Measuring renal cortical cell-specific mitochondrial metabolism

Abstract

The metabolic health of the kidney is a primary determinant of the risk of progressive kidney disease. Our understanding of the metabolic processes that fuel kidney functions is limited by the kidney's structural and functional heterogeneity. As the kidney contains many different cell types, we sought to determine the intra-renal mitochondrial heterogeneity that contributes to cell-specific metabolism. To interrogate this, we utilized a recently developed mitochondrial tagging technique to isolate kidney cell-type specific mitochondria. Here, we investigate mitochondrial functional capacities and the metabolomes of the early and late proximal tubule (PT) and the distal convoluted tubule (DCT). The conditional MITO-Tag transgene was combined with Slc34a1-CreERT2, Ggt1-Cre, or Pvalb-Cre transgenes to generate mouse models capable of cell-specific isolation of hemagglutinin (HA)-tagged mitochondria from the early PT, late PT, or the DCT, respectively. Functional assays measuring mitochondrial respiratory and fatty acid oxidation (FAO) capacities and metabolomics were performed on anti-HA immunoprecipitated mitochondria from kidneys of ad libitum fed and 24-hour fasted male mice. The renal MITO-Tag models targeting the early PT, late PT, and DCT revealed differential mitochondrial respiratory and FAO capacities which dynamically changed during fasting conditions. The renal MITO-Tag model captured differential mitochondrial metabolism and functional capacities across the early PT, late PT, and DCT at baseline and in response to fasting.

Keywords: cellular metabolic heterogeneity; kidney; metabolism; mitochondria; tubular epithelium.

Figure 1.. A model to study renal tubular mitochondrial heterogeneity.

A. Schematic of the heterogeneous renal tubular epithelium. Inset highlights the juxtaposition of proximal tubular (PT) and distal convoluted tubular (DCT) cells. B. Immunostaining with sodium glucose co-transporter 2 (SGLT2, early PT S1-S2 segment), Lotus lectin (LTL, PT S1-S2>S3) and sodium chloride co-transporter (NCC, DCT). Scale bar = 100 μm. C. MITO-Tag mouse models to target the Early PT, Late PT, and DCT. D. Immunostaining with anti-GFP (anti-HA MITO-Tag includes GFP construct), SGLT2, LTL, and NCC of Slc34a1-MT, Ggt1-MT, and PvAlb-MT kidneys. Scale bar = 50 μm.

Figure 2.. Isolation of intact kidney cell specific mitochondria

A. Electron microscopy of proximal tubule and distal convoluted tubules at low and high magnification of wild-type (WT, Cre negative), Slc34a1-MT, Ggt1-MT, and PvAlb-MT kidneys. Low magnification, 20000X. High magnification, 50000X. Scale bar = 1 μm. B. Immunoblot of protein samples of whole kidney homogenate (HM) and mitochondria isolated via anti-HA immunoprecipitation from Slc34a1-MT, Ggt1-MT, and PvAlb-MT kidneys. C. Immunoblot of protein samples of whole kidney homogenate (HM), mitochondria isolated from whole kidney (whole tissue mito), and mitochondria isolated via anti-HA immunoprecipitation from Slc34a1-MT, Ggt1-MT, and PvAlb-MT kidneys. D. Immunostaining with anti-GFP (MITO-Tag), Calnexin (endoplasmic reticulum), PMP70 (peroxisome), and LTL of Slc34a1-MT, Ggt1-MT, and PvAlb-MT kidneys. Scale bar = 50 μm.

