Tubular ACSM3 controls fatty acid metabolism and safeguards against acute kidney injury in male mice

Abstract

Kidney tubular epithelial cells exceptionally exhibit high energy demands and preferentially metabolize long-chain fatty acids via fatty acid oxidation (FAO), where the impairment of FAO represents a hallmark of acute kidney injury (AKI). However, the role of medium-chain fatty acid metabolism in kidney injury remains unexplored. Here, we identify that tubular acyl-CoA synthetase medium-chain family member 3 (ACSM3), the key enzyme responsible for medium-chain fatty acid activation, is significantly down-regulated in damaged kidneys of distinct AKI male mouse models and acute tubular necrosis patients. Unexpectedly, tubule-specific ACSM3 deletion improves renal dysfunction, pathological damage, and metabolic disturbances in AKI male mice. Mechanistically, tubular ACSM3 deficiency preserves free fatty acid pool and reduces medium-chain fatty acids utilization, where these unused medium-chain fatty acids as ligands can activate peroxisome proliferator-activated receptor alpha (PPARα) and further upregulate PPARα-associated fatty acid metabolic genes to repair injured kidneys. Notably, dietary supplementation of medium-chain fatty acids confers protective effects against AKI in male mice. Our findings highlight tubular ACSM3 as a potential therapeutic target to control renal fatty acid metabolism and provide preclinical evidence that medium-chain fatty acid supplementation safeguards against AKI.

Fatty acid oxidation impairment represents a hallmark of acute kidney injury (AKI). Here the authors report that tubular deficiency of ACSM3, an enzyme involved in medium chain fatty acid activation, is protective against kidney injury in male mouse models of AKI potentially via modulating PPARα activity.

Introduction

Acute kidney injury (AKI) is a clinically significant syndrome characterized by rapid renal function decline, with increasing global incidence and mortality¹. AKI results from diverse causes, including nephrotoxins (e.g., cisplatin), infections, and obstruction, yet treatments remain limited due to incomplete mechanistic insights^2,3. Currently, it is widely accepted that the pathophysiological core of AKI centers on severe injury to proximal tubule epithelial cells (PTECs), featured by the impairment of fatty acid β-oxidation (FAO)^4–6. Restoring FAO has emerged as a promising therapeutic approach to against AKI^7–9.

Fatty acids are the preferred macronutrient to support kidney energy metabolism. Although PTECs typically rely on long-chain fatty acids (LCFA, C > 12) as their primary energy source, the role of medium-chain fatty acids (MCFA, C8–C12) remains unexplored in cells^10,11. A fundamental difference characterizes the mitochondrial shuttle mechanism, where LCFA oxidation is a rate-limited process via carnitine palmitoyltransferase 1 (CPT-1) activity. However, medium-chain fatty acid oxidation can be independent of L-carnitine in the liver and kidney, attributable to their shorter aliphatic chain structure^12,13. Biochemically, FAO initiation is activated by acyl-CoA synthetases (ACSs), which are categorized into short-, medium-, and long-chain subtypes based on their substrate specificity. Among these, medium-chain acyl-CoA synthetases (ACSMs) convert MCFA into their corresponding CoA esters and thereby facilitates their downstream fatty acid oxidation^14–16. Despite their pivotal role in MCFA metabolism, the function of ACSMs in AKI constitutes a critical knowledge gap in the field.

In this study, we first discovered that tubular ACSM3 was significantly decreased in the kidneys of AKI male mouse models and patients with acute tubular necrosis (ATN). Further, we developed the Acsm3 tubule-specific knockout (Acsm3-CKO) and global knockout (Acsm3^-/-) mice to establish cisplatin and ischemia-reperfusion injury (IRI) induced AKI models. We demonstrate that tubular ACSM3 controls fatty acid metabolism via MCFAs-mediated peroxisome proliferator-activated receptor alpha (PPARα) activation and safeguards against AKI.

Results

Tubular ACSM3 is significantly decreased in kidneys of AKI mice and ATN patients

Fatty acid metabolism was the most significantly enriched pathway among the 4,185 differentially expressed genes found by transcriptomic analysis of two AKI models (cisplatin and IRI-induced). (Fig. 1A, B). Strikingly, Acsm3 was among the more dramatically changed genes in the pathway of fatty acid metabolism across all models (Fig. 1C, Supplementary Fig. 1A). Under physiological condition, ACSM3 localizes to tubular mitochondria to regulate MCFA metabolism (Fig. 1D). After cisplatin and IRI injury, Acsm3 mRNA level and protein expression were decreased in AKI models (Supplementary Fig. 1B, C), and its cytoplasmic localization in proximal tubules was revealed by immunohistochemistry (IHC) and immunofluorescence (IF) (Fig. 1E, G; Supplementary Fig. 1D). Publicly available single-nucleus/single-cell RNA sequencing data^17,18 from IRI-induced AKI models both demonstrated ACSM3 downregulation specifically in the proximal tubule (PT) (Supplementary Fig. 1E, F). Clinically, ATN patients (Supplementary Table 1) had the considerably lower levels of ACSM3 in contrast to control (Fig. 1F, H). These findings suggested the potential role of tubular ACSM3 in AKI pathogenesis.

Fig. 1. Tubular ACSM3 is decreased in kidneys of AKI mice and acute tubular necrosis patients.

