DUSP26 protects against acute kidney injury by dephosphorylating p53 at serine 312

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
Acute kidney injury (AKI) remains a leading cause of morbidity and mortality, yet the molecular pathways driving kidney tubule damage in AKI are not fully understood. Here, we report dual-specificity phosphatase 26 (DUSP26) as a critical regulator of kidney tubule injury in AKI. In our study, DUSP26 expression was markedly reduced in kidney biopsies from AKI patients of both sexes and in male murine models of cisplatin nephrotoxic and ischemic AKI. This down-regulation was driven by hypermethylation of the gene promoter of DUSP26 in kidney tubular cells. Loss of DUSP26 exacerbated tubular damage, whereas knock-in of DUSP26 specifically in kidney proximal tubule cells conferred protection. Mechanistically, DUSP26 directly bound to p53 to dephosphorylate it at serine 312, dampening the transcriptional activity of p53 towards cell death genes. Pharmacologic inhibition of DUSP26 sensitized kidneys to AKI, whereas DUSP26 overexpression was protective. Pharmacologic inhibition of DUSP26 also exacerbated ischemia-reperfusion injury in the liver. These findings uncover DUSP26 as a key phosphatase guarding against tissue injury by dephosphorylating p53 at serine 312, and highlight the DUSP26-p53 axis as a promising therapeutic target.

Introduction
Acute kidney injury (AKI) is a major kidney disease characterized by a rapid decline in renal function that is associated with high morbidity, mortality, and healthcare costs1,2. Intrarenal causes account for the majority of clinical cases of AKI, with the primary causes being ischemic injury or nephrotoxic injury3. Ischemic AKI frequently occurs in clinical settings such as major surgery, hypotension, myocardial infarction, dehydration, and kidney transplantation4. At the pathological level, AKI is marked by injury and death of renal tubular epithelial cells5, which are particularly vulnerable to hypoxia, toxins, proteinuria, and metabolic stress6,7. Importantly, AKI is now recognized as a major contributor to the development of chronic kidney disease (CKD), yet no effective therapies currently exist to substantially mitigate AKI or accelerate kidney recovery after AKI8. Emerging single-nucleus transcriptomic analyses of human AKI have revealed heterogeneous injury states of kidney tubular cells, underscoring the need to delineate the molecular regulators of epithelial cell fate in AKI9.

Among the regulatory mechanisms of AKI, the dynamic balance of protein phosphorylation and dephosphorylation—play a central role in modulating protein activity and signaling pathways10. While kinase-mediated phosphorylation events have been extensively studied in AKI, implicating pathways such as JAK/STAT and MAPK11-15, much less is known about the role of phosphatases. In this regard, there are reports about classical dual-specificity phosphatases (DUSPs) in oxidative stress, inflammation, and pyroptosis in AKI16-18. However, the contribution of non-classical DUSPs and the full landscape of phosphatase-mediated regulation in AKI remains poorly defined. DUSP26 is an atypical (lacks the conserved ERK-binding motif) dual-specificity phosphatase with a conserved catalytic domain that enables it to target a range of substrates beyond canonical MAPKs19. Previous studies have shown that DUSP26 may dephosphorylate p38 while paradoxically enhancing JNK and ERK phosphorylation and signaling, indicating a complex regulatory profile20,21. Reflecting its broad biological significance, DUSP26 has been implicated in diverse pathologies ranging from malignancies (such as neuroblastoma22,23) to metabolic diseases (including diabetes24 and diabetic cardiomyopathy25), underscoring its context-dependent roles in cell signaling and disease progression. In search of potential therapeutic targets in AKI, we performed integrative bioinformatic analyses of publicly available AKI datasets from the Gene Expression Omnibus (GEO) and applied machine learning algorithms including LASSO regression and support vector machines (SVM). These analyses highlighted DUSP26 as a promising candidate gene associated with AKI.

In this study, we systematically investigated the functional role and regulatory mechanisms of DUSP26 in AKI, focusing on its downstream targets and signaling effects in kidney proximal tubular epithelial cells. Our findings reveal a previously unrecognized role of DUSP26 in attenuating tubular injury in AKI through selective dephosphorylation of p53 at serine 312, offering mechanistic insights into AKI pathogenesis and pointing to a potential therapeutic axis for intervention.

