A Platform for Mitochondrial Profiling in Enriched Kidney Segments Under Thermodynamic Control

Abstract

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.

Figure 1.. Segment-resolved isolation and high-resolution respirometry of nephron mitochondria.

(A) Schematic of the sieving and adhesion-based method for collecting enriched fractions of proximal tubules (PT), distal tubules (DT), and glomeruli (Glom) from mouse kidney cortex. (B–D) Transmission electron microscopy (TEM) images showing preserved mitochondrial ultrastructure in PT, DT, and Glom-enriched fractions. (E) RT-qPCR validation of segmental enrichment using Nphs1 (nephrin), Slc5a2 (Sodium-glucose translocator 2; SGLT2), and Aqp2 (Aquaporin 2) as markers. (F) Substrate-uncoupler-inhibitor titration (SUIT) protocol used for assessing mitochondrial respiratory capacity, where substrates. (G) Representative oxygen consumption tracing from the Oroboros O2K system. (H) Pilot experiment showing measurable oxygen flux from pooled kidneys of a single mouse (~18.75 μg total protein). (I) Pilot experiment showing measurable oxygen flux from a single kidney of a single mouse (~9 μg total protein).

Figure 2.. Adenine diet elicits segment-specific mitochondrial dysfunction and loss of ATP production.

(A) Schematic of the adenine diet (AD) model used to induce kidney disease, consisting of alternating concentrations of 0.15% and 0.25% adenine over four weeks. (B–D) AD results in systemic effects, including reductions in body, kidney, and liver mass. (E) Representative Masson’s trichrome images of cortical kidney sections from chow- and AD-fed mice reveal marked fibrosis and tubular atrophy. (F–I) HistoLens-based morphometric analysis of glomerular structure shows a trend toward increased luminal area but no significant changes in H&E stain area, nuclear count, or nuclear size. (J) Conventional isolation of mitochondria from whole kidney cortex confirms a ~75% reduction in oxygen consumption (JO₂) in AD-fed mice. (K–M) Segment-enriched respirometry reveals severe impairment in PT (~90% reduction), moderate impairment in DT (~75%), and preserved respiration in Glom. (N–Q) NADPH-based ATP production assays show disproportionately reduced ATP generation in PT, with relative preservation in DT and Glom, indicating impaired phosphorylation efficiency in PT mitochondria following adenine-induced injury.

Figure 3.. Conceptual framework for using the creatine kinase (CK) clamp to modulate mitochondrial energetics and ATP production.

(A) Schematic overview of the CK clamp system. CK catalyzes the reversible exchange of phosphate between phosphocreatine (PCr) and adenosine diphosphate (ADP), enabling dynamic buffering of ATP and ADP concentrations. Stepwise titration of PCr drives changes in the free energy of ATP hydrolysis $\left(\Delta \text{G}{\prime }_{\text{ATP}}\right)$ and mimics physiological transitions in energy demand. (B) Diagram showing the relationship between metabolite concentrations (ATP, ADP, PCr, Cr) and $\left(\Delta \text{G}{\prime }_{\text{ATP}}\right)$ under different energetic states. High ATP and PCr levels maintain a low energy demand (less negative $\Delta \text{G}{\prime }_{\text{ATP}}$), whereas accumulation of ADP and Cr reflects high demand (more negative $\Delta \text{G}{\prime }_{\text{ATP}}$), increasing the thermodynamic pull on ATP-producing reactions. (C) Theoretical tracing of oxygen consumption rate (JO₂) during sequential PCr additions. Increasing PCr reduces available ADP and progressively lowers JO₂, illustrating how the CK clamp puts a “brake” on oxidative phosphorylation. This coordinated shift in energy state simultaneously suppresses mitochondrial ATP production, as lower ADP availability limits ATP synthase turnover. (D) Force-flux plot showing the linear relationship between JO₂ and $\Delta \text{G}{\prime }_{\text{ATP}}$. The slope defines mitochondrial thermodynamic conductance, reflecting the efficiency of the respiratory system in meeting energetic demand. A steeper slope indicates higher respiratory responsiveness and greater capacity to sustain ATP production under changing thermodynamic load.

Figure 4.. Segment-specific effects of adenine diet on mitochondrial thermodynamic conductance.

Oxygen consumption (JO₂) from mitochondria isolated from whole kidney cortex (A), proximal tubules (C), distal tubules (E), and glomeruli (G) from chow- and adenine diet (AD)-fed mice under creatine kinase (CK) clamp conditions. Sequential additions of phosphocreatine (PCr) modulate $\Delta \text{G}{\prime }_{\text{ATP}}$ while oligomycin and FCCP are used to define ATP synthase-dependent and maximal uncoupled respiration, respectively. Corresponding force-flux relationships plotting JO₂ against $\Delta \text{G}{\prime }_{\text{ATP}}$ in each group. In whole mitochondria (B) and PTs (D), AD feeding reduces both respiratory capacity and slope, indicating impaired electron transport chain (ETC) conductance. DT (F) and Glom segments (H) show preserved or less affected conductance. (I) Summary of ETC conductance derived from linear regression of JO₂ vs. $\Delta \text{G}{\prime }_{\text{ATP}}$. AD-fed mice exhibit significantly reduced conductance in whole mitochondria and PTs, but not in DT or Glom, confirming that mitochondrial energetic responsiveness is selectively impaired in proximal tubules.

Figure 5.. Segment-specific effects of adenine diet on mitochondrial membrane potential (ΔΨmt) and phosphorylation coupling.

Mitochondrial membrane potential $\left(\Delta {\Psi }_{\text{mt}}\right)$ measurements during creatine kinase (CK) clamp titration in isolated mitochondria (A), proximal tubules (C), distal tubules (E), and glomeruli (G) from chow- and adenine diet (AD)-fed mice. $\Delta {\Psi }_{\text{mt}}$ was monitored using TMRM fluorescence (577/552 nm ratio) across a physiologic range of $\Delta \text{G}{\prime }_{\text{ATP}}$. Stepwise PCr additions, followed by oligomycin and potassium cyanide (KCN), allowed for the delineation of bioenergetically coupled and uncoupled states. $\Delta {\Psi }_{\text{mt}}$ as a function of $\Delta \text{G}{\prime }_{\text{ATP}}$ across energy states. AD-fed mice show significant impairment in $\Delta {\Psi }_{\text{mt}}$ dynamics in PT (D), indicating defective coupling of membrane potential to energetic demand. DT (F) and Glom (H) retain more stable polarization. (I) Phosphorylation coupling efficiency across nephron segments, calculated from the slope of $\Delta {\Psi }_{\text{mt}}$. AD feeding selectively reduces coupling in PT mitochondria, while DT and Glom maintain functional responsiveness to changes in energy demand.