PGC-1α Regulates Mitochondrial Function Blocking Chronic Kidney Disease Podocyte Injury

Podocytes are highly differentiated glomerular epithelial cells with limited potential to divide. They are primarily responsible for maintaining the integrity of the glomerular basement membrane, and abnormal podocyte morphology and podocyte dysfunction are involved in proteinuria. Moreover, podocyte depletion is a major mechanism driving glomerulosclerosis, which leads to end-stage renal disease (ESRD).Mitochondria are complex intracellular organelles that are responsible for various metabolic functions, including energy production via oxidative phosphorylation. Mitochondria are also a major source of reactive oxygen species (ROS), and the overproduction of ROS damages mitochondrial DNA (mtDNA) and the oxidation respiratory chain, which ultimately causes mitochondrial dysfunction (MtD). In general, MtD is characterized by increased ROS production, the accumulation of impaired mtDNA, and progressive respiratory chain dysfunction. We previously demonstrated that MtD is an early event in aldosterone-induced podocyte injury. However, the role of MtD in podocyte loss remains unknown.PGC-la (Peroxisome proliferator-activated receptor-y coactivator-la), the first identified cofactor of the nuclear hormone receptor PPAR and other nuclear hormone receptors, potently modulates mitochondrial biogenesis and function. PGC-la is also an important regulator of diverse metabolic pathways in response to environmental and physiological changes. The overexpression of PGC-1α increases oxidative capacity in cultured myotubes by improving lipid metabolism and increasing the expression of genes involved in mitochondrial function and biogenesis. The overexpression of PGC-la also decreases apoptosis in adipose-derived stem cells by reducing intracellular and mitochondrial ROS levels. In vivo, specific PGC-1α overexpression in the muscle increases exercise performance and peak oxygen uptake. However, the role of PGC-1α in podocyte loss has not been defined.Recent clinical and experimental studies have shown that aldosterone (Aldo) is a potent inducer of proteinuria and proteinuria significantly decreases after Aldo inhibitors treatment in many glomerular diseases, Aldo has recently attracted further attention for podocytes as novel targets of Aldo. Circulating Aldo is associated with hypertension, chronic kidney disease, obesity, metabolic syndrome, concentric left ventricular hypertrophy, and may predict cardiovascular, renal, and metabolic disease.We previously demonstrated Aldo induces excess production of reactive oxygen species (ROS) and oxidative stress in podocyte, and injures podocytes via MtD resulting in disruption of glomerular filtration barrier, proteinuria, and progression of chronic kidney disease in mice model. More, evidence shown mineralocorticoid receptor (MR) activation plays a pivotal role in the pathogenesis of chronic kidney disease in metabolic syndrome, and that MR may be activated both Aldo dependently and independently.In the present study, we employed transgenic approaches to fully investigate the role of the PGC-la/mitochondria axis in modulating Aldo-induced podocyte loss and phenotypic alteration. In detail, we examined the ability of 1) PGC-1α overexpression in mice to protect against podocyte loss and MtD,2) MtD to confer Aldo-induced podocyte loss, and 3) PGC-la overexpression in cells to block Aldo-induced MtD and podocyte detachment.Part 1 Expression of PGC-la in renal biopsy tissue of children with chronic kidney diseaseObjective:To explore the expression of PGC-1α in kidneys of patients with podocyte disease.Methods:We choose 20 cases of patients with podocyte diseases, male (12,66%) and female (8,34%), with an average age of 5 years and 9 months. The types of these patients involved:10 cases of FSGS,10 cases of MCD,6 cases of Normal control. The normal renal tissues, were from the 6 cases of children with normal urinary protein, who received the kidney tumor resection. We examined PGC-la expression in renal biopsy specimens by immunohistochemistry, and WT-1、 a3-intergrin expression by immunofluorescence.Results:Immunohistochemical staining showed that PGC-la of kidney tissue biopsies in children with podocyte disease significantly reduced compared with normal kidney tissue, especially in glomerular. PGC-la expression was mainly located in the renal tubular and glomerular. PGC-1α expression decreased significantly in FSGS group compared with MCD group. PGC-1α expression was positively correlated with the decrease of a3-integrin and WT-1.Conclusion:PGC-la expression in glomerular of chlidren with podocyte disease was significant reduced and FSGS group was more lessened than MCD group. PGC-la expression was positively correlated with the decrease of a3-integrin and WT-1.Part 2 Conditional podocyte PGC-la deletion aggravate aldosterone induced mitochondrial dysfunction and podocyte injuryObjective:To further explore PGC-la deletion on podocyte on mitochondrial dysfunction and podocyte injuryMethods:Differents concentration of 25、50、100 nmol/L Aldo were treated cultured