Comprehensive Profiling of In Situ Mitochondrial Bioenergetics in Enriched Murine Permeabilized Nephron Segment

Background: Mitochondrial dysfunction is a hallmark of kidney disease, yet tools to assess oxidative phosphorylation in enriched nephron segments ex vivo are limited. We developed and validated a comprehensive approach to measure mitochondrial bioenergetics in isolated mitochondria (Mito) and enriched glomeruli (Glom), proximal tubules (PT), and distal tubules (DT) freshly harvested from mouse kidneys, using a sieving and adhesion-based isolation strategy combined with high-resolution respirometry and fluormetry to measure oxygen consumption (JO2), Gibbs free energy of ATP synthesis (ΔG’ATP), ATP production (JATP), and mitochondrial membrane potential (ΔΨmt). Methods: Fresh mouse kidneys were processed using differential sieving and selective adhesion to isolate enriched nephron subsegments, validated by qPCR and EM. Tissues were permeabilized using digitonin and subjected to high-resolution respirometry and fluorometry under SUIT and CK clamp protocols. Adenine diet (AD) was used to show the sensitivity of our measurements with a model of CKD. Results: PTs exhibited significantly higher ADP-stimulated JO2 (18,331 ± 131 pmol O2/sec/mgprotein) compared to DTs (4,107 ± 883 pmol O2/sec/mgprotein) and Glom (2,317 ± 764 pmol O2/sec/mgprotein) and Mito (1953 ± 131 pmol O2/sec/mgprotein). PT (3,484 ± 949 kcal/mol/O2) were more responsive to changes in ΔG’ATP compared to DT (660 ± 353 kcal/mol/O2), Glom (685 ± 516 kcal/mol/O2), or Mito (1,113 ± 319 kcal/mol/O2). Likewise, PT (0.0218 ± 0.009 kcal/mol/ΔΨmt) membrane potential was more adaptable to energetic demands than DT (0.0001 ± 0.002 kcal/mol/ΔΨmt) , Glom (-0.0003 ± 0.002 kcal/mol/ΔΨmt) , or Mito (0.0115 ± 0.0115 kcal/mol/ΔΨmt). AD particularly impacted PT more than DT, Glom, or Mito. Conclusion: Our method enables bioenergetic profiling of nephron segments with high resolution and physiological relevance. PTs demonstrate JO2 and adaptability energy demand, likely reflecting their role in reabsorptive workload. In contrast, DTs and Glom operate within a narrower bioenergetic continuum. The PT's dynamic bioenergetic program may underlie its vulnerability in renal disease. This provides a powerful tool for mechanistic discovery and therapeutic development. Funding: NIDDK Support