Abstract: FR-PO941
Towards Clinical Assays for Evaluating Circulating Permeability Factors in Nephrotic Syndrome
Session Information
- Glomerular Diseases: Podocyte Biology - II
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Veissi, Susan, Amalia Children's Hospital, Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands
- Smeets, Bart, Radboud University Medical Center, Nijmegen, Netherlands
- Van Wijk, Joanna, Vrije Universiteit University Hospital Amsterdam, Amsterdam, Netherlands
- Jeronimus-Klaasen, Annelies, Amalia Children's Hospital, Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands
- Van den heuvel, Lambertus P.W.J., Amalia Children's Hospital, Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands
- Schreuder, Michiel F., Amalia Children's Hospital, Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands
Background
Circulating permeability factors (CPFs) have been implicated as one of the causes of nephrotic syndrome (NS). Evidence for CPFs comes mainly from clinical observations and animal studies, since reliable in vitro assays are lacking. Therefore, we aim to study the presence and pathogenic relevance of CPFs in plasma of NS patients during active disease and remission using conditionally immortalized human podocytes (ciHPOD) and primary human glomerular microvascular endothelial cells (GMVECs) in vitro.
Methods
Podocytes (ciHPOD) and primary endothelial cells (GMVECs) were incubated with plasma from NS patients in relapse and remission as well as from a non-renal control patient. Cell viability, podocyte motility, podocyte actin cytoskeleton architecture, and reactive oxygen species (ROS) formation at the presence or absence of ROS scavenger, dimethylthiourea, were investigated by CCK-8 assay, scratch-assay, immunofluorescence stainings, and CM-H2DCFDA probing, respectively.
Results
Plasma from active NS patients, but not from remission or control patients, caused excessive ROS formation in podocytes, but not in endothelial cells. Immunofluorescence microscopy revealed severe derangement of the podocyte’s actin cystoskeleton in response to active NS plasma. Moreover, the motility of podocytes seemed to be reduced in the presence of active NS plasma, but not in the presence of remission or control plasma. Prolonged incubation of podocytes, but not endothelial cells, led to cell death only when active NS plasma was present. Furthermore, the ROS scavenger dimethylthiourea abolished the ROS formation and the podocyte’s actin cytoskeleton dearangement and cell death in response to active NS plasma, suggesting that ROS plays an important role in podocyte injury in NS.
Conclusion
We provide a high-throughput and sensitive assay to measure ROS in response to NS plasma, providing a new framework for monitoring in vivo CPF activity that could be used for diagnostics or disease monitoring purposes. Moreover, our findings suggest that the inhibition of ROS formation or facilitating rapid ROS scavenging might exert beneficial effects in patients with NS.