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Kidney Week

Abstract: SA-PO064

A Novel ATP Imaging System Using the Kidney Slice Culture Reveals ATP Dynamics in Whole Kidney Under Pathophysiological Conditions

Session Information

  • AKI: Mechanisms - III
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Yamamoto, Shigenori, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Yamamoto, Shinya, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Takahashi, Masahiro, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Okubo, Akihiro, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Yamamoto, Masamichi, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
  • Yanagita, Motoko, Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
Background

Mitochondrial disorders and ATP depletion play the central role in the pathogenesis of renal diseases. Recently, we generated a mouse line (GO-ATeam2 mouse), which expresses FRET-based ATP biosensor systemically, and reported spatiotemporal ATP dynamics during ischemia-reperfusion (IR) using two-photon microscopy. However, observation from kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP production pathways in each segment.

Methods

We established the novel ex vivo ATP imaging system using slice culture of GO-ATeam2 mouse kidney. We evaluated ATP production pathways in each segment by the administration of oligomycin as an oxidative phosphorylation (OXPHOS) inhibitor and phloretin as a glycolysis inhibitor. We also analyzed ATP dynamics during ex vivo IR and cisplatin nephropathy models.

Results

We, for the first time, succeeded in visualizing ATP dynamics in whole kidney. After oligomycin administration, ATP in proximal tubules (PTs) decreased most rapidly and severely, followed by the decrease in podocytes. On the other hand, after phloretin administration, ATP decreased in podocytes most severely, but less apparently in other segments, indicating PTs are strongly dependent on OXPHOS for ATP production and podocytes rely on both OXPHOS and glycolysis. We further confirmed that ex vivo IR model could recapitulate ATP dynamics in vivo: ATP recovery after reperfusion in PTs varied depending on the length of ischemia, whereas ATP in distal tubules (DTs) recovered well even after long ischemia. After cisplatin administration, ATP in PTs decreased first, followed by the decrease in DTs. The administration of higher concentration of cisplatin resulted in more rapid and severer ATP depletion. Cisplatin accumulation in kidney slices was confirmed using mass spectrometry, which was attenuated by the administration of cimetidine, an OCT2 inhibitor. Cimetidine administration led to ATP recovery in PTs, but not in DTs, suggesting DT injury is not OCT2-mediated. Finally, we confirmed MA-5, a mitochondria protection reagent, delayed cisplatin-induced ATP decrease.

Conclusion

This novel system could provide valuable information of energy dynamics and pathogenesis of renal diseases, and might be useful for drug screening.