Decker, S. T.; Opurum, P. C.; Choi, R. H.; Paula, V. L.; Kurian, A. S.; Stewart, D.; Nikolova, L. S.; Sanchez, A.; Al-Rabadi, L.; Ramkumar, N.; Fisher-Wellman, K. H.; Funai, K.

Mitochondrial function varies widely across kidney nephron segments, yet conventional approaches lack the resolution and control needed to assess cell-type-specific bioenergetics in situ. We present a methodological platform that enables segment-resolved profiling of mitochondrial respiration, conductance, and membrane potential in freshly isolated mouse nephron segments. Combining mechanical sieving and adhesion-based enrichment with permeabilized high-resolution respirometry, we adapted the creatine kinase clamp to quantify oxygen flux and mitochondrial membrane potential across defined free energies. Using this approach, we found that proximal tubules exhibit high respiratory conductance and dynamic mitochondrial polarization, while distal tubules and glomeruli maintain static membrane potential and low conductance. In a model of adenine-induced nephropathy, only proximal tubule mitochondria showed marked reductions in respiration and ATP production. This segment-specific dysfunction was not detectable in bulk mitochondrial isolates. Our approach provides thermodynamically anchored, segment-resolved insight into mitochondrial adaptation under physiological and pathological conditions. It is broadly applicable to other tissues with metabolic heterogeneity and compatible with disease models, genetic tools, and pharmacological interventions. This platform bridges a critical gap between conventional respirometry and functional mitochondrial phenotyping in native tissue structures.