Dicarboxylic Acid Dietary Supplementation Protects against AKI

Abstract

Significance statement: In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage.

Background: Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism.

Methods: Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed.

Results: Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 .

Conclusions: DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.

Graphical abstract

Figure 1

DC₈ promotes succinylation in kidneys only, whereas DC₁₂ does it in both kidneys and liver. (A) Schematic showing mouse supplementation regimen with 10% DC₈ w/w, 10% DC₁₀ w/w, or 10% DC₁₂ w/w for 1 week. (B) Western blotting showing the expression pattern of succinyllysine in the livers and kidneys of the supplemented animals. (C) No differences were observed in the liver or kidney morphology of con, 10% DC₁₂-, and 10% DC₈-fed animals. (D) Five days on termination of the medium-chain fatty acid–enriched diet, succinylation expression is vanished from the body, evidencing no risk on long-term accumulation. N=2. Scale bars represent ×5 magnification.

Figure 2

DC₁₂ and DC₈ supplementation protect male mice from ischemia-reperfusion–induced AKI. (A) Schematic showing mouse supplementation regimen with 10% DC₈ w/w or 10% DC₁₂ w/w, as well as the IRI procedure and euthanasia. (B) Serum creatinine and BUN levels in IRI and Nx animals (N=3–9). (C) H&E staining in IRI animals, evidencing morphological changes in dicarboxylic acid–supplemented animals, such as a decline in tubular cast formation, dilation, and mitigated brush border loss, especially in DC₈ animals. Arrows point to different degrees of tubular cell injury, such as tubular dilation, tubular cast formation, and loss of brush borders. Semiquantitative scoring (0–4) for tubular injury was performed in a blind fashion for tubular dilation, proteinaceous cast formation, and loss of brush border (N=5–9). Scale bars represent ×10 and ×20 magnification, respectively. Results are expressed as mean±SD. Prism 9.0.0 software (GraphPad) was used for statistical analysis. Analysis was performed using one-way ANOVA with Tukey post hoc test. Significance was given by a P value <0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. H&E, hematoxylin and eosin; IRI, ischemia-reperfusion injury.

Figure 3

DC₁₂ and DC₈ supplementation mitigate tubular injury and prevent ischemia-reperfusion–induced AKI. (A) TUNEL staining. (B) Renal expression of NGAL, LTL, and DAPI. The grayscale values were quantified by drawing ROIs, around LTL-positive tubules using ImageJ, and dividing by the number of DAPI-positive nuclei per tubule (N=4). Arrows point to protein expression of NGAL. Scale bars represent ×10 magnification in images (A and B) and a zoomed area in (B′). Results are expressed as mean±SD. Prism 9.0.0 software (GraphPad) was used for statistical analysis. Analysis was performed using one-way ANOVA with Tukey post hoc test. Significance was given by a P value < 0.05. *P < 0.05, ****P < 0.0001. LTL, Lotus tetragonolobus lectin; NGAL, neutrophil gelatinase–associated lipocalin; ROIs, regions of interest; TUNEL, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling.

Figure 4

DC₈-fed animals are protected from cisplatin-induced AKI. (A) Schematic of supplementation regimen, cisplatin injection, and euthanasia of animals. (B) Serum creatinine and renal NGAL expression. (C) H&E staining and tubular injury score (scale bars represent ×5 and ×20 magnification, respectively). Arrows point to different degrees of tubular cell injury, such as tubular dilation, tubular cast formation, and loss of brush borders. Semiquantitative scoring (0–4) for tubular injury was performed in a blind fashion for tubular dilation, proteinaceous cast formation, and loss of brush border. (D) Immunofluorescence for NGAL, LTL, and DAPI. The grayscale values were quantified by drawing ROIs, around LTL-positive tubules using ImageJ, and dividing by the number of DAPI-positive nuclei per tubule. (Scale bars represent ×5 magnification.) N=7 per group. Results are expressed as mean±SD. Prism 9.0.0 software (GraphPad) was used for statistical analysis. Analysis was performed using the t test. Significance was given by a P value < 0.05. *P < 0.05.

Figure 5

DC₈ preserves mitochondrial function during IRI. (A–F) Mass spectrometry was used to quantify changes in mitochondrial protein abundance (A–C) and in protein succinylation (D–F, normalized to protein abundance), N=4. All volcano plots depict log₂ of the indicated FCs on the x axis plotted against log₁₀ of the q value on the y axis. Each dot represents a peptide; blue indicates significantly downregulated peptides, gold represents upregulated peptides, and gray represents peptides with nonsignificant change. Percentages given are the percent of total peptides for the indicated quadrant of the graph. (G and H) Oroboros high-resolution respirometry was used to evaluate mitochondrial respiration in freshly harvested kidney lysates (N=5). To allow combination of graphs across multiple days of experiments, each trace was normalized to the maximum uncoupled respiration for con kidneys. *P < 0.05 for con IRI versus con Nx. FC, fold change.

Figure 6

DC₈ remodels the peroxisomal proteome and succinylome. (A–F) Mass spectrometry was used to quantify changes in peroxisomal protein abundance (A–C) and in protein succinylation (D–F, normalized to protein abundance), N=4. All volcano plots depict log₂ of the indicated FCs on the x axis plotted against log₁₀ of the q value on the y axis. Each dot represents a peptide; blue indicates significantly downregulated peptides, gold represents upregulated peptides, and gray represents peptides with nonsignificant change. Percentages given are the percent of total peptides for the indicated quadrant of the graph.

Figure 7

Peroxisomal FAO enzymes demonstrate massively increased succinylation in the kidneys of DC₈-fed animals. Key enzymes involved in the different steps of the peroxisomal FAO pathway were hypersuccinylated in DC₈ IRI in comparison to con IRI N=4 per group. ABC, ATP-binding cassette; CoA, coenzyme A; FAO, fatty acid oxidation.

Figure 8

DC₈ protects peroxisomes during AKI and increases peroxisomal FAO. (A and B) Immunofluorescence for the peroxisomal markers PMP70 and PEX5. Scale bars represent ×5 and ×20 magnification, respectively. The grayscale values were quantified by drawing ROIs, around tubules identified through morphology using ImageJ, and dividing by the number of DAPI-positive nuclei per tubule (N=5). Results are expressed as mean±SD. Prism 9.0.0 software (GraphPad) was used for statistical analysis. (C and D) FAO flux was measured with ¹⁴C-palmitate (C₁₆) in Nx kidneys at baseline. Peroxisomal FAO was defined as the etomoxir-resistant portion. ***P < 0.001 by t test. (E and F) FAO flux with two peroxisome-specific substrates, ¹⁴C-DC₁₂ and ¹⁴C-C₂₄, measured in kidney lysates after IRI. *P < 0.05, **P < 0.01 by t test.