A Venn diagram showing the overlap of differentially expressed genes (DEGs) identified from comparisons of the two AKI models. Created in BioRender. Fp, Z. (2026) https://BioRender.com/3edvic1. B The most significantly enriched KEGG pathways of the 4,185 common DEGs identified in RNA sequencing analysis. C Volcano plot of genes related to fatty acid metabolism, the most significantly enriched KEGG pathway. D Subcellular localization of ACSM3 under physiological conditions was observed using laser confocal microscopy in TCMK-1 cells, along with a schematic diagram of ACSM3 function, Bar = 5 µm. Created in BioRender. Fp, Z. (2026) https://BioRender.com/9fj2b3a. E, G Immunohistochemical analysis of ACSM3 expression in cisplatin- and ischemia-reperfusion injury-induced AKI in C57 mice using paraffin-embedded kidney sections (n = 4 mice per group). Bars = 50 µm. F, H Immunohistochemical analysis of ACSM3 expression in kidney tissues from patients diagnosed with acute tubular necrosis and adjacent non-tumor tissues from renal cancer patients (n = 10 patients per group). Bars = 25 µm. The ACSM3-positive area was quantified using Image J software in five randomly selected fields per specimen. CP cisplatin, Veh vehicle, IRI ischemia-reperfusion injury, ATN acute tubular necrosis. Data are presented as mean ± SEM. Statistical significance was determined by two-sided t-test (G, H). Source data are provided as a Source Data file.

Tubular ACSM3 deficiency safeguards against AKI in mice

To examine the role of ACSM3 in AKI, we generated tubule-specific knockout (Acsm3-CKO) and global knockout (Acsm3^-/-) male mice (Supplementary Figs. 2, 3). Compared to Acsm3^f/f mice in the cisplatin-induced AKI model (Fig. 2A), Acsm3-CKO significantly reduced serum creatinine (sCR) and blood urea nitrogen (BUN) (Fig. 2B, C), attenuated histopathological damage by H&E and PAS staining (Fig. 2D), as well as decreased tubular injury marker NGAL and KIM-1 expression (Fig. 2E). Metabolic profiling revealed comprehensive improvements in lipid homeostasis in injured kidneys of the ACSM3-deficient mice, including reduced triglyceride (TG) accumulation (Fig. 3A), decreased lipid peroxidation as measured by malondialdehyde (MDA) (Fig. 3B), and diminished intrarenal lipid deposition (Fig. 3C). Furthermore, these ACSM3-deficient AKI mice exhibited the preserved mitochondrial ultrastructure (Fig. 3D) as well as increased FAO capacity and improved cellular energy status (Fig. 3E). ACSM3 deficiency in primary renal tubular cells significantly mitigated cisplatin-triggered lipid accumulation and reactive oxygen species (ROS) production (Fig. 3F, G). In addition, tubular ACSM3 deficiency also improved kidney function, pathological damage, and metabolic disorder in IRI-induced AKI mice (Supplementary Figs. 4, 5). Acsm3 global knockout did not exhibit any overt metabolic disturbances or extra-renal pathological alterations under baseline physiological conditions (Supplementary Fig. 6). In cisplatin and IRI-induced AKI mice, global knockout of ACSM3 also mitigated renal injury, enhanced fatty acid oxidation and improved lipid deposition. (Supplementary Figs. 7–9). To test the adaptive nature of ACSM3 downregulation, we performed gain-of-function experiments. Conversely, ACSM3 overexpression in renal tubular epithelial cells exacerbated cisplatin-induced injury, with the increase of tubular damage markers (KIM-1, NGAL) and inflammation (IL-1β, MCP1) (Supplementary Fig. 10).

Fig. 2. Tubular ACSM3 deficiency attenuated cisplatin-induced kidney injury.

A Schematic of the cisplatin (CP)-induced acute kidney injury (AKI) experiment: mice were intraperitoneally injected with 20 mg/kg CP to induce AKI, while the control group received vehicle. Samples were collected 72 hours post-treatment. Created in BioRender. Fp, Z. (2026) https://BioRender.com/rnih90x. B, C Serum creatinine (sCR) and blood urea nitrogen (BUN) levels were measured using an automated biochemistry analyzer (n = 6 mice per group). D Paraffin-embedded kidney sections were stained with hematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS). The tubular injury scores and pathologic cast counts were evaluated in five randomly chosen fields per kidney (n = 6 mice per group). Bars = 50 μm. “▲“ indicated typical pathological cast injury. E The mRNA and protein expression levels of NGAL and KIM-1 were detected by RT-qPCR and Western blot, with β-Actin used as a loading control. Band densities were quantified using ImageJ software (n = 4/6 mice per group). CP cisplatin, Veh vehicle. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s two-sided multiple comparison test (B, C, E) or two-sided t-test (D). Source data are provided as a Source Data file.

Fig. 3. Tubular ACSM3 deficiency ameliorates cisplatin-induced lipid metabolism dysregulation.

A, B Kidney triglyceride (TG) and malondialdehyde (MDA) levels in mice were measured using a commercial assay kit (n = 6 mice per group). C Lipid accumulation was assessed by Oil Red O staining and quantified using ImageJ software in five randomly selected fields per sample. Bars = 50 μm. D Mitochondrial damage and lipid droplet changes in mouse kidneys were observed by transmission electron microscopy. Bars = 500 nm. “∆“ indicated lipid droplets. “★“ indicated typical mitochondrial injury. E Relative NAD + /NADH, Acetyl-CoA levels, and ATP in kidneys were measured using a commercial assay kit (n = 6 mice per group). F Lipid droplets accumulation in primary renal tubular epithelial cells (PTECs) were visualized using Nile red staining. Bars = 50 μm. G Intracellular reactive oxygen species levels in PTECs were assessed using the fluorescent probe DCFH-DA. Bars = 50 μm. CP, cisplatin; Veh, vehicle; WT, wild-type; ROS, reactive oxygen species. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test. All tests were two-sided. Source data are provided as a Source Data file.