Figure 3.. MITO-Tag isolated kidney cell-specific mitochondria are viable and respirating

Kidney mitochondria were isolated via traditional methods (whole) or via anti-HA immunoprecipitation from Slc34a1-MT (Early PT), Ggt1-MT (Late PT), and PvAlb-MT (DCT) kidneys. Data shown as mean±SD. A. Mitochondrial outer membrane (MOM) permeability measured by oxygen consumption rate (OCR) using Complex II substrate (5 mM Succinate) with cytochrome c treatment after treatment with an uncoupler (CCCP). Data shown as ratio of cytochrome c OCR versus CCCP OCR. n=5–6/group. B. Mitochondrial membrane potential measured by TMRE fluorescent intensity. n=7–8/group. C-F. Mitochondrial respiration measured by oxygen consumption rate (OCR) before (State IV) and after 4 mM ADP (State III). C. Complex I activity of Slc34a1-MT (Early PT), Ggt1-MT (Late PT), and PvAlb-MT (DCT) mitochondria isolated from fed mice. n=7–10/group. D. Complex II activity of Slc34a1-MT (Early PT), Ggt1-MT (Late PT), and PvAlb-MT (DCT) mitochondria isolated from fed mice. n=5–6/group. E. Complex II activity of whole kidney mitochondria isolated from fed or fasted mice. n=5/group. F. Complex II activity of Slc34a1-MT (Early PT), Ggt1-MT (Late PT), and PvAlb-MT (DCT) mitochondria isolated from fed or fasted mice. n=5–9/group. A,B. One-way ANOVA with Dunnett’s multiple comparisons test, ns, not significant. Bartlett’s test for homogeneity p = 0.0729. C-F. Two-way ANOVA with Sidak’s multiple comparisons test, *P<0.05, **P<0.01.

Figure 4.. Differential and fasting-induced changes in mitochondrial metabolite composition in the proximal tubule and the distal convoluted tubule.

Mitochondria isolated via anti-HA immunoprecipitation from Slc34a1-MT (Early PT), Ggt1-MT (Late PT), and PvAlb-MT (DCT) kidneys from fed or 24-hour fasted mice were submitted for targeted screening metabolomics by LC/MS-MS, n=3/group. Analysis performed via Metaboanalyst, details described in Methods. A. Principal components analysis (PCA) of metabolites across the early PT, late PT, and DCT in fed and fasted conditions. B. Two factor PCA of metabolites across the early PT, late PT, and DCT in fed and fasted conditions. C. Relative abundance (RA) of total acylcarnitines, individual acylcarnitines and acetylcarnitine, and free carnitine. Data shown normalized to total ion count (TIC). Data shown as mean±SD. Two-way ANOVA with Sidak’s multiple comparisons test, *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

Figure 5.. Differential fatty acid oxidation capacities in late proximal tubular and distal convoluted tubular mitochondria in response to fasting.

A. Diagram of fatty acid oxidation. Created in BioRender. Huen, S. (2025) https://BioRender.com/9rvibxd B. Mitochondria were isolated via traditional whole kidney tissue isolation (whole) or anti-HA immunoprecipitation from Ggt1-MT (Late PT) and PvAlb-MT (DCT) kidneys from fed or 24-hour fasted mice. FAO capacity measured by OCR using 40 μM palmitoyl-CoA as a substrate, before (State IV) and after 4 mM ADP (State III), n=5–6/group. C. Single cell RNA-sequencing data from Ransick et al. (5) of select FAO genes visualized via Kidney Cell Explorer at https://cello.shinyapps.io/kidneycellexplorer/. 3/4: S1 PT female/male, 5/6: S2 PT female/male, 7/8: S3 PT female/male, 18: DCT. D. Whole kidney mRNA expression shown relative to Rpl13a. n=11–13/group. E. Representative immunoblot of protein samples of mitochondria isolated via anti-HA immunoprecipitation. F. Densitometry of (E), n=5/group, no comparison was significantly different. G. After ADP-stimulation, OCR measured after treatment with antimycin A, which specifically inhibits mitochondrial function, is the non-mitochondrial contribution to OCR. Percentages shown on bar graph represent the percent contribution of non-mitochondrial OCR. Related to Figure 5B. B,F. Data shown as mean±SD. Two-way ANOVA with Sidak’s multiple comparisons test, ***P<0.001, ****P<0.0001. D. Data shown as mean±SD. Unpaired t-test, **P<0.01.