WT1 in the adult kidney : podocyte maintenance and the epithelial-mesenchymal balance

Abstract

Glomerular diseases are the leading cause of end stage kidney disease worldwide. Podocyte injury plays a key role in the initiation and development of such diseases, which follow a progressive course due to the limited capacity of podocytes to regenerate. Podocytes are highly specialised, terminally differentiated cells, which play a vital role in the glomerular filtration barrier. They are also the main sites of expression of the Wilms Tumour Suppressor gene, WT1, in the adult. WT1 is a complex gene, which plays an essential role in renal development by controlling the process of mesenchymal to epithelial transition that forms the nephron. Adult podocytes maintain both epithelial and mesenchymal features and continue to express high levels of WT1. Little is known about the role of WT1 in adult podocytes as previous studies have been limited due to the confounding developmental effects and embryonic lethality of existing animal models. This thesis sought to investigate the hypothesis that WT1 is an essential gene in adult kidney and plays a fundamental role in the adult podocyte. Given its role in nephron development, WT1 loss was hypothesised to result in dedifferentiation and an alteration of the epithelial-mesenchymal balance in the podocyte, affecting its specialised function. Using an inducible, conditional animal model of Wt1 loss, Wt1 was deleted from the adult, confirming its essential role in adult kidney. Wt1 deletion resulted in severe podocyte injury and failure of the glomerular filtration barrier, as well as loss of expression of key podocyte genes. Preliminary analysis suggests this was not simply due to podocyte apoptosis and/or detachment, supporting a role for Wt1 in podocyte differentiation. This was corroborated by in vitro studies that demonstrated a requirement for Wt1 for podocyte differentiation. Significantly, Wt1 loss resulted in a marked change in the expression of epithelial and mesenchymal markers in podocytes, with upregulation of mesenchymal characteristics, in keeping with a transitional stage consistent with an earlier developmental form. To investigate the mechanism behind these findings a conditionally immortalised podocyte cell line was generated as a model of Wt1 loss in vitro. In order to confirm and specifically analyse the podocyte phenotype, BAC recombineering was utilised to produce a promoter-reporter transgene construct to attempt to generate a fluorescent-labelled, podocyte specific animal model of Wt1 loss. The findings of this thesis establish that Wt1 is essential for adult podocyte function, and appears to be a key upstream regulator of podocyte differentiation. Extension of this work may allow the identification of potential targets to promote podocyte differentiation and/or regeneration in the setting of acquired and progressive glomerular disease.