To address the long-term impact of ACSM3 deficiency, we established a renal IRI-induced AKI-to-CKD transition model in Acsm3-CKO mice (Supplementary Fig. 11A). At 21 days post-IR, Acsm3-CKO mice showed significantly decreased uACR compared to Acsm3^f/f controls (Supplementary Fig. 11B). In addition, the level of sCR and BUN in mean value were reduced in Acsm3-CKO mice (Supplementary Fig. 11C, D). Additional analyses further revealed attenuated tubular injury and fibrosis (Supplementary Fig. 11E–L). Collectively, these results demonstrate that tubular ACSM3 deficiency confers long-term renoprotection in the AKI-to-CKD transition model, with the protective effect being more pronounced in the ameliorating of tubular injury, albuminuria, and fibrosis than in the normalizing of sCR and BUN levels at this time point.

Tubular ACSM3 deficiency controls fatty acid metabolism in the injured kidneys

Through integrating the RNA sequencing and metabolomic analyses, we identified that fatty acid metabolism was the significant enrichment pathway in cisplatin-injured kidneys of Acsm3-CKO mice (Fig. 4A–C). Notably, mitochondrial and peroxisomal fatty acid oxidation were markedly improved cisplatin-injured kidneys of Acsm3-CKO mice, as evidenced by upregulated mRNA levels (e.g., Cpt-1ɑ, Acsl3, Acox1, Acad9, Abca2, Acaa2 and Abcd1) and protein expression levels (CPT-1ɑ, ACSL3, and ACOX1) (Fig. 4D–F, Supplementary Fig. 12). Here, we found that tubular ACSM3 deficiency suppressed the expression of key glycolysis-related genes, including Hk2, Pkm, and Pfkfb3 (Supplementary Fig. 13). Although the free fatty acid pool showed no significant change in injured kidneys (Supplementary Fig. 13A), the utilization of certain long and very long-chain fatty acids was enhanced (Supplementary Fig. 14B, C), which was verified by the increase of their corresponding acyl-CoAs (e.g., C16:0-, C18:0-, and C22:6-CoA) as well as TCA cycle metabolite acetyl-CoA (Supplementary Fig. 14D). In addition, the increase of CPT-1α, ACSL3 and ACOX1 in renal IRI model of Acsm3-CKO mice (Supplementary Fig. 15) and Acsm3^-/- mice (Supplementary Fig. 16A, B), as well as cisplatin-stimulated primary tubular cells (Supplementary Fig. 16C) also confirmed that ACSM3 deficiency restores fatty acid metabolism and energy homeostasis against kidney injury.

Fig. 4. Tubular ACSM3 deficiency improves fatty acid oxidation in injured kidneys.

A Mechanistic investigations were performed using RNA sequencing-based transcriptomics and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics in Acsm3-CKO mice. Created in BioRender. Fp, Z. (2026) https://BioRender.com/ajfj1nf. B Venn diagrams of differentially expressed genes (DEGs) from RNA-seq analysis for the comparisons between the indicated comparisons. C Top 10 significantly enriched KEGG pathways (FDR < 0.05) from 3,541 DEGs. D Heatmap of lipid metabolism-related DEGs from RNA-seq analysis, with functional localization in metabolic processes: fatty acid synthesis, fatty acid β-oxidation (mitochondrial and peroxisomal β-oxidation), fatty acid transport, and fatty acid regulation. Created in BioRender. Fp, Z. (2026) https://BioRender.com/weefi6q. E The mRNA levels of Cpt-1ɑ, Acsl3, and Acox1 in kidneys were detected by RT-qPCR (n = 6 mice per group). F Protein expression levels of CPT-1ɑ, ACSL3, and ACOX1 in kidney tissues were quantified by Western blot using β-actin as a loading control. Veh vehicle, CP cisplatin. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test. Source data are provided as a Source Data file.

Furthermore, RNA sequencing analysis revealed significant enrichment of the lipid droplets (LDs) decomposition pathway in injured kidneys of Acsm3-CKO mice, evidenced by upregulation of Pnpla2 (encoding ATGL, adipose triglyceride lipase, the rate-limiting enzyme of lipolysis) and downregulation of the LDs stabilizer Plin2 (Fig. 5A, B). These findings were confirmed at both mRNA and protein levels (Fig. 5C). Direct functional assays further demonstrated that ACSM3 deficiency significantly enhanced total lipase activity and glycerol levels in cisplatin-injured kidneys, which are confirmed by increased lipolytic flux (Supplementary Fig. 17). Consistent with this enhanced lipolysis, electron microscopy showed that the number and size of LDs were decreased in injured kidneys of Acsm3-CKO mice in contrast to control mice (Fig. 5D). IF staining also indicated the reduction of Plin2 expression in cisplatin-injured kidneys of Acsm3-CKO mice (Fig. 5E). Notably, BODIPY 493/503 staining revealed the fewer LDs in ACSM3-deficient tubular cells after cisplatin treatment (Fig. 5F). This reduction in lipid accumulation was further corroborated by a live-cell trafficking assay, which was demonstrated that the fluorescent fatty acid (Red C12) was more efficiently directed toward mitochondria (MitoTracker Green) in ACSM3-deficient cells (Fig. 5G). The similar renoprotective effects were confirmed in the IRI-AKI mouse model (Supplementary Fig. 18). Overall, these results shows that ACSM3 deficiency promotes LDs catabolism via lipolysis in AKI mice.