podocytes. To generate of the podocyte specific deletion of PGC-la knockout mice, homozygous mice with PGC-1αfloxflox were crossed with NPHS2 cre(+) mice to get PGC-1αflox/+NPHS2cre(+) mice. Then PGC-laflox/+NPHS2cre(+) mice were crossed with PGC-lafloxflox mice to get PGC-laflox/floxNPHS2cre(+) male mice(PO-PGC-la-CKO). All male littermates with Cre transgene negative served as a control (WT). CKO and WT mice weighing 25-30 g were treated with Aldo using osmotic subcutaneously implanted mini-pumps (300ng/kg/d), after 14D, mice were sacrificed. By western blotting and qRT-PCR, the expression of a3-integrin, PGC-la, and markers of podocyte proteins Nephrin, fibroblast marker proteins MMP9, a-SMA and desmin were detected; the cell adhesion assay detection kit was used to examze podocyte adhesion function.Urinary albumin and serum creatinine and blood urea nitrogen (BUN) levels were detected as above. Glomerular morphology was explored by PAS staining and Electron microscopy. DNA from Cells and tissues was extracted and Mitochondrial DNA (mtDNA) copy numbers were determined by PCR. Luciferase enzymatic was used to detect ATP content, kit was used to detect the activity of the mitochondrial respiratory chain (Ⅰ,ⅡI,Ⅲ,Ⅳ).Results:Aldosterone in a dose and time-dependent decrease podocyte PGC-1α expression and induced podocyte phenotype changes:increased MMP9, a-SMA and desmin and reduce the expression of P-cadherin and nephrin. Aldosterone also time and dose dependently inhibited the a3-integrin expression and reduce podocyte adhesion.50μmol/L ethidium (EtBr) was used to induce mitochondrial dysfunction in podocyte, characterlyzed by increasement of reactive oxygen species and decreasement of mtDNA copy number and mitochondrial membrane potential mitochondrial dysfunction, mitochondrial dysfunction further increases MMP9, a-SMA and desmin and inhibit P-cadherin and expression of nephrin. After aldosterone infusion 14d (300ng/kg/d),24 h urinary albumin increased significantly in CKO mice compared with WT mice; electron microscope was used to observe the fusion of foot processes, CKO mice exhibited more seriously foot process fusion and reduction of a3-integrin and nephrin aggravated in CKO mice. Phenotypic changes also increased significantly in CKO mice, WT-1 number decreases more significantly in CKO mice after Aldo infusion.Conclusion:These findings demonstrate that conditional podocyte PGC-1α deletion aggravated podocyte depletion after aldo infusion, possibly by maintaining normal mitochondrial function.Part3 PGC-la overexpression on aldosterone-induced podocyte injury and role of mitochondriaObjective:The effect of PGC-la overexpression on aldosterone-induced podocyte injury and role of mitochondriaMethods:Overexpression of PGC-1 a by Ad-PGC-la transfection,10-week-old PGC-1α transgenic (TG) and wild-type (WT) male mice weighing 25-30 g were treated with Aldo using osmotic subcutaneously implanted mini-pumps. The pumps delivered a continuous infusion of Aldo (300μg/kg/d) (Alzet, Durect, Cupertino, CA) for 14 days. The a3-integrin expression and podocyte detachment MMP-9, a-SMA, desmin, P-cadherin, and nephrin were determined in protein level. The ATP content was detected by luminescence assay kit and mitochondrial membrane potential was examined by JC-1 staining. DNA from Cells and tissues was extracted and mtDNA were determined by PCR. ROS production was detected by DCFDA staining and analyzed by flow cytometry. Global PGC-la transgenic mice were generated, and PGC-1α expression was determined by RT-PCR and western blotting from different tissues, such as kidney, spleen, liver. Glomerular morphology was explored by PAS staining and Electron microscopy. Mitochondria from glomeruli were isolated using a kit purchased from Sigma following the manufacturer’s protocol. The ATP content and complex Ⅰ,Ⅱ, Ⅲ and Ⅳ enzyme activities of isolated mitochondria were measured using microplate assay kits. Urinary albumin was measured by a mouse Albumin ELISA according to the manufacturer’s instructions. Serum creatinine and blood urea nitrogen (BUN) levels were measured using commercially available kits following the manufacturer’s instructions.Results:We confirmed the overexpression of PGC-1α in podocytes entirely reversed podocyte detachment and phenotypic changes induced by Aldo via improving mitochondrial function. PGC-1α overexpression in podocyte protects phenotypic change and podocyte depletion and the involvement of mitochondria in this process. We then induced glomerular disease by aldosterone (Aldo) infusion. Chronic Aldo infusion caused significant podocyte loss and remarkable podocyte phenotypic alterations. Strikingly, the increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in wild-type mice but not in PGC-la transgenic mice. Western blot analysis and immunofluorescence shown overexpression of PGC-la prevented both podocyte loss and podocyte phenotypic changes in mice. Furthermore, Aldo-induced MtD was markedly attenuated in PGC-la transgenic mice.Conclusion:PGC-la overexpression protects podocyte phenotypic change and podocyte depletion through blocking MtD.