Abstract 1491: Notch1 Inhibition Interferes with Differentiation of Glomerular Progenitor Cells into Podocytes

Abstract

The objective of this work was to identify a population of glomerular progenitor cells (GPCs) capable of generating podocytes which are specialized epithelial cells that stabilize glomerular capillaries and participate in the barrier function of the glomerular filter. The stem cell antigen prominin-1 was detected in GPCs located within the glomerular tuft of the mouse kidney; these cells expressed also the Notch1 receptor. The Notch1 pathway mediates cell fate decision by regulating the expression of transcription factors that maintain stemness or promote differentiation. Upon ligand binding, the Notch1 receptor is cleaved by γ-secretase and the active intracellular domain of Notch1 (N1ICD) translocates to the nucleus where it triggers transcription of Notch1-dependent genes. A consistent co-localization of N1ICD and the transcription factor WT-1 was demonstrated in synaptopodin-positive cells suggesting that Notch1 activation induced the differentiation of GPCs into podocytes. To strengthen this possibility, experiments were conducted in newborn mice because early postnatally there is a dramatic increase in kidney volume and in the number of glomeruli. The function of Notch1 in the generation of podocytes was established by interfering with the Notch1 pathway through the administration of a γ-secretase inhibitor to neonatal mice; γ-secretase inhibition opposes the release and translocation of N1ICD to the nucleus and the initiation of transcription. Over a period of 4–7 days, treated mice showed a 50% reduction in the number of glomeruli. However, the size of glomeruli was preserved. Importantly, in glomeruli of treated mice, the fraction of WT-1-positive cells that co-expressed N1ICD was decreased 50%. Additionally, podocytes showed an immature phenotype lacking synaptopodin in 30% of glomeruli. Quantitative RT-PCR documented that γ-secretase inhibition attenuated the expression of prominin-1, the early developmental kidney transcription factor Pax2 and the podocyte lineage marker WT-1. In conclusion, we have documented that prominin-1-positive GPCs acquire the podocyte phenotype and the Notch1 pathway controls glomerular number during postnatal development by modulating podocyte differentiation.