Fig. 5. Tubular ACSM3 deficiency promotes lipolysis in injured kidneys.

A Heatmap of lipid droplet decomposition pathway-related genes derived from RNA sequencing analysis. B Schematic diagram of key steps in the two major pathways of lipid droplet degradation: lipolysis and lipophagy. Created in BioRender. Fp, Z. (2026) https://BioRender.com/f6g2k1n. C The mRNA levels of ATGL (Pnpla2), and Plin2 in kidneys were detected by RT-qPCR and protein levels of ATGL and Plin2 in kidneys were analyzed by Western blot (n = 4 mice per group). D Lipid droplets size and lipolysis in kidneys were observed by transmission electron microscopy. Bars = 200 nm. “○“ indicated larger fused lipid droplets. “∆“ indicated smaller lipid droplets (n = 4 mice per group). E Immunofluorescence was used to observe changes in Plin2 in kidneys. Bars = 50 μm. F, G Primary RTECs from WT and Acsm3^-/- mice were pulsed with Red C12 fluorescent fatty acid, cisplatin-treated, stained for LDs (BODIPY 493/503, Green) and mitochondria (MitoTracker, Green), with representative images showing Red C12 (red) distribution. Bars = 5 μm. WT wild-type, CP cisplatin, Veh vehicle, LDs lipid droplets, RTEC renal tubular epithelial cells, TG triglyceride, TAG triacylglycerol, MAG monoacylglycerol, HSL hormone-sensitive lipase, MGL monoacylglycerol lipase, ATGL adipose triglyceride lipase. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test (C) or two-sided t-test (D). Source data are provided as a Source Data file.

Tubular ACSM3 deficiency confines MCFA utilization to activate PPARα-mediated fatty acid metabolism against AKI

As known, ACMS3 is a key enzyme of MCFA metabolism, and how does tubular ACSM3 deficiency control fatty acid metabolism against injured kidneys? Here, our RNA sequencing analysis revealed the significant enrichment of the PPAR signaling pathway (Fig. 4C), and subsequent protein interaction analysis identified PPARα as a central regulator of fatty acid metabolism in kidneys of Acsm3-CKO AKI mice (Fig. 6A). Transcription experimental and factor prediction validation confirmed that Ppara was upregulated and strongly related to these key FAO genes (Cpt-1a, Acsl3, Acox1) (Supplementary Fig. 19; Fig. 6B, C). Importantly, PPARα from cytoplasm relocated into tubular cell nucleus in injured kidneys of Acsm3-CKO mice (Fig. 6D, E). Metabolically, tubular ACSM3 deficiency led to the accumulation of MCFAs (C8:0, C10:0, and C12:0) and a concomitant reduction in their activated CoA esters, indicating a blockade in MCFA metabolic flux (Fig. 6F). To delineate the downstream consequences, we assessed key lipid-handling proteins. Consistent with this metabolic disruption, the expression of medium-chain acyl-CoA dehydrogenase (MCAD), a key MCFA β-oxidation enzyme, was significantly downregulated. In contrast, the levels of fatty acid transporters CD36 and FATP2 remained unchanged, indicating that altered fatty acid uptake is not a primary mechanism (Supplementary Fig. 20). We next investigated how the accumulated MCFAs exerted their protective effects. Molecular docking, biophysical binding assays, and in vivo immunoprecipitation-lipidomics confirmed the direct binding and activation of PPARα by MCFAs, with lauric acid (C12:0) serving as the predominant agonist (Fig. 6G, H; Supplementary Fig. 21). Consequently, in a luciferase reporter assay, MCFAs [C8:0, C10:0, GTCC (glyceryl tricaprylate/caprate, C8:0/C10:0), C12:0] enhanced PPARα transcriptional activity (Fig. 6I, Supplementary Fig. 22).

Fig. 6. Tubular ACSM3 deficiency upregulated FAO and lipolysis through MCFAs-PPARα in injured kidneys.

A Protein-protein interaction (PPI) network prediction based on 3,540 commonly differentially expressed genes identified from RNA sequencing analysis. B Volcano plot of differentially expressed genes related to fatty acid metabolism in RNA-seq analysis, along with changes in several nuclear transcription factors. C The mRNA levels of Pparα in kidneys were detected by RT-qPCR (n = 6 mice per group). D Protein levels of nuclear PPARα (nPPARα) and cytoplasmic PPARα (cPPARα) in kidneys were analyzed by Western blot. E Immunofluorescence was used to observe changes in nuclear PPARα in kidneys. Bars = 50 μm. F Levels of MCFAs and MCFAs-CoAs in kidneys were assessed by UPLC-MS (n = 4 mice per group). G Molecular docking analysis of MCFAs with PPARα ligand-binding domain. H Heatmap of lipidomics analysis following PPARα immunoprecipitation (IP) showing relative abundance of fatty acids bound to PPARα in kidney tissues from cisplatin-treated Acsm3^f/f and Acsm3-CKO mice. I Dual-luciferase reporter assay validated MCFAs-mediated activation of PPRE (peroxisome proliferator response element) in 293 T cells (n = 6 independent cell cultures per group). Veh vehicle, CP cisplatin, MCFAs medium-chain fatty acids, GTCC glyceryl tricaprylate/caprate (C8:0/C10:0), FAO fatty acid oxidation. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test. Source data are provided as a Source Data file.

