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Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury

Nature volume 565, pages 96–100 (2019)Cite this article

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An Author Correction to this article was published on 15 May 2019

This article has been updated

Abstract

Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood1,2. Nitric oxide-based cellular signalling is generally mediated by protein S-nitrosylation, the oxidative modification of Cys residues to form S-nitrosothiols (SNOs). S-nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S-nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively3,4. In Saccharomyces cerevisiae, the classic metabolic intermediate co-enzyme A (CoA) serves as an endogenous source of SNOs through its conjugation with nitric oxide to form S-nitroso-CoA (SNO-CoA), and S-nitrosylation of proteins by SNO-CoA is governed by its cognate denitrosylase, SNO-CoA reductase (SCoR)5. Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1)5 with an unknown physiological role. Here we report that the SNO-CoA–AKR1A1 system is highly expressed in renal proximal tubules, where it transduces the activity of eNOS in reprogramming intermediary metabolism, thereby protecting kidneys against acute kidney injury. Specifically, deletion of Akr1a1 in mice to reduce SCoR activity increased protein S-nitrosylation, protected against acute kidney injury and improved survival, whereas this protection was lost when Enos (also known as Nos3) was also deleted. Metabolic profiling coupled with unbiased mass spectrometry-based SNO-protein identification revealed that protection by the SNO-CoA–SCoR system is mediated by inhibitory S-nitrosylation of pyruvate kinase M2 (PKM2) through a novel locus of regulation, thereby balancing fuel utilization (through glycolysis) with redox protection (through the pentose phosphate shunt). Targeted deletion of PKM2 from mouse proximal tubules recapitulated precisely the protective and mechanistic effects of S-nitrosylation in Akr1a1−/− mice, whereas Cys-mutant PKM2, which is refractory to S-nitrosylation, negated SNO-CoA bioactivity. Our results identify a physiological function of the SNO-CoA–SCoR system in mammals, describe new regulation of renal metabolism and of PKM2 in differentiated tissues, and offer a novel perspective on kidney injury with therapeutic implications.

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Fig. 1: Knockout of AKR1A1 protects against AKI in a SNO-dependent manner.
Fig. 2: PKM2 is a major locus of regulation by the SNO-CoA–SCoR system.
Fig. 3: S-nitrosylation of renal PKM2 inhibits its activity by blocking tetramer formation.
Fig. 4: Inhibition of PKM2 by S-nitrosylation increases flux through the PPP and protects from AKI.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. All datasets generated and/or analysed in the current study are available from the corresponding author upon reasonable request. Supplementary Fig. 1 contains scanned complete images of western blots. All experimental data from mice models are provided as Source Data.

Change history

  • 15 May 2019

    Change history: In Fig. 1j of this Letter, one data point was inadvertently omitted from the graph for the acute kidney injury (AKI), double knockout (−/−), S-nitrosothiol (SNO) condition at a nitrosylation level of 25.9 pmol mg−1 and the statistical significance given of P = 0.0221 was determined by Fisher’s test instead of P = 0.0032 determined by Tukey’s test (with normalization for test-day instrument baseline). Figure 1 and its Source Data have been corrected online.

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Acknowledgements

We thank H. Fujioka for mitochondrial analyses, J. Mikulan and A. Kresak for histology and immunostaining, C. Geng for assistance with statistics, and J. Reynolds, D. Hess, R. Premont and D. Seth for discussions. This work is supported by National Institutes of Health grants DK119506, HL075443, HL128192 and HL126900.

Reviewer information

Nature thanks H. Christofk, C. Lowenstein and G. Remuzzi for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Rongli Zhang, Puneet Anand

Authors and Affiliations

  1. Institute for Transformative Molecular Medicine, Department of Medicine, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, OH, USA

    Hua-Lin Zhou, Rongli Zhang, Puneet Anand, Colin T. Stomberski, Zhaoxia Qian, Alfred Hausladen & Jonathan S. Stamler

  2. Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Liwen Wang

  3. Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Eugene P. Rhee

  4. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Eugene P. Rhee

  5. Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Samir M. Parikh & S. Ananth Karumanchi

  6. Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Samir M. Parikh & S. Ananth Karumanchi

  7. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    S. Ananth Karumanchi

  8. Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH, USA

    Jonathan S. Stamler

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Contributions

H.-L.Z., P.A. and J.S.S. designed the study. H.-L.Z. carried out most of the experiments and analysed the results. R.Z. performed AKI surgery. P.A. prepared samples for iTRAQ LC–MS/MS and metabolomics. C.T.S. prepared samples for the photolysis-chemiluminescence assay. A.H. and P.A. purified SCoR from bovine kidney. Z.Q. handled mice. L.W. performed quantitative iTRAQ LC–MS/MS. E.P.R. and S.M.P. contributed to project conception and carried out metabolomics analyses. S.A.K. contributed to project conception and performed histological stains. H.-L.Z. and J.S.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Jonathan S. Stamler.