Results
1. Down-regulation of DUSP26 in human AKI.
To identify key regulators of AKI, we performed integrative transcriptomic analyses across three publicly available AKI-related datasets from the Gene Expression Omnibus (GEO). Following batch effect correction (Fig. 1A), we identified differentially expressed genes (DEGs) between AKI and control samples (Fig. 1B). To prioritize functionally relevant candidates, we applied two machine learning algorithms-least absolute shrinkage and selection operator (LASSO) regression and support vector machine-recursive feature elimination (SVM-RFE). LASSO regression identified optimal variables based on minimal binomial deviance (Fig. 1C), while SVM-RFE ranked genes according to 10-fold cross-validation error (Fig. 1D). Cross-comparison of both approaches revealed a set of overlapping candidate genes (Fig. 1E), among which DUSP26 emerged as a robustly downregulated gene in AKI samples (Fig. 1F). Receiver operating characteristic (ROC) curve analysis further confirmed the predictive value of DUSP26 for distinguishing AKI from controls (Fig. 1G). To validate its relevance at the protein level, we performed immunofluorescence staining on human kidney biopsy specimens. The clinical data of the AKI patients are presented in Supplementary Table 1. Immunofluorescence analysis revealed decreased DUSP26 expression in kidney biopsy specimens from AKI patients compared to controls, as visualized by co-staining with the proximal tubule marker LTL (Fig. 1H). Quantitative analysis demonstrated significantly lower DUSP26 expression (IOD/area) in LTL-positive tubules from AKI patients (Fig. 1I). Notably, DUSP26 expression levels in LTL-positive tubules showed a strong inverse correlation with acute tubular necrosis (ATN) scores and a strong positive correlation with estimated glomerular filtration rate (eGFR) (Fig. 1J). These findings identify DUSP26 as a downregulated gene in human AKI and suggest its potential role in maintaining kidney tubular integrity.

2. DUSP26 is downregulated in cisplatin- and ischemia-induced acute kidney injury in mice.
To explore whether DUSP26 downregulation observed in human AKI also occurs in experimental models, we examined its expression in mouse kidneys following cisplatin-induced nephrotoxic injury. Immunoblotting revealed a marked reduction in DUSP26 protein at 48 hours post-injection, which remained significantly suppressed at 72 hours (Fig. 2A, B). Given the reports about transcriptional repression of DUSP26 in cancer 20, we next assessed Dusp26 mRNA that showed a significant decrease in cisplatin-treated mice kidneys (Fig. 2C). Immunofluorescence analysis further confirmed DUSP26 decrease in cisplatin AKI across both renal cortex and outer medulla (Fig. 2D), consistent with tubular injury localization. Supporting these in vivo findings, cisplatin also induced a marked decrease in DUSP26 expression in BUMPT cells (a mouse kidney proximal tubule cell line), with significant suppression at 12 hours and further reduction by 36 hours (Supplementary figure 2A, B).
To determine whether DUSP26 downregulation is a generalizable feature of AKI, we assessed DUSP26 expression in a bilateral renal ischemia-reperfusion injury (IRI) model. Mice subjected to 30 minutes of bilateral ischemia followed by 48 or 72 hours of reperfusion exhibited significant reductions in renal DUSP26 protein (Fig. 2E, F), accompanied by decreased Dusp26 mRNA expression (Fig. 2G). Immunofluorescence further demonstrated diminished DUSP26 staining within tubular epithelial cells of the injured kidneys (Fig. 2H). We next extended these findings to an in vitro model of hypoxia/reoxygenation (H/R) injury. BUMPT cells cultured under hypoxic conditions for 8 hours followed by various durations of reoxygenation (2-10 hours) exhibited time-dependent increases in apoptosis, as indicated by cleavage of caspase-3 (c-Cas3; Supplementary figure 2C). Notably, DUSP26 protein levels declined progressively from 6 to 10 hours of reoxygenation (Supplementary figure 2D), and its expression showed a strong inverse correlation with c-Cas3 activation (Supplementary figure 2E). Together, these results demonstrate that DUSP26 is consistently downregulated across multiple models of AKI, both in vivo and in vitro, and its loss is closely associated with renal tubular cell injury and apoptosis.

