M BAKHTIAR1, YM WANG1, G ZHANG1, J KARUNIA1, A WILARAS1, Y CHEN2, S ALEXANDER1, H MCCARTHY1
1Centre For Kidney Research, Children’s Hospital At Westmead, Sydney, Australia, Sydney, Australia, 2Molecular Oncology Laboratory; Children’s Cancer Research Unit; Kids Research Institute, Sydney, Australia , Sydney, Australia
Background: In steroid resistant nephrotic syndrome (SRNS) without a genetic aetiology, pathogenesis is considered secondary to a circulating factor. Utilising a conditionally immortalised human podocyte (ciPod) cell line, we studied the effect of human active disease plasma on podocytes and the role of protease activated receptors (PARs).
Methods: Expression of the four PARs (PAR1-4) in ciPod was evaluated through RT-PCR. FITC-phalloidin staining was used to qualitatively assess patient’s plasma-mediated actin cytoskeleton changes. Cell viability assays using the xCELLigenceTM Real-Time Cellular Analysis system (ACEA Biosciences) were performed to quantify podocyte damage. Differentiated ciPod were pre-treated with PAR1 selective antagonist RWJ56110 (Tocris) for 30 minutes, and then treated with fresh media with or without 10% patient plasma. Response to 0 µM, 10 µM, 20 µM or 30 µM RWJ56110 was measured for 24 hours. Percentage change in cell index compared to baseline was used for statistical analysis of cell viability.
Results: The ciPod cell line displayed PAR1 and PAR2 expression. 10% patient plasma treatment for 24 hours (p=0.0024, n = 2) led to a significant loss in viability compared to the control group and immunofluorescence demonstrated weaker actin filament staining at both 24 and 48 hours. Pre-treatment with RWJ56110 reduced the podocyte damage in a dose-dependent manner: 30 µM RWJ56110 yielding a less significant reduction in cell viability after 24 hours compared with the no patient plasma, no drug control (p=0.0206, n=2).
Conclusions: Our study confirms that SRNS plasma impacts the survival of human podocytes in vitro. This effect may be ameliorated by blockade of PAR1. Further studies are required to support these findings and to extend the mechanistic role of PAR1 and PAR2 in podocyte damage.
Mahnoor Bakhtiar completed a Bachelor of Medical Science (Honours, Class I) from the University of Sydney in 2017. Her honours’ project at the Centre for Kidney Research (Children’s Hospital at Westmead) looked into novel podocyte-based assays for assessing potential biomarkers and therapeutics in idiopathic steroid-resistant nephrotic syndrome. She has a strong interest in understanding the nature of circulating factor disease and gene therapy in steroid-resistant nephrotic syndrome.