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Extended data figures and tables

Extended Data Fig. 1 Identification of enzymes involved in the SNO-CoA–SCoR system.

a, b, Enzymatic mechanism by which the SNO-CoA–SCoR system regulates protein S-nitrosylation. a, Equilibrium between SNO-CoA and S-nitrosylated proteins. b, AKR1A1 (SCoR) mediates protein denitrosylation. c, AKR1A1 was purified to homogeneity using the indicated steps. AKR1A1 protein at each stage was calculated on the basis of activity in each eluate pool or original crude lysate. Image is representative of two independently performed experiments with similar results. d, e, Expression of iNOS, nNOS and eNOS in sham-treated versus injured kidneys of wild-type mice; injury induced by I/R. Images are representative of three independently performed experiments with similar results. For gel source data, see Supplementary Fig. 1. f, Expression of eNOS before and after AKI; normalized to GAPDH as in e (n = 8 mice per group). Results are presented as mean ± s.d. Two-tailed Student’s t-test was used to detect significance. g, Schema illustrating generation of Akr1a1−/− mice. h, PCR amplification of the Akr1a1 gene with genomic DNA isolated from the tails of Akr1a1+/+, heterozygous Akr1a1+/− and homozygous Akr1a1−/− mice. Image is representative of three independently performed experiments with similar results.

Source data

Extended Data Fig. 2 SCoR activity and role in protection.

a, b, Expression of AKR1A1 after I/R-induced AKI. Expression of AKR1A1 is normalized to GAPDH in b (n = 6 mice per group). c, NADPH-dependent SNO-CoA metabolizing activity measured in kidney extracts from sham-treated wild-type mice or wild-type mice subjected to I/R-induced AKI (n = 9 mice per group). d, Serum creatinine and BUN in sham-treated kidneys from Akr1a1+/+, Akr1a1−/−, Akr1a1−/−Enos−/− and Enos−/− mice (Akr1a1+/+: n = 41 mice; Akr1a1−/−: n = 35 mice; Akr1a1−/−Enos−/−: n = 10 mice; Enos−/−: n = 10 mice). Note lower scales (y axis) compared to Fig. 1e, f. e, Haematoxylin and eosin stain of sham-treated kidneys from Akr1a1+/+, Akr1a1−/− and Akr1a1−/−Enos−/− mice. Images are representative of two independently performed experiments with similar results. Scale bars, 50 μm. f, Pathological scores of tubular lysis, loss of brush border and sloughed debris (n = 5 mice per group). Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to detect significance. g, Mortality of Akr1a1+/+, Akr1a1−/−, Akr1a1−/−Enos−/− and Enos−/− mice 24 h after AKI (Akr1a1+/+: 35 mice; Akr1a1−/−: 36 mice; Akr1a1−/−Enos−/−: 12 mice; Enos−/−: 8 mice). Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to detect significance in d, f. Two-tailed Student’s t-test was used to detect significance in b, c.

Source data

Extended Data Fig. 3 Additional models of AKI.

