ROLES OF THE RETINOBLASTOMA PROTEIN RB AND P53 IN THE CONTROL OF RENAL STEM AND PROGENITOR CELLS’ PROPERTIES IN VITRO - DIRECT IMPLICATIONS ON KIDNEY DEVELOPMENT-

Abstract

Nephron formation necessitates the presence of a tight balance between self-renewal and differentiation of NPCs (Nephron Progenitor cells). Proper nephrogenesis during embryonic development is, therefore, critical for proper renal function in the adult kidney. Thus, a better understanding of the molecular framework (genes and pathways) regulating renal cell development including nephron stem and progenitor cells (NSPCs) is of major significance and will provide a better insight about disease pathogenesis. The Retinoblastoma protein, pRb, is known for its central role in controlling cell cycle progression at the G1-S phase checkpoint. Various studies have also highlighted other functions carried by the Rb pathway such as the regulation of progenitors’ differentiation and migration as well as survival of their mature progeny in different tissues including the brain and the retina. Recent work from our laboratory revealed, for the first time, that Rb is also required for proper kidney development and control of nephrogenesis. Hence, we found that Rb negatively regulate nephron progenitors’ proliferation, and is indispensable for the survival of immature nephrons in vivo. On the other hand, the role of the tumor suppressor gene, p53, is well investigated in kidney development. Several studies have shown that p53 is needed, in a dose-dependent manner, to regulate ureteric bud branching, self-renewal and maintenance of nephron stem/progenitor cells inside the cap mesenchyme as well as terminal maturation of nephron structures.To gain a better understanding of the roles of Rb and p53 in the control of NSPCs’ development, we have investigated here how each tumor suppressor gene regulates the properties of these cells in vitro. We used a well-established protocol, adapted from Brown et. 2011, to isolate and culture renal cells derived from the cap mesenchyme (by mild enzymatic digestion and purification) after inducing a deletion of Rb and p53 during mid-development (Aaron C Brown et al., 2011). Then, we assessed renal cell growth, morphology as well as their rate of proliferation and their expression of key developmental markers such as Cited1 and Six2 by immunocytochemistry after 2-3 days in culture. To delete Rb and p53, we generated pregnant females carrying Nestin- CreERT2-YFP; Rbfl/fl and Nestin-CreERT2-YFP; p53fl/fl embryos along with heterozygous or wild type control littermates, separately. Then, we induced Rb-p53 gene deletion by single tamoxifen treatments administered to these females by oral gavage at E10.5 followed by sacrifice at E17.5. Our results showed successful Cre recombination and gene deletion in cultured renal cells, the majority of which belonged to the cap mesenchyme lineage and expressed the NSPCs’ markers Cited1 and Six2. Accordingly, more than half of the isolated renal cells co-expressed Nestin and GFP and showed similar morphology between different genotypes after 2-3 days in culture. However, we detected, in Rb-/- cultures, a significant and proportional increase in cell proliferation as assessed by cell density, Ki67 labeling, and BrdU incorporation compared with controls. This indicated that Rb negatively controls the proliferation of renal cells including NSPCs in primary culture and is consistent with our recent in vivo findings showing enhanced proliferation inside the nephrogenic zone following the loss of Rb. Moreover, we found that loss of p53 significantly compromises renal cell growth in culture as manifested by the sharp reduction in cell density compared with p53+/+ cultures. This finding is unlikely associated with a proliferation defect given that the rate of proliferation was not affected by the loss of p53. Hence, the mechanisms mediating this effect require further investigation and may be linked to a survival defect among other defects as seen in our in vivo study. This is the first study to assess the roles of Rb and p53 in the control of renal stem and progenitor cells’ proliferation in vitro. Complementary work in the future may shed more light on how these genes regulate NSPCs’ differentiation and survival in culture. Altogether, these findings will help provide a better understanding of the cellular and molecular pathways implicating the Rb and p53 pathways in kidney development.