Real-Time ATP Imaging Reveals the Metabolic State During Branching Nephrogenesis

Background: Energy metabolism is fundamental to organ development, yet its role in determining nephron formation remains poorly understood. Disturbances in the intrauterine environment are known to affect nephron endowment and increased risk of hypertension and chronic kidney disease later in life. However, very little is known about the mechanisms that determine nephron number and metabolic status during nephrogenesis. Methods: We focused on cytosolic adenosine 5-triphosphate (ATP) levels to examine energy metabolism in embryonic kidneys. To explore the spatiotemporal dynamics of the cytosolic ATP level, we used transgenic mice expressing a cytosolic ATP-FRET biosensor, GO-ATeam2, which enabled ex vivo live imaging of metanephric kidneys at single-cell resolution. Results: We performed real-time ex vivo ATP imaging of embryonic kidneys of GO-ATeam2 mice. During branching nephrogenesis, ATP levels of ureteric bud (UB) tip cells are significantly lower than those of the UB stalk and cap mesenchyme (CM) cells. Glycolytic inhibition in the early phase of metanephric kidney (embryonic day E12.5) severely suppressed UB branching with a dose-dependent reduction in ATP levels in both UB and CM cells. Time-course observations revealed that the ATP reduction by glycolytic inhibitor was faster and more prominent in UB cells than in CM cells. In addition, glycolytic inhibition significantly reduced the number of branch segments and tips of UB as well as the expression of specific markers in UB and CM cells. Electron microscopy revealed loosening of lateral cell-cell adhesion and disorganized alignment of CM cells, which were accompanied by decreased expression of N-cadherin. These effects were not observed with the inhibition of oxidative phosphorylation. Conclusions: UB branching was heavily dependent on glycolysis, and UB cells in the early branching phase were more sensitive to glycolytic inhibition than mesenchyme cells are. These results highlight the significance of metabolic regulation in branching nephrogenesis.