Mapping the Molecular Atlas of Podocyte Response to Glucocorticoids Using Multi-Omics Analyses

Journal of the American Society of Nephrology, vol. 34, pp. 1014

Abstract

Background: Glucocorticoids are the mainstay of therapy for idiopathic nephrotic syndrome (iNS). Glucocorticoids have been found to directly influence podocyte behaviour and morphology, but the exact mechanism is unknown. Understanding this mechanism may help to identify targets for more effective treatment options with fewer side effects compared to glucocorticoids. We established an animal and kidney organoid model of steroid-sensitive NS to dissect the molecular mechanisms of glucocorticoids on podocytes using single-cell ATAC and RNA sequencing. Methods: We used the puromycin aminonucleoside (PAN) rat model using one intravenous injection of PAN (10mg/100g) and daily intraperitoneal injections of prednisolone (1.5mg/100g). Human induced pluripotent stem cells were used to make kidney organoids, which were injured to various degrees using Adriamycin (ADR; ranging 0.5-2.5 ug/mL) or PAN (ranging 25-100ug/mL) and rescued with prednisolone (ranging 5-100 ug/mL). Single-cell sequencing was performed using the 10X genomics platform. Results: Increased proteinuria, foot process effacement and increased podocyte specific desmin-protein expression were observed after PAN injection in rats. The injury was partially rescued by prednisolone treatment, whereas higher doses of prednisolone showed more proteinuria and desmin expression. Prednisolone treatment was not able to rescue more severe injury (by higher PAN injection). At the moment we are analysing single-cell ATAC and RNA sequencing data to identify responses in the podocytes to prednisolone. ADR and PAN can both be used to injure podocytes in kidney organoids, confirmed by western blot for autophagy markers and immunofluorescence for podocyte markers and caspase 3. Currently, rescue with prednisolone is tested before single-cell RNA sequencing is used to map molecular changes in the rescued podocytes. Conclusions: We established a partially glucocorticoid-responsive in vivo model of NS and are currently performing single-cell sequencing to unravel mechanisms involved in prednisolone-induced podocyte recovery. Interestingly, high doses of prednisolone have adverse effects, and when the injury is too severe it cannot be rescued with prednisolone. In human kidney organoids, podocytes can be injured with ADR or PAN and recovery is being tested at the moment.

Authors 5

1. Martijn van den Broek

   Radboud University Nijmegen · Radboud University Medical Center · Amalia Kinderziekenhuis

   Affiliation as printed

   Department of Pathology, Radboud University Medical Center, Nijmegen, Netherlands;

   Department of Pediatric Nephrology, Radboud University Medical Center, Amalia Children's Hospital, Nijmegen, Netherlands;

2. Bartholomeus T. van den Berge

   Radboud University Nijmegen · Radboud University Medical Center

   Affiliation as printed

   Department of Nephrology, Radboud University Medical Center, Nijmegen, Netherlands;

   Department of Pathology, Radboud University Medical Center, Nijmegen, Netherlands;

3. Michiel F. Schreuder

   Radboud University Nijmegen · Radboud University Medical Center · Amalia Kinderziekenhuis

   Affiliation as printed

   Department of Pediatric Nephrology, Radboud University Medical Center, Amalia Children's Hospital, Nijmegen, Netherlands;

4. Bart Smeets

   Radboud University Nijmegen · Radboud University Medical Center

   Affiliation as printed

   Department of Pathology, Radboud University Medical Center, Nijmegen, Netherlands;

5. Jitske Jansen Aachen Institute of Experimental Medicine and Systems Biology

   RWTH Aachen University

   Affiliation as printed

   Institute of Experimental Medicine and Systems Biology. RWTH Aachen University, Aachen, Germany