Tubular mitochondrial pyruvate carrier disruption elicits redox adaptations that protect from acute kidney injury

Abstract

Objective: Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity.

Methods: We generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal ¹³C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI.

Results: MPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival.

Conclusions: Our findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Keywords: Acute kidney injury; Hormesis; Metabolomics; Mitochondrial metabolism; Oxidative stress.

Figure 1

Mpc1 is expressed in proximal and distal tubular segments and is decreased in AKI. (A) Bar graph comparing kidney Mpc1 mRNA levels after vehicle treatment or cisplatin-, ischemia reperfusion (IR)-, or rhabdomyolysis (Rhabdo)-induced AKIs. Samples were collected 72 h after cisplatin injury and 24 h after IR and rhabdomyolysis injuries. (n = 6/group; ∗∗∗p < 0.001 by unpaired t test). (B) Representative Western blot of kidney MPC1 protein abundance 24 h after rhabdomyolysis-induced AKI. VDAC was blotted as a loading control and ponceau staining of the membrane is shown. (n = 5, ∗∗p < 0.01 by unpaired t test). (C) Quantification of VDAC normalized MPC1 protein abundance after rhabdomyolysis-induced AKI. (n = 5, ∗∗p < 0.01 by unpaired t test). (D) Representative immunostaining images of MPC1 (red), lotus tetragonolobus lectin (LTL, green, proximal tubule marker), or peanut agglutinin (PNA, green, distal tubule marker), and DAPI (blue) in whole kidney, outer cortex (OC) and cortico-medullary junction (CM) kidney sections 30 h following vehicle treatment or rhabdomyolysis-induced AKI. (Images captured at 15× magnification; whole kidney scale bar = 1,000 μm; OC and CM scale bar = 50 μm). (E) Representative fluorescence image of kidney sections of mT/mG/Ggt1-Cre mice confirming GFP + renal tubular epithelial cells (green, #, GFP) and tdTomato + non-RTEC cells (red, ∗, tdT) stained with Dapi (blue). (Scale bar = 100 μm). (F) Bar graph comparing Mpc1 mRNA levels in flow-sorted Non-RTEC (tdTomato+) and RTEC (GFP+) cells 24 h after vehicle treatment or rhadbomyolysis-induced AKI. (n = 5/group, ∗∗∗p < 0.001 by unpaired t test). (G) Representative Western blot of MPC1 and VDAC protein abundance in flow-sorted Non-RTEC (tdTomato+) and RTEC (GFP+) cells 24 h after rhabdomyolysis-induced AKI. β-ACTIN was blotted as a loading control. Data are presented as means ± SEM.

Figure 2

Generation and characterization of MPC TubKO mice. (A) Schematic illustrating the generation of tubular Mpc1 null allele mice (MPC TubKO). (B–C) Bar graphs showing body weights (B) and serum cystatin C concentration (C) in WT and MPC TubKO mice. (n = 5/group, 8-week-old mice). (D) Bar graph comparing mouse kidney Mpc1 mRNA levels in WT and MPC TubKO mice. (n = 4/group, 7 - 12-week-old mice, ∗∗p < 0.01 by unpaired t test). (E–G) Representative Western blot of kidney MPC1 and MPC2 protein abundance (E) and quantification of normalized MPC1 (F) and MPC2 (G) protein levels in WT and MPC TubKO mice. Tubulin was blotted as loading control and used as the protein quantification normalizer. Ponceau staining of the membrane is shown. (n = 4–6/group, 7 - 12-week-old mice, ∗∗p < 0.01 and ∗∗∗p < 0.001 by unpaired t test). (H) Representative immunostaining images of kidney MPC1 (green) and lotus tetragonolobus lectin (LTL, green, proximal tubule marker) or peanut agglutinin (PNA, green, distal tubule marker) in the whole kidney (WK), outer-cortex (OC), and cortico-medullary junction (CM) in WT and MPC TubKO mice. (Images taken at 4× (WK) or 20× (OC and CM) magnification, scale bar = 500 μm). Data are presented as means ± SEM.