As suggested that ACSM3 deficiency exerted renoprotection via PPARα activity, we conducted the rescued experiment using a PPARα antagonist GW6471 (Fig. 7A). The GW6471 aggravated renal function (Fig. 7B; Supplementary Fig. 23A), and histological damage (Fig. 7C) in cisplatin-injured Acsm3-CKO mice. GW6471 further deteriorated lipid metabolism disorder (Supplementary Fig. 23B), TG, Acetyl-CoA, and ATP levels (Fig. 7D, E; Supplementary Fig. 23C), and concurrently suppressed FAO/lipolysis-related gene and protein expression (Supplementary Fig. 23D) in cisplatin-injured Acsm3-CKO mice. Additionally, ACSM3 deficiency alleviated cisplatin-induced cell injury (Supplementary Fig. 24A), improved lipid metabolism (Supplementary Fig. 24B, C), educed LDs accumulation (Fig. 7F; Supplementary Fig. 24D), enhanced mitochondrial bioenergetics (Fig. 7G) and membrane potential (Fig. 7H) in cisplatin-stimulate tubular cells. All these protective effects were counteracted by the PPARα antagonist GW6471. These results demonstrated that ACSM3 deficiency protected against AKI through the MCFAs-PPARα-fatty acid metabolism axis (Fig. 7I).

Fig. 7. PPARα antagonist GW6471 counteracted the protective effect of tubular ACSM3 deficiency in cisplatin-induced kidney injury.

A Schematic of the experimental design. Created in BioRender. Fp, Z. (2026) https://BioRender.com/nb03v0o. B Serum creatinine (sCR) (n = 6 mice per group). C Hematoxylin & eosin (H&E) staining of kidney sections. Bars = 25 μm (n = 6 mice per group). D Kidney triglyceride (TG) levels in mice were measured using a commercial assay kit (n = 6 mice per group). E Kidney Acetyl-CoA levels in mice were measured using a commercial assay kit (n = 6 mice per group). F BODIPY493/503 staining of lipid droplets in primary renal tubular epithelial cells (PTECs). G Mitochondrial oxygen consumption rate (OCR) (n = 8 independent cell cultures per group). H JC-1 assay for membrane potential. I Proposed mechanism: ACSM3 deficiency alleviates AKI. Created in BioRender. Fp, Z. (2026) https://BioRender.com/sb45bhy. WT wild-type, Veh vehicle, CP cisplatin, MCFAs medium-chain fatty acids. Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test. Source data are provided as a Source Data file.

Dietary supplementation of MCFAs protects against AKI in mice

Based on our findings, we therapeutically performed MCFAs supplementation for AKI mice (Fig. 8A). The MCFAs comprised GTCC (C8:0/C10:0) and C12:0. Guided by dose-optimization and safety assessments, a dose of 5 mg/kg was selected for studies, as it effectively attenuated kidney injury without adverse effects on liver function or other major organs (Supplementary Figs. 25, 26). Specifically, treatment with MCFAs significantly improved renal function (Fig. 8B), mitigated tubular damage (Fig. 8C-E), and restored lipid homeostasis (Fig. 8F-I; Supplementary Fig. 27B). MCFAs also activated PPARα nuclear translocation and increased PPARα-targeted CPT-1α, ACSL3, ACOX1, and ATGL at both transcriptional and protein levels in injured kidneys of cisplatin-induced mice(Fig. 8J; Supplementary Fig. 27A, C). In cisplatin-stimulated renal tubular TCMK-1 cells, MCFAs markedly attenuated lipid accumulation (Supplementary Fig. 27D), ameliorated metabolic perturbations (Supplementary Fig. 27E), potentiated PPARα signaling (Supplementary Fig. 27F), and rescued mitochondrial function (Supplementary Fig. 27G). Collectively, these data demonstrate that dietary supplementation of MCFAs improves fatty acid metabolism and protects against AKI in mice.

Fig. 8. Dietary MCFA supplementations protects against cisplatin-induced AKI mice.

A Experimental design for MCFAs supplementation. Created in BioRender. Fp, Z. (2026) https://BioRender.com/bau3f92. B Serum creatinine (sCR) and blood urea nitrogen (BUN) levels (n = 6 mice per group). C, D Hematoxylin & eosin (H&E) staining of kidney sections. (n = 6 mice per group). Bars = 50 μm. “▲“ indicated a typical pathological cast injury. E The mRNA levels of NGAL and KIM-1 in kidneys were detected by RT-qPCR (n = 6 mice per group). F, H Kidney triglyceride (TG), acetyl-CoA, and ATP levels were measured using commercial assay kit (n = 6 mice per group). G Oil red O-stained lipid accumulation (n = 4 mice per group). I Lipid droplets in the kidneys were observed by transmission electron microscopy. J Protein levels of nuclear PPARα and fatty acid oxidation-related proteins in the kidneys were analyzed by Western blot. CP cisplatin, Veh vehicle, MCFAs medium-chain fatty acids, GTCC glyceryl tricaprylate/caprate (C8:0/C10:0). Data are presented as mean ± SEM. Statistical significance was determined by ANOVA with Tukey’s two-sided multiple comparison test. Source data are provided as a Source Data file.

