into three groups: Sham group, Aldo group, and Rosi-treated group. All mice were sacrificed at day 15. The proteinuria was evaluated by urine protein: urine creatinine. Histology of kidney was tested by Periodic acid Schiff (PAS) staining, and the podocytes morphology was investigated by transmission electron microscopy. Mice podocytes were cultured in vitro, and was treated with Rosi, Aldo, Aldo+Rosi, Aldo+EPL, Aldo+ROT separately. Moreover, the podocytes were transfected transiently with pcDNA3.1-PPARγor PPARγsiRNA vector and than treated with Rosi, Aldo, Aldo + Rosi separately. The expression of Nephrin, P-cadherin, ZO-1, Fibronectin and Desmin were detected by real-time PCR and western blot. ROS production was determined by DCFDA fluorescence. Mitochondria ultramicrostructure was displayed by electromicrograph morphometry. The ATP content was measured with ATP lite-glo, a luciferase-based luminescence assay kit and the mitochondrial membrane potential was determined by JC-1 staining. Mitochondrial DNA (mtDNA) copy number was detected by real-time PCR. And expression of PGC-1αand TFAM were also detected by real-time PCR and western blot. The urinary F2-isoprostane was detected by ELISA and renal cortex lipid peroxides was assessed by lipid extraction and spectrophotometric.Results: (1) Rosi pretreatment inhibited Nephrin decrease induced by Aldo, and PPARγwas also expressed in podocytes, Aldo (100nmol/L) decreased the expression of PPARγ, while Rosi (5μmol/L) pretreatment restored the PPARγexpression; (2) Rosi pretreated inhibited the decrease of Nephrin, P-cadherin and ZO-1, while inhibited the increases of Fibronectin and Desmin induced by Aldo; (3) In vivo, Rosi reduced proteinuria, ameliorated the renal histological damage, and restored the expression of podocytes specific proteins; (4) Aldo-induced ROS production was markedly inhibited by mitochondrial complex I inhibitor rotenone, mineralocorticoid receptor antagonist EPL and PPARγagonist Rosi; (5) Mineralocorticoid receptor antagonist EPL prevented the damage of mitochondria ultramicrostructure, the mitochondrial membrane potential, the mtDNA and ATP synthesis as well as the expression of PGC1-αand TFAM; (6) Both PPARγagonist Rosi and overexpression of PPARγrestored the mitochondria ultramicrostructure, upregulated the mitochondrial membrane potential, the mtDNA and ATP synthesis as well as the expression of PGC-1αand TFAM. Moreover, when the expression of PPARγwas inhibited by siRNA, the protective effect of Rosi was disappeared; (7) In vivo, Rosi effectively inhibited the urine F2-isoprostane and renal cortex malondialdehyde as well as the ROS production. And Rosi also ameliorated the downregulation of mitochondrial membrane potential, the mtDNA and ATP synthesis induced by Aldo, and also upregulated the PGC-1αand TFAM expression.Conclusions: MR mediates the pathogenic actions of Aldo in podocytes, includes the overproduction of mitochondrial ROS and induction of mitochondrial dysfunction. Moreover, Rosi prevented Aldo-induced podocytes injury via inhibiting the mitochondrial ROS production and restoring the mitochondrial function mainly in PPARγ-dependent manner.Objective: To observe the protective effects of Huai Qihuang (HQH) on adriamycin (ADR) induced podocyte injury, and explore the effect of HQH on mitochondrial function.Methods: Eighteen male SD rats were randomly divided into three groups; include control group, ADR group and HQH treated group. All rats were sacrificed at day 15. 24h proteinuria was detected at day 7 and day 15. Histology of kidney was tested by Periodic acid Schiff (PAS) staining; the podocyte ultramicrostructure was investigated by transmission electron microscopy. The expression of Nephrin, Podocin, PGC-1αand TFAM were examined by real-time PCR and western blot. The macrophage infiltration was detected by immunohistochemistry and western blot. The levels of serum TNF-αand IL-1βwere evaluated by the enzyme linked immunosorbent assay (ELISA). Moreover, mice podocytes were cultured in vitro and expression of Nephrin, Podocin, PGC-1αand TFAM were examined by real-time PCR and western blot. The mtDNA as well as podocyte TNF-αand IL-1βmRNA were also detected by real-time PCR. The TNF-αand IL-1βin the supernatant of cultured podocytes were also detected by ELISA.Results: (1) ADR rats showed heavy proteinuria, detachment and effacement of podocyte foot process, while HQH treatment partly reduced proteinuria, ameliorated the detachment and effacement of podocyte foot process; (2) ADR rats displayed the decreased expression of Nephrin and Podocin, while HQH treatment increased expression of Nephrin and Podocin; (3) Compared to ADR rats, HQH treatmentinhibited the the infiltration of macrophage and increase of serum cytokines TNF-αand IL-1β; (4) HQH upregulated the expression of PGC-1αand TFAM, which were downregulated in ADR rats; (5) In vitro, HQH pretreatment significantly restored the expression of Nephrin, Podocin, PGC-1α, TFAM and mtDNA copy numbers, while HQH also reduced the levels of TNF-αand IL-1βboth in podocytes and in the supernatant of cultured podocytes.Conclusion: ADR causes proteinuria and podocyte injury, induces inflammation and inhibits the mitochondrial biogenesis function, while HQH reduces proteinuria and podocyte injury, inhibits inflammation and increases the mitochondrial biogenesis function.