Kidney Week

Abstract: FR-PO1070

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

Session Information

• Health Maintenance, Nutrition, and Metabolism
  November 07, 2025 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Health Maintenance, Nutrition, and Metabolism

• 1500 Health Maintenance, Nutrition, and Metabolism

Authors

• Decker, Stephen, University of Utah, Salt Lake City, Utah, United States

Stephen Decker, PhD

• Opurum, Precious Chinonyerem, University of Utah, Salt Lake City, Utah, United States

Precious Chinonyerem Opurum, PhD

• Paula, Venisia L., University of Utah, Salt Lake City, Utah, United States

Venisia L. Paula

• Stuart, Deborah, University of Utah, Salt Lake City, Utah, United States

Deborah Stuart

• Ramkumar, Nirupama, University of Utah, Salt Lake City, Utah, United States

Nirupama Ramkumar, MD, MPH, FASN

• Funai, Katsuhiko, University of Utah, Salt Lake City, Utah, United States

Katsuhiko Funai, PhD

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 (ΔΨ_mt).

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/mg_protein) compared to DTs (4,107 ± 883 pmol O₂/sec/mg_protein) and Glom (2,317 ± 764 pmol O₂/sec/mg_protein) and Mito (1953 ± 131 pmol O₂/sec/mg_protein). 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/ΔΨ_mt) membrane potential was more adaptable to energetic demands than DT (0.0001 ± 0.002 kcal/mol/ΔΨ_mt) , Glom (-0.0003 ± 0.002 kcal/mol/ΔΨ_mt) , or Mito (0.0115 ± 0.0115 kcal/mol/ΔΨ_mt). 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

Digital Object Identifier (DOI)

• doi: 10.1681/ASN.2025c9h9bcan