Discussion

In this study, tubular ACSM3 expression was significantly downregulated in both AKI male mice and patients, while tubular ACSM3 deficiency safeguards against cisplatin- and IRI-induced AKI. Mechanistically, ACSM3 deletion leads to the accumulation of MCFAs, which act as ligands to activate PPARα signaling and improve fatty acid metabolism. These findings for the first time establish tubular ACSM3 and dietary MCFAs supplementation as a novel target and therapeutic strategy against AKI.

Notably, these kidney-specific findings stand in striking contrast to ACSM3’s established roles in other pathologies. ACSM3 displays paradoxical regulatory roles across disease states. ACSM3 modulates metabolic diseases by promoting adipocyte MCFA catabolism¹⁹, enhancing thermogenesis to alleviate type 2 diabetes²⁰, and preserving hepatic homeostasis through inhibition of MCFAs-p38 MAPK axis²¹. Intriguingly, ACSM3 exhibits tissue-specific oncogenic properties by promoting prostate cancer progression through FAO-dependent ferroptosis resistance¹⁶, yet demonstrates tumor-suppressive effects in ovarian cancer²² and melanoma²³ via AMPK activation and PI3K/Akt inhibition, respectively. Emerging single-cell and spatial transcriptomic analyses consistently revealed the PTECs-specific ACSM3 downregulation in renal IRI in murine models^17,24, findings corroborated by proteomic datasets from both aristolochic acid- and IRI-induced AKI in mice^25,26. Contrary to expectations, our studies demonstrate that ACSM3 deficiency confers renal protection against AKI. We propose that ACSM3 downregulation represents an adaptive metabolic remodeling mechanism during AKI that restores lipid homeostasis to mitigate tubular epithelial lipotoxicity and cellular dysfunction. Remarkably, Acsm3 knockout potentiates this favorable effect by enhancing metabolic regulation, thereby uncovering its pivotal role in AKI pathogenesis and highlighting its therapeutic potential.

ACSM3, functioning as an acyl-CoA synthetase that primarily catalyzes the conversion of MCFA into their corresponding acyl-CoAs for subsequent β-oxidation. Our data demonstrates that renal tubule-specific ACSM3 deficiency significantly reduces activated medium-chain (C8-C10) acyl-CoA levels and concomitantly increases renal free MCFAs accumulation. RNA-seq analysis further revealed significant enrichment of PPAR signaling pathways following Acsm3 ablation. Consistent with existing evidence that MCFA serve as endogenous ligands for nuclear receptors and fatty acid sensors, particularly PPARα^27–29, our integrated metabolomic, biophysical, and functional analyses also confirm that MCFAs accumulating due to ACSM3 deficiency directly bind to and activate PPARα. Collectively, these findings establish a signaling axis of “ACSM3-MCFA-PPARα” in AKI.

This metabolic shift is functionally critical. PPARα, acting as the master transcriptional regulator of lipid metabolism, exerts multifaceted protective effects in renal pathophysiology by directly upregulating genes in FAO and lipolysis, while coordinately suppressing de novo lipogenesis and inflammatory pathways^30–32. This view is supported by evidence from diverse experimental models demonstrating that renal PPARα activation not only mitigates tubular damage in cisplatin- and IRI-induced AKI but also alleviates inflammatory responses in septic AKI^33–39. Furthermore, PPARα activation sustains mitochondrial FAO capacity, thereby preventing metabolic stagnation and halting the progression from AKI to CKD⁴⁰. Its indispensable role is further substantiated by genetic studies: proximal tubule-specific PPARα overexpression confers resistance to kidney injury^41,42, whereas its functional decline precipitates renal lipid overload and accelerates fibrosis⁴³. In line with these findings, our study demonstrates that pharmacological blockade of PPARα using the specific antagonist GW6471 significantly attenuates the renoprotective effects mediated by ACSM3 ablation. This functional experiment definitively establishes that the protective benefits of ACSM3 deficiency are dependent on PPARα activation. Together, these results support our proposed mechanistic model wherein ACSM3 deficiency induces MCFAs accumulation and activates PPARα signaling, ultimately leading to the improvement of lipid metabolic disorders.

As the central transcriptional regulator of lipid homeostasis, PPARα drives a coordinated protective response by upregulating key FAO genes such as Cpt-1α and Acox1, thereby enhancing LCFA catabolism. This transcriptional regulation aligns with our observed metabolic reprogramming, characterized by elevated LCFA‑CoA species and increased expression of β‑oxidation enzymes, including CPT‑1α, reflecting a physiologically adaptive shift toward heightened LCFA utilization in renal tubular cells, which are known to depend primarily on LCFAs for energy generation⁴⁴. This metabolic adjustment establishes that ACSM3 deficiency safeguards AKI through PPARα-mediated augmentation of mitochondrial fatty acids catabolism^31,32. Concurrently, PPARα activation promotes lipolysis to reduce lipid accumulation. This aligns with our observed downregulation of lipid droplet-associated proteins such as PLIN2, indicating enhanced lipid droplet degradation and thereby effectively mitigating renal lipotoxicity^31,32,45. These collective findings delineate an ACSM3-MCFAs-PPARα signaling axis where accumulated MCFAs activate PPARα to coordinate metabolic rescue, a therapeutic paradigm we successfully replicated through exogenous MCFAs supplementation. This innovative approach simultaneously corrects the lipid dysmetabolism and bioenergetic impairment characteristic of AKI by strategically harnessing endogenous protective mechanisms.

