Hilary Cassidy1, Jennifer Slyne2, Michael Higgins2, Robert Radford3, Peter J Conlon4, Alan J Watson5, Michael P Ryan2, Tara McMorrow2, Craig Slattery6. 1. UCD Centre for Toxicology, School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland. 2. UCD Centre for Toxicology, School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. 3. Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, United States of America. 4. Department of Nephrology & Transplantation, Beaumont Hospital, Beaumont, Dublin, Ireland. 5. Department of Nephrology, St. Vincent's Hospital, Elm Park, Dublin 4, Ireland; School of Medicine and Medicinal Sciences, University College Dublin, Belfield, Dublin 4, Ireland. 6. UCD Centre for Toxicology, School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. Electronic address: craig.slattery@ucd.ie.
Abstract
BACKGROUND: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated. METHODS: Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort. RESULTS: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells. CONCLUSION: The accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.
BACKGROUND: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated. METHODS:Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort. RESULTS: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells. CONCLUSION: The accumulation of NGAL in supernatants in vitro and in the urine of CANpatients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.
Authors: Kritleen K Bawa; Saffire H Krance; Nathan Herrmann; Hugo Cogo-Moreira; Michael Ouk; Di Yu; Che-Yuan Wu; Sandra E Black; Krista L Lanctôt; Walter Swardfager Journal: J Neuroinflammation Date: 2020-03-14 Impact factor: 8.322