a, Serum ascorbate in Akr1a1+/+ and Akr1a1−/− mice fed with chow containing 1% ascorbic acid for six weeks (n = 4 mice per group). b, c, Serum creatinine and BUN in injured kidneys from Akr1a1+/+ and Akr1a1−/− mice fed with chow containing 1% ascorbic acid for six weeks (n = 4 mice per group); injury by I/R. d, e, Serum creatinine and BUN in sham-treated or injured kidneys from female Akr1a1+/+ and female Akr1a1−/− mice (sham-treated Akr1a1+/+: n = 11 mice; sham-treated Akr1a1−/−: n = 12 mice; injured Akr1a1+/+: n = 25 mice; injured Akr1a1−/−: n = 31 mice). Injury by I/R. f, g, Serum creatinine and BUN in saline-treated or LPS-treated male Akr1a1+/+ and Akr1a1−/− mice (saline-treated Akr1a1+/+: n = 7 mice; saline-treated Akr1a1−/−: n = 5 mice; LPS-treated Akr1a1+/+: n = 11 mice; LPS-treated Akr1a1−/−: n = 11 mice). h, i, Serum creatinine and BUN in saline-treated or LPS-treated female Akr1a1+/+ and Akr1a1−/− mice (saline-treated Akr1a1+/+: n = 6 mice; saline-treated Akr1a1−/−: n = 3 mice; LPS-treated Akr1a1+/+: n = 12 mice; LPS-treated Akr1a1−/−: n = 15 mice). j, Endogenous S-nitrosylation of PKM2 in saline-treated or LPS-treated male Akr1a1+/+ and Akr1a1−/− mice. Data are representative of three mice per genotype. Without ascorbate (–Ascorbate) is control for SNO. k, Quantification of SNO-PKM2. SNO normalized to PKM2 (input) (n = 4 mice per group). l, Activity of endogenous pyruvate kinase in saline- or LPS-treated kidneys from Akr1a1+/+ and Akr1a1−/− mice (n = 3 mice per saline-treated group; n = 5 mice per LPS-treated group). Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to detect significance a, d–i, k, l. Two-tailed Student’s t-test was used to detect significance in b, c.

Source data

Extended Data Fig. 4 PK interactions, activity and expression.

a, Interaction between AKR1A1 and PKM2. Myc–PKM2 and V5–AKR1A1 were co-overexpressed in HEK-293 cells. Immunoprecipitation with anti-Myc rabbit antibody; immunoblotting with V5 antibody. Image is representative of two independently performed experiments with similar results. b, Activity of recombinant PKM2, PKM1 and PKLR proteins after SNO-CoA treatment (n = 3 independent experiments). Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to assess significance. c, Expression of PKM2, PKM1 and PKLR in the kidneys of Akr1a1+/+ and Akr1a1−/− mice after 24 h of I/R-induced AKI. Image is representative of two independently performed experiments with similar results. d, Quantification of expression of PKM2, PKM1 and PKLR in c (n = 6 mice per group). Result is presented as mean ± s.d. Two-tailed Student’s t-test was used to detect significance.

Source data

Extended Data Fig. 5 Characterization of SNO-PKM2.

a, Endogenous SNO level of PKM2 Cys-mutants in eNOS-overexpressing HEK-293 cells. Image is representative of two independently performed experiments with similar results. b, Mutation of C152 to alanine affects the SNO level of PKM2 in eNOS-overexpressing HEK-293 cells. Image is representative of two independently performed experiments with similar results. c, Quantification of expression of Myc–PKM2(WT), Myc–PKM2(C49A) and Myc–PKM2(C152A) in eNOS-overexpressing HEK-293 cells. Normalized to the expression of GAPDH (n = 5 independent experiments). d, Quantification of SNO-PKM2 in eNOS-overexpressing HEK-293 cells. SNO is normalized to PKM2 (input) (n = 5 independent experiments). e, Relative mRNA levels of Myc–PKM2(WT), Myc–PKM2(C49A) and Myc–PKM2(C152A) in eNOS-overexpressing HEK-293 cells (n = 3 independent experiments). Results presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to detect significance.

Extended Data Fig. 6 Pkm gene, structure and expression.

a, Alternative splicing of Pkm gene. C423 and C424 are encoded by PKM2-specific exon 10. b, Ribbon structure of tetrameric PKM2 analysed by MacPyMOL. Four pairs of C423 and C424 in tetrameric PKM2 are highlighted in red. c, Expression of PKM1 and PKM2 in fifteen different tissues from uninjured mice. Image is representative of two independently performed experiments with similar results.