While our study provides a mechanistic framework from acute injury to chronic progression, several translational and mechanistic nuances invite further exploration. First, the exclusivity of PPARα as the mediator and the cell-type specificity of this axis within the kidney need definitive validation. Second, bridging our findings to the clinic requires identifying predictive biomarkers based on ACSM3-MCFA and optimizing MCFA dosing regimens for human physiology. Finally, the systemic metabolic consequences of long-term MCFA modulation merit careful evaluation to ensure holistic therapeutic safety. Collectively, our study highlights that tubular ACSM3 as a novel drug target to control fatty acid metabolism and MCFA supplementation as a feasible strategy to safeguard AKI.

Methods

Experimental animals

The animal study protocol was approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (#20230919002). Male C57BL/6 J mice (8-10 weeks old, 22–25 g) were obtained from GemPharmatech. Only male mice were utilized in this study, as this decision was grounded in robust preliminary data and established data (Supplementary Fig. 2) indicating that renal ACSM3 expression is biologically negligible in female mice, thereby ensuring the model’s focus on the functional role of ACSM3 in AKI⁴⁶. Briefly, for global Acsm3-knockout (Acsm3^−/−) mice, Acsm3 gene was targeted using the CRISPR/Cas9 system. For ubular epithelial cell conditional Acsm3-knockout (Acsm3-CKO) mice, Acsm3-floxed (Acsm3^f/f) mice were generated and then crossed with mice expressing Cre recombinase under the control of the cadherin 16 (Cdh16) promoter (Supplementary Fig. 3)⁴⁷. Mice were housed under specific pathogen-free conditions (20–26 °C, 40–70% humidity, 12-h light/dark cycles) with ad libitum access to food and water.

Animal models and procedures

Cisplatin-induced AKI: wild-type (WT), Acsm3^−/−, Acsm3^f/f, and Acsm3-CKO mice (8–10 weeks old) were randomly assigned to experimental groups. AKI was induced by a single intraperitoneal injection of 20 mg/kg cisplatin (#479306, Sigma) or saline (vehicle). Mice were euthanized 72 hours post-injection, and serum/kidney samples were collected^3,48. IRI-induced AKI: under isoflurane anesthesia, bilateral renal arteries were clamped for 30 minutes, followed by reperfusion. Sham-operated mice underwent identical procedures without vascular clamping. Postoperatively, all mice received intraperitoneal saline (100 μL, 37 °C)⁴⁹. Ischemia-reperfusion induced AKI-to-CKD: mice were subjected to unilateral renal ischemia for 23 minutes followed by reperfusion to induce progressive kidney injury. Phenotypic assessments were performed at the chronic phase (21 days post-injury).

Pharmacological treatments

The PPARα antagonist GW6471 (#HY-15372, MedChemExpress) was administered via intraperitoneal injection at a dose of 20 mg/kg, 30 minutes prior to cisplatin injection or IRI surgery. GW6471 was first dissolved in dimethyl sulfoxide (DMSO) and then diluted in sterile saline, with the final DMSO concentration not exceeding 5%^50,51.To model cisplatin-induced nephrotoxicity in vitro, primary proximal tubule cells were exposed to cisplatin following a 1-hour pretreatment with 2 mM GW6471 or vehicle⁵².

The MCFAs octanoic acid (C8:0, #124-07-2), decanoic acid (C10:0, #334-48-5), and their structured triglyceride, glyceryl tricaprylate/caprate (GTCC, #65381-09-1, typically composed of 50-80% C8:0 and 20-50% C10:0), lauric acid (C12:0, #143-07-7) were all sourced from Biohybrid Pharmatech. Prior to administration, each MCFA was freshly prepared as an emulsion by dissolving in a vehicle containing 5% Tween-80 and 10% polyethylene glycol 400 (PEG 400) in sterile saline, with gentle vortexing and sonication to ensure homogeneity. All compounds were administered via oral gavage at a molar dose of 5 mg/kg, delivered in a final volume of 100 μL. For the cisplatin-induced AKI model, mice received treatment twice daily for three consecutive days to ensure sustained metabolic modulation during nephrotoxin exposure. In the renal IRI model, a single dose was administered 30 minutes prior to surgery to provide acute preconditioning.

Histological staining and renal tubular injury score

Kidney tissues were fixed in 10% neutral-buffered formaldehyde, embedded in paraffin, and sectioned at 4 µm thickness. Sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Tubular injury was assessed in ≥10 random fields of the renal cortex/outer medulla per sample using a semi-quantitative scoring system(0–4): 0, normal/minimal injury; 1, <25% tubules affected; 2, 25–50%; 3, 50–75%; 4, >75% of tubules showing epithelial swelling, necrosis, or dilation. Scoring was performed independently by two blinded observers^53,54.

RNA sequencing analysis

Total RNA was extracted from four representative kidney samples per group. Following quality control assessment, RNA-seq library preparation and sequencing were performed by Lianchuan Biotechnology.

