Study of the role of Rb and, the combined roles of Rb and p53 pathways, during kidney development

Abstract

Introduction: Establishment of renal nephron endowment occurs exclusively during embryonic development and is critical for proper renal function in the adult. Therefore, a better understanding of the signals and pathways that regulate the balance between nephron progenitor cell (NPC) proliferation and differentiation is essential. Studies of renal development in mice helped advance our understanding of the pathways governing some aspects of kidney development in humans given the relative conservation of the molecular mechanisms involved. The mammalian kidney maintains a population of self-renewing (Cited1+; Six2+) stem cells until termination of nephrogenesis. The Retinoblastoma protein, Rb, and the p53 pathways play key roles in the control of cell proliferation, senescence and apoptosis in many organs. Previous studies have uncovered a requirement for p53 in the regulation of renal development, specifically the maintenance of self-renewal capacity of the (Cited1+; Six2+) population and terminal differentiation in nephrons. However, it is still unknown whether Rb plays a role during kidney development. In the present study, we have examined and described for the first time a specific requirement for Rb during renal development, and the existence of crosstalk between the Rb and p53 pathways that are indispensable for proper kidney development and function in mice.

Methods: Using tamoxifen-inducible Nestin-CreERT2-YFP/YFP mice, and, single or double Rb;p53 floxed/floxed (fl/fl) mice, we induced conditional deletion(s) of Rb alone, p53 alone or both genes that are targeted to Nestin-positive cells and their progeny. Recombined renal cells include cap mesenchyme stem cells and progenitors and their progeny, endothelial cells in immature glomeruli, and podocytes of mature glomeruli. Tamoxifen was administered by oral gavage to pregnant females at E10.5 (the onset of nephrogenesis) and at E13.5. Embryonic kidneys were collected from single and double mutant embryos as well as wild type and heterozygote littermate controls at distinct developmental stages. Histological analyses and gene expression studies were conducted to characterize the renal phenotypes in the absence of Rb and/or p53.

Results: We assessed stem and progenitor cell proliferation, differentiation and survival in embryonic kidneys carrying single or combined deletion(s) in Rb and/or p53 at distinct stages during nephrogenesis. Our results showed that Rb negatively controls NPCs proliferation, and is needed for nephron survival around birth. Hence, compared with Rb littermate controls, loss of Rb let to increased and ectopic proliferation in the (Cited1-;Six2+) progenitor population, but without affecting self-renewal of the stem cell pool (Cited1+; Six2+), nor subsequent nephron differentiation. However, this enhanced nephrogenesis was offset by a severely compromised survival in the tubule epithelia of the immature nephron at later stages in the absence of Rb. In p53fl/fl treated embryos, we identified multiple renal defects including renal hypoplasia, underdeveloped nephronic structures with severe nephron deficit, and expansion of glomerular Bowman’s space in comparison with wild-type littermate controls. Unexpectedly, combined deletions of both Rb and p53 exacerbated the observed survival defects in Rb mutant embryos, as manifested by a more severe renal hypoplasia with almost complete loss of immature glomeruli at birth.

Conclusion: This is the first study to uncover a novel role for Rb in the control of NPC proliferation (rate of nephrogenesis), and a critical requirement for Rb in immature nephron survival around birth. It also highlights the existence of distinct as well as synergistic functions between the Rb and p53 pathways during kidney development, with direct implications on renal function. These findings about the roles of the Rb pathway and its downstream effector pathways will bring new insights on human renal development, and help understand better the pathogenesis associated with some cases of kidney disease and injury in humans.