Extended Data Fig. 7 Role of PKM2 in AKR1A1-mediated protection.

a, Expression of endogenous and overexpressed PKM2 in HEK-293 cells. Image is representative of two independently performed experiments with similar results. b, Activity of Myc–PKM2(WT) and Myc–PKM2(C423/424A) after NO (DETANO; 500 μM) treatment in HEK-293 cells (n = 3 independent experiments). c, The total amount of GSH and GSSG in injured kidneys from Akr1a1+/+, Akr1a1−/− and Akr1a1−/−Enos−/− mice (n = 4 mice per group); injury induced by I/R. d, The amount of 6PG, a key PPP intermediate, in HEK-293 cells expressing Myc–PKM2(WT) and Myc–PKM2(C423/424A) after NO (DETANO; 500 μM) treatment (n = 4 independent experiments). e, Amount of serine in injured kidneys from Akr1a1+/+ and Akr1a1−/− mice (n = 10 mice per I/R-injured Akr1a1+/+ group; n = 11 mice per I/R-injured Akr1a1−/− group). f, Amount of serine in HEK-293 cells expressing Myc–PKM2(WT) and Myc–PKM2(C423/424A) after NO treatment (DETANO; 500 μM) (n = 4 independent experiments). g, Amount of GHB in serum of Akr1a1+/+ and Akr1a1−/− mice (n = 7 mice per group). No injury. Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc test was used to detect significance in b and f. Two-tailed Student’s t-test was used in e.

Source data

Extended Data Fig. 8 Mechanism of kidney injury.

a, Mitochondrial morphology in tubule cells after sham operation or injury induced by I/R from Akr1a1+/+ and Akr1a1−/− mice as assessed by electron microscopy. Red arrows, mitochondrial swelling. Scale bars, 1 μm. b, Quantification of swollen mitochondria versus total mitochondria in sham-treated or I/R-injured kidneys from Akr1a1+/+ and Akr1a1−/− mice. Results are presented as mean ± s.d. One-way ANOVA with Tukey post hoc was used to detect significance. c, The ratio of ADP to ATP in HEK-293 cells expressing Myc–PKM2(WT) or Myc–PKM2(C423/424A) after NO treatment (DETANO; 500 μM) (n = 4 independent experiments). d, Amounts of TCA cycle intermediates (aconitate, isocitrate, succinate, fumarate and malate) in sham-treated or injured kidneys from Akr1a1+/+ and Akr1a1−/− mice (n = 10 mice per sham-treated Akr1a1+/+ or I/R-injured Akr1a1+/+ group; n = 11 mice per sham-treated Akr1a1−/− or I/R-injured Akr1a1−/−group). Results are presented as mean ± s.d. There were no significant differences in c, d using one-way ANOVA with Tukey’s post hoc test.

Source data

Extended Data Fig. 9 Characterization of Pkm2−/− mice.

a, Schema illustrating generation of renal epithelial cell-specific Pkm2−/− mice. b, Survival curve following I/R-induced AKI (23 wild-type mice; 20 Pkm2−/− mice). Survival was analysed using Kaplan–Meier estimation. Gehan–Breslow–Wilcoxon test was used to detect significance. c, PEP in injured kidneys from wild-type and Pkm2−/− mice (n = 6 mice per group); injury induced by I/R. d, Pyruvate in injured kidneys from wild-type and Pkm2−/− mice (n = 6 mice per group). Results in c, d are presented as mean ± s.d. Two-tailed Student’s t-test was used to detect significance.

Source data

Extended Data Fig. 10 Expression of PKM1, PKM2 and PKLR after AKI.

a, Immunostaining showing expression of PKM2 in sham or injured kidneys of wild-type mice on the indicated days after surgery; injury induced by I/R. Images are representative of two independently performed experiments with similar results. b, Western blot showing expression of PKM2, PKM1 and PKLR in sham or injured kidneys of wild-type mice; injury induced by I/R. Images are representative of two independently performed experiments with similar results. c, Quantification of expression of PKM2, PKM1 and PKLR in b (n = 3 mice). Results are presented as mean ± s.d. One-way ANOVA with Tukey’s post hoc test was used to detect significance.

Source data

Supplementary information

Supplementary Figures

This file contains scanned complete images of western blots

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Supplementary Tables

This file contains Supplementary Tables 1-3. Supplementary Table 1: SNO-proteins enriched ≥1.4 fold in injured Akr1a1-/- kidneys vs. Akr1a1+/+, detected by SNO-RAC-coupled quantitative iTRAQ MS. Supplementary Table 2: AKR1A1 interactome in mouse kidney by IP-MS. Supplementary Table 3: Shared targets identified in both the S-nitrosoproteome and AKR1A1 interactome

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Zhou, HL., Zhang, R., Anand, P. et al. Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury. Nature 565, 96–100 (2019). https://doi.org/10.1038/s41586-018-0749-z

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