title: Smad3/Nox4-mediated mitochondrial dysfunction plays a crucial role in puromycin aminonucleoside-induced podocyte damage.
abstract: Podocyte depletion due to apoptosis is the key hallmark of proteinuric kidney disease progression. Recently, several studies reported that mitochondrial (mt) dysfunction is involved in podocyte injury, while the underlying molecular mechanisms remain elusive. This study investigated the potential proximal signaling related to in vitro and in vivo mitochondrial dysfunction in a puromycin aminonucleoside (PA)-induced podocyte injury model. PA time- and dose-dependently resulted in cultured mouse podocyte damage, presenting with an increase of apoptotic cells and induction of activated caspase3/9. PA also caused mitochondrial damage and dysfunction based on the downregulation of the mtDNA level, decrease of transcriptional factors mtTfa and Nrf-1, decrease of CoxI, II and IV, and reduction of the oxygen consumption level and mitochondrial membrane potential level as well as excessive production of cellular ROS. Additionally, antioxidant MnSOD and catalase levels were decreased in mitochondrial fractions, and reduction of complex I and IV activity was also observed in PA-stimulated podocytes. Furthermore, an obvious translocation of p-Smad3 from the cytosol to nuclei and induction of mitochondrial Nox4 were detected following PA application. The PA-induced shift of cytochrome c was observed from mitochondria to the cytoplasm. Induction of Nox4 by PA administration was significantly repressed by Smad3-shRNA, while Nox4-shRNA showed no effect on PA-induced p-Smad3 activation. Notably, both Smad3 and Nox4 silencing significantly prevented the reduction of the mtDNA level, restored mitochondrial function, and decreased cellular apoptosis in PA-stimulated podocytes. A similar mitochondrial dysfunction was obtained in a PA-injected nephropathy rat, which was effectively inhibited by treatment with the antiproteinuric drug prednisone. In addition, Dab2 knockdown decreased albumin uptake and influx whereas it showed no effect on cellular apoptosis in PA-stimulated podocytes. In conclusion, our findings demonstrated that Smad3-Nox4 axis-mediated mitochondrial dysfunction is involved in PA-induced podocyte damage likely via increasing ROS generation and activating the cytochrome c-caspase9-caspase3 apoptotic signaling pathway. Dab2 may be required for the increased permeability of podocytes following injury.
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Pediatrics, First Hospital of Jilin University, Changchun 130031, China.
authors.forename: Yanbo
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pneumology, Second Hospital of Jilin University, Changchun 130041, China.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China.
authors.forename: Qifeng
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Gu
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Renal-Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pediatrics, Peking University First Hospital, Beijing 100034, China. Electronic address: qingfeng@mail.med.upenn.edu.
authors.forename: Qingfeng
authors.initials: Q
authors.lastname: Fan
authors.affiliation: Department of Nephrology, First People's Hospital of Kunshan, Affiliated Kunshan Hospital of Jiangsu University, Kunshan, Jiangsu 215300, China. Electronic address: ks_yjm@163.com.
authors.forename: Jianming
authors.initials: J
authors.lastname: Ye
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D055785:Gene Knockdown Techniques; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D011241:Prednisone / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011692:Puromycin Aminonucleoside; D017207:Rats, Sprague-Dawley; D051900:Smad3 Protein / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000418:Albumins; D051017:Apoptosis Regulatory Proteins; D004272:DNA, Mitochondrial; C478773:Dab2 protein, mouse; D051900:Smad3 Protein; D011692:Puromycin Aminonucleoside; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D011241:Prednisone
keywords: Mitochondrial dysfunction; NADPH oxidative (Nox4); Podocyte damage; Proteinuria; Puromycin aminonucleoside (PA); Smad3