Figure 3

Tubular Mpc1 deletion decreases cellular pyruvate oxidation and perturbs ETC function. (A-C) Line graph showing the oxygen consumption rate of LLC-PK1 cells provided with (A) pyruvate, (B) glutamine, or (C) palmitate + glucose as substrates. Inhibitors were added as indicated. Veh., Vehicle; Oligo., Oligomycin; Rot./Anti.A, Rotenone/Antimycin A;, Eto., Etomoxir. (n = 5–12, †p < 0.1, ∗p < 0.05, ∗∗∗p < 0.001 by two-way ANOVA with the Holm-Sidak multiple comparison test within measurement). (D) Line graph showing the extracellular acidification rate of LLC-PK1 cells provided with glucose and pyruvate. Inhibitors added as indicated. (n = 12). (E) Representative Western blot of kidney ETC marker Complex I (CI), NDUFB8; Complex II (CII), SDHB; Complex III (CIII), UQCRC2; Complex IV (CIV), MTCO1; and Complex V (CV), ATP5A protein abundances in WT and MPC TubKO mice. (n = 4/group, 6 - 8-week-old mice). (F) Bar graph comparing the quantified VDAC protein level in WT and MPC TubKO mice. (n = 5/group, 6 - 8-week-old mice). (G-J) Bar graphs comparing the whole-kidney enzymatic activities of citrate synthase (G), Complex I (H), Complex II (I), and Complex III (J) in WT and MPC TubKO mice. (n = 4/group, 6-week-old mice, ∗p < 0.05 by unpaired t test). Data are presented as means ± SEM.

Figure 4

Tubular MPC disruption leads to upregulation of oxidant defense systems. (A-D) Bar graphs showing kidney metabolite levels in WT and MPC TubKO mice. Pyruvate, lactate, and alanine (A), TCA cycle metabolites (B), GSH synthesis substrates glycine, cysteine, and glutamate (C), and 2-hydroxybutyrate, a marker of GSH turnover (D). (n = 6/group, 8 - 12-week-old mice, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by unpaired t test). (E) Schematic illustrating mitochondrial antioxidant defense system including MnSOD, manganese superoxide dismutase; GSH, glutathione; GSSG, oxidized glutathione; GPX, glutathione peroxidase; GR, glutathione reductase; Prx, peroxiredoxin; TRR, thioredoxin reductase; Trx_red, reduced thioredoxin; and Trx_ox, and oxidized thioredoxin. (F-G). Bar graphs comparing kidney total GSH (GSH + GSSG) (F) and the % of GSSG of total GSH (GSH + GSSG) (G) in WT and MPC TubKO mice. (n = 5/group, 12 - 14-week-old mice, ∗p < 0.05 by unpaired t test). (H) Bar graph showing MitoSOX oxidation in the presence and absence of antimycin A (AA) of isolated WT and MPC TubKO tubular epithelial cell. The y-axis shows the fold mean fluorescence intensity (MFI). (n = 5/group, 12-week-old mice, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA with the Holm-Sidak multiple comparison test). (I) Representative immunohistochemistry images of kidney 3NT staining in WT and MPC TubKO mice. (Images taken at 40× magnification, scale bar = 100 μm). (J) Bar graph comparing kidney 3NT quantification in WT and MPC TubKO mice. (n = 8–11, 12 - 14-week-old mice, ∗∗p < 0.01 by unpaired t test). (K-M) Bar graphs showing kidney enzyme activities of MnSOD (K), GR (L), and TRR (M) in WT and MPC TubKO mice. (n = 7–8/group, 12 - 14-week-old mice, ∗p < 0.05 and ∗∗∗p < 0.001 by unpaired t test). (N) Bar graph comparing the kidney NADPH:NADP ratio in WT and MPC TubKO mice. (n = 6/group, 8 - 12-week-old mice, ∗∗p < 0.01 by unpaired t test). Data are presented as means ± SEM.

