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.