Lipoyl CoA analysis (UPLC-MS)

Kidney samples were homogenized in acidified extraction buffer (isopropanol: 50 mM KH2PO4:BSA, 25:25:1) containing 19:0-CoA internal standard. Sequential extractions were performed with petroleum ether (3×) and chloroform:methanol (1:2). Long-chain acyl-CoAs were collected from supernatant, while short-chain CoAs were extracted from pellets using trichloroacetic acid. Combined extracts were analyzed by UPLC-MS/MS (Shimadzu 40X3B-Sciex QTRAP 6500 + ) in methanol:water (9:1, 0.05% acetic acid).

Metabolomics analysis (UPLC-MS)

Kidney samples were homogenized with zirconia beads in methanol/water containing internal standard. After derivatization (30 °C, 60 min) and evaporation, samples were reconstituted in 50% methanol. Quantification was performed by UPLC-MS/MS using derivatized standard curves.

Dual-Luciferase reporter assay

HEK-293T cells at 90% confluence were seeded in 24-well plates. At 50-70% confluency, cells were transfected with reporter plasmids. Transfection efficiency was monitored via fluorescent markers for 24-48 h. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase reporter assay kit (#RG009S, Beyotime).

Oxygen consumption rate (OCR) assay

PTECs were seeded in XF24 cell culture microplates (#100777-004, Agilent) and incubated in Seahorse XF Base Medium (#103334-100, Agilent) supplemented with 2 mM glutamine, 1 mM pyruvate, and 10 mM glucose for 1 h in a CO₂-free incubator. OCR was measured using a Seahorse XFe24 Extracellular Flux Analyzer (#S7800, Agilent) with the XF Cell Mito Stress Test Kit (#103015-100, Agilent). Mitochondrial function was assessed through sequential injection of: 5 µM oligomycin, 3 µM FCCP, and 1 µM rotenone/antimycin A.

Surface plasmon resonance (SPR) binding assay

SPR assays were performed by Sino Biological Inc. using a Biacore 8 K instrument (Cytiva). PPARα was immobilized on a CM5 chip and tested against fatty acids (lauric, octanoic, decanoic acid). Alternatively, biotinylated fatty acids were immobilized on a Streptavidin (SA) chip and probed with PPARα. Under these conditions, only lauric acid-biotin showed a binding signal (~978 RU), while octanoic acid-biotin and decanoic acid-biotin showed no significant binding.

Bio-Layer Interferometry (BLI) affinity measurement

BLI assays were performed by Sino Biological Inc. using an Octet RED384 system (Sartorius). Attempts to capture His-tagged PPARα on His1K biosensors were unsuccessful. Alternatively, biotinylated fatty acids were immobilized on SA biosensors. Only lauric acid-biotin was successfully immobilized and assayed with PPARα, yielding a binding affinity (KD) of 22.5 nM. Octanoic acid-biotin and decanoic acid-biotin could not be immobilized on the SA sensors under the experimental conditions.

Immunoprecipitation–Lipidomics for MCFA Profiling

PPARα-associated lipids were isolated from kidney lysates by immunoprecipitation and subjected to lipidomics. Eluted lipids were analyzed by LC–MS/MS for MCFAs. Samples were extracted, derivatized with 3-nitrophenylhydrazine, separated on a C18 column, and quantified using isotope-labeled internal standards⁵⁵.

Statistics and reproducibility

Data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA) and R software (version 4.3.1). For two-group comparisons, unpaired two-sided t-tests were used. For multi-group comparisons, one-way ANOVA followed by Tukey’s two-sided multiple comparison test was used. A p-value < 0.05 was considered statistically significant, and exact p-values are provided in the figure legends where possible. All experiments were repeated independently at least three times with similar results, including all representative images. For all experiments, n represents the number of biologically independent samples per group. No technical replicates were used for statistical derivation. Mice were randomly assigned to experimental groups using a random number generator, and investigators were blinded to group allocation during data collection and analysis.

Supplementary Methods

Detailed protocols for immunohistochemistry, immunofluorescence, lipid and metabolic assays, Oil red O staining, lipid droplet staining, JC-1 staining, cellular ROS assay, western blotting, RT‑qPCR, glycerol and lipase activity assays, and quantitative image analysis are provided in the Supplementary information.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

L.M., L.Lin, and P.Z. designed experiments. F.Z., L.F., T.X., J.L., Q.W. and F.G. performed experiments. L.M., F.Z., L.F., T.X., J.L., L.Li, Z.T., and F.G. analyzed the data. F.Z., L.F., T.X., P.F., and L.M. wrote the draft of the manuscript and edited it. All authors have read and approved the article.

Peer review

Peer review information

Nature Communications thanks Ronaldo Araujo, who co-reviewed with Raisa Brito Santos and the other, anonymous, reviewers for their contribution to the peer review of this work. [A peer review file is available].

Funding

This work was supported by the National Natural Science Foundation of China (U24A220667, 82570868, and 82370737), the 1.3.5 project for disciplines of excellence from West China Hospital of Sichuan University (ZYGD23015), the Clinical Research Projects from Health Commission of Sichuan Province Technology Projects in 2023 (23LCYJ015), and the 2024 Central Guiding Local Science and Technology Development Fund Project from Sichuan Provincial Department of Science and Technology (2024ZYD0070).

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. Raw RNA-seq data are publicly accessible via the NCBI BioProject database (PRJNA1275624) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1275624), and all processed data for each figure and Supplementary Fig. are provided as Source Data files with this paper. No data are available except by request. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-026-76637-7.