Figure 5

MPC TubKO mice decrease glucose oxidation, increase PPP activity, and upon injury are better able to switch to glycolytic metabolism. (A) Schematics illustrating pentose phosphate pathway (PPP) (top) and ¹³C-enrichment patterns into glycolysis, the PPP, and the TCA cycle from ¹³C-glucose (bottom). MPC, mitochondrial pyruvate carrier; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; OAA, oxaloacetate. (B) Schematics illustrating the experimental design for in vivo uniformly labeled [U–¹³C]-glucose tracing experiments under control and rhabdomyolysis-induced AKI conditions. (C-M) Bar graphs showing kidney ¹³C-isotopologue enrichments and metabolite abundances 30 min after [U–¹³C]-glucose bolus injection in control (CTRL) and rhabdomyolysis-injured (Rhabdo) WT and MPC TubKO mice. (C-E) Pentose phosphate pathway metabolites (C) Glucose 6-phosphate, (D) 6-phosphogluconate, and (E) Ribulose/Ribose 5-phosphate. (F–H) Glycolytic intermediate metabolites (F) Fructose 6-phosphate, (G) Pyruvate, and (H) Lactate. (I-M) TCA cycle intermediate metabolites (I) Acetyl-CoA, (J) Citrate, (K) Fumarate, (L) Malate, and (M) Aspartate. (n = 7/group, 7 - 8-week-old mice, ∗p < 0.05 and ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA with the Holm-Sidak multiple comparison test (% Enrichment) or by unpaired t test (Fold abundance)). Data are presented as means ± SEM.

Figure 6

MPC TubKO mice are protected from rhabdomyolysis induced kidney injury. (A-B) Schematics illustrating the time course of the rhabdomyolysis-induced AKI model (A) and the interconnectedness of the pentose pathway and cellular antioxidant defense systems (B). G6PD, glucose 6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; GR, glutathione reductase; TRR, thioredoxin reductase. (C–F) Bar graphs showing kidney enzyme activities following vehicle treatment or rhabdomyolysis-induced AKI (Rhabdo). G6PD (C), 6PGDH (D), GR (E), and TRR (F). (n = 4/group for vehicle treatment, n = 12–13/group for Rhabdo, 8 - 12-week-old mice, ∗p < 0.05 and ∗∗p < 0.01 by two-way ANOVA with the Holm-Sidak multiple comparison test). (G) Representative immunostaining images of kidney protein-glutathionylation (pink) and Dapi (blue) following vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (Scale bar = 100 μm, n = 4/group for vehicle treatment, n = 12–13/group for Rhabdo, 8 - 12-week-old mice). (H) Bar graph quantifying kidney protein-glutathionylation following vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 4/group for vehicle treatment, n = 12–13/group for Rhabdo, 8–12 week-old mice, ∗p < 0.05 by two-way ANOVA with the Holm-Sidak multiple comparison test). Data presented as means ± SEM.

Figure 7

Tubular MPC1 genetic deletion protects from rhabdomyolysis induced kidney injury. (A) Line graph showing the survival curve of WT and MPC TubKO mice following rhabdomyolysis-induced AKI (Rhabdo). (n = 10–11/group, 8 - 12-week-old mice, ∗p < 0.05 by Mantel–Cox log-rank test). (B–C) Bar graphs showing serum cystatin C (B), and blood urea nitrogen (C, BUN) levels prior to (D0) and on day 1 (D1, 24-hours) and day 2 (D2, 48 h) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 10–11/group, 8–12 week-old mice, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-way ANOVA followed by the Holm-Sidak multiple comparison tests). (D-E) Bar graphs showing kidney Ngal (D) and Kim1 (E) mRNA levels one day (24 h) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 4/group for vehicle treatment, n = 12–13/group for Rhabdo, 8 - 12-week-old mice, ∗p < 0.05 by two-way ANOVA with the Holm-Sidak multiple comparison test). (F-G) Bar graphs showing quantification of histologically assessed tubular injury score (F) and tunel positive tubular cells (G) one day (24 h) after vehicle treatment or rhabdomyolysis-induced AKI in WT and MPC TubKO mice. (n = 4/group for vehicle treatment, n = 12–13/group for Rhabdo, 8 - 12-week-old mice, ∗∗p < 0.01 and ∗∗∗p < 0.001 by two-way ANOVA with the Holm-Sidak multiple comparison test). (G-J) Heatmaps showing Spearman correlation between variables analyzed following vehicle treatment or rhabdomyolysis-induced AKI. Correlation calculated in WT mice comparing Mpc1 mRNA levels, tubular injury, Ngal and Kim1 mRNA levels, tunel score, and tubular GSH with AKI (H). Spearman correlation performed in WT (I) and MPC TubKO (J) mice comparing GSH and antioxidant defense system markers following rhabdomyolysis-induced AKI. Data are presented as means ± SEM.