Fig. 6.

Functional characterization of identified metabolite effects on nephron progenitor biology. (A) Embryonic kidneys at E11.5 were cultured for 72 h under different concentrations of pyruvate supplementation and immunostained for calbindin (red) to visualize the entire ureteric bud epithelium. Normal kidney culture medium contained 1 mM pyruvate, which served as a control for other concentrations. Scale bar: 200 µm. (B) P1 control (wild-type) and Pycr1/2 double-knockout (dko) kidneys were immunostained for PAX2 (green, marker of nephron progenitors and precursors) or SIX2 (green, marker of nephron progenitors) in combination with calbindin (red, marker of ureteric bud epithelium). White arrows indicate the regions where PAX2- and SIX2-positive NP cells should be located but were significantly diminished in the absence of Pycr1/2. White asterisks point to the differentiating nephron precursors, which show only very weak SIX2 signal in control kidneys, but to which SIX2 localizes almost exclusively in Pycr1/2 double-knockout kidneys. Scale bars: 50 µm (calbindin and Pax2 wild type); 200 μm (calbindin and Six2 Pycr1/2 double-knockout; also applies to calbindin and Pax2 Pycr1/2 double-knockout and to calbindin and Six2 wild type). (C) Quantification of ureteric tip numbers (total tip) and cortical nephron progenitor cells (C-NPCs) per each tip in the cortex of control and Pycr1/2 double-knockout kidneys (n=2 kidneys/genotype, a total of 15 randomly selected medullar sections were counted). Data are average±s.d.