Bhavya Khullar1, Renu Balyan2, Neelam Oswal2, Nidhi Jain2, Amita Sharma1, Malik Z Abdin3, Arvind Bagga4, Shinjini Bhatnagar1, Nitya Wadhwa1, Uma Chandra Mouli Natchu1, Anna George2, Satyajit Rath1,2, Vineeta Bal1,2, Shailaja Sopory5. 1. Pediatric Biology Center, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, 121001, India. 2. National Institute of Immunology, New Delhi, India. 3. Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India. 4. Division of Paediatric Nephrology, Department of Paediatrics, All India Institute of Medical Sciences, New Delhi, India. 5. Pediatric Biology Center, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, 121001, India. ssopory@thsti.res.in.
Abstract
BACKGROUND: The induction of CD80 on podocytes has been shown in animal models of podocyte injury and in certain cases of nephrotic syndrome. In a lipopolysaccharide (LPS)-induced mouse model of albuminuria, we have recently shown a signalling axis of LPS-myeloid cell activation-TNFα production-podocyte CD80 induction-albuminuria. Therefore, in this report, we investigated the cellular and molecular consequences of TNFα addition and CD80 expression on cultured podocytes. METHODS: A murine podocyte cell line was used for TNFα treatment and for over-expressing CD80. Expression and localization of various podocyte proteins was analysed by reverse transcriptase-polymerase chain reaction, western blotting and immunofluorescence. HEK293 cells were used to biochemically characterize interactions. RESULTS: Podocytes treated with LPS in vitro did not cause CD80 upregulation but TNFα treatment was associated with an increase in CD80 levels, actin derangement and poor wound healing. Podocytes stably expressing CD80 showed actin derangement and co-localization with Neph1. CD80 and Neph1 interaction was confirmed by pull down assays of CD80 and Neph1 transfected in HEK293 cells. CONCLUSION: Addition of TNFα to podocytes causes CD80 upregulation, actin reorganization and podocyte injury. Overexpressed CD80 and Neph1 interact via their extracellular domain. This interaction implies a mechanism of slit diaphragm disruption and possible use of small molecules that disrupt CD80-Neph1 interaction as a potential for treatment of nephrotic syndrome associated with CD80 upregulation.
BACKGROUND: The induction of CD80 on podocytes has been shown in animal models of podocyte injury and in certain cases of nephrotic syndrome. In a lipopolysaccharide (LPS)-induced mouse model of albuminuria, we have recently shown a signalling axis of LPS-myeloid cell activation-TNFα production-podocyte CD80 induction-albuminuria. Therefore, in this report, we investigated the cellular and molecular consequences of TNFα addition and CD80 expression on cultured podocytes. METHODS: A murine podocyte cell line was used for TNFα treatment and for over-expressing CD80. Expression and localization of various podocyte proteins was analysed by reverse transcriptase-polymerase chain reaction, western blotting and immunofluorescence. HEK293 cells were used to biochemically characterize interactions. RESULTS: Podocytes treated with LPS in vitro did not cause CD80 upregulation but TNFα treatment was associated with an increase in CD80 levels, actin derangement and poor wound healing. Podocytes stably expressing CD80 showed actin derangement and co-localization with Neph1. CD80 and Neph1 interaction was confirmed by pull down assays of CD80 and Neph1 transfected in HEK293 cells. CONCLUSION: Addition of TNFα to podocytes causes CD80 upregulation, actin reorganization and podocyte injury. Overexpressed CD80 and Neph1 interact via their extracellular domain. This interaction implies a mechanism of slit diaphragm disruption and possible use of small molecules that disrupt CD80-Neph1 interaction as a potential for treatment of nephrotic syndrome associated with CD80 upregulation.
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