3. Promoter methylation mediates DUSP26 downregulation in proximal tubular cells following injury.
Although limited information is currently available regarding the transcriptional regulation of DUSP26, existing studies suggest that its expression is primarily modulated at the mRNA level19. Recent studies have highlighted DNA methylation as a critical epigenetic mechanism underlying tubular cell dysfunction and gene repression during AKI26. Notably, DUSP26 downregulation has been linked to promoter DNA methylation in cancer cells, where treatment with 5-Aza-2'-deoxycytidine (5-Aza, a DNA methyltransferase inhibitor) can restore DUSP26 expression20. To explore whether similar epigenetic regulation occurs in kidney tubular injury, we analyzed the promoter region of the mouse Dusp26 gene using the MethPrimer database. This analysis revealed a prominent CpG island, suggesting potential susceptibility to DNA methylation-mediated transcriptional repression (Fig. 3A). To directly evaluate promoter methylation, we performed methylation-specific PCR (MSP) following bisulfite conversion of genomic DNA. Specific primer pairs were designed to distinguish methylated (M) and unmethylated (U) alleles within the CpG island of the Dusp26 promoter (Fig. 3B). MSP revealed increased promoter methylation in both cisplatin-treated and hypoxia/reoxygenation (H/R)-treated BUMPT cells compared with controls, while 5-Aza treatment markedly reduced the methylation signal in both injury models (Fig. 3C). We next examined the kinetics of Dusp26 re-expression following 5-Aza treatment. qPCR analysis showed that in cisplatin-injured cells, Dusp26 mRNA levels remained low at early time points and significantly increased only after 12–24 hours of 5-Aza exposure (Fig. 3D). Western blotting further validated that DUSP26 protein levels were restored after 5-Aza treatment (Fig.3E).
We next assessed the downstream functional consequences of this restoration. While 5-Aza treatment successfully restored DUSP26 expression (Fig. 3E) and subsequently reduced the phosphorylation of p53 at Ser312 (p-p53(S312)), it paradoxically failed to suppress, and in fact significantly increased, levels of cleaved caspase-3 (c-Cas3) (Supplementary figure 8). This suggests that the global, non-specific demethylating activity of 5-Aza likely activates other pro-apoptotic pathways, masking the specific benefit of DUSP26 restoration. To further investigate the mechanism driving this hypermethylation, we assessed promoter occupancy of both the maintenance methyltransferase DNMT1 and the de novo methyltransferases DNMT3A and DNMT3B. ChIP–qPCR analysis revealed significantly increased enrichment of DNMT1 and DNMT3A at the Dusp26 promoter in cisplatin-treated cells compared with controls, whereas DNMT3B enrichment was not significantly altered (Fig. 3F). Consistent with this finding, total protein levels of the DNMTs themselves were upregulated. Western blot analysis confirmed a time-dependent increase in the expression of DNMT1, DNMT3A, and DNMT3B in cisplatin-treated BUMPT cells (Supplementary figure 6A, 6B). To confirm whether this epigenetic silencing also occurs in vivo, we analyzed kidney tissue from a mouse model of IRI-induced AKI. Quantitative, site-specific analysis using targeted bisulfite sequencing revealed a widespread increase in methylation levels across the Dusp26 promoter in IRI-AKI mice compared to controls (Fig. 3G, 3H). Notably, quantification of individual CpG sites confirmed significantly higher methylation at multiple specific loci in the AKI group (Fig. 3I). Collectively, these data demonstrate that DUSP26 is silenced in vitro through promoter hypermethylation accompanied by recruitment of the de novo methyltransferase DNMT3A together with DNMT1, and this epigenetic repression is recapitulated in an in vivo mouse model of AKI.

4. DUSP26 functions as a protective factor against acute injury in proximal tubular epithelial cells.
To assess the functional role of DUSP26 in acute tubular injury, we initially modulated its expression in BUMPT cells and examined apoptotic markers following cisplatin and H/R challenges. Knockdown of Dusp26 with specific siRNAs significantly increased cleaved caspase-3 (c-Cas3) levels after cisplatin treatment, as demonstrated by immunoblotting and corresponding quantification (Fig. 4A, B). Consistently, Dusp26-knockdown cells exhibited markedly higher c-Cas3 expression following 8 hours of H/R than control cells (Fig. 4C, D). Moreover, NSC87877 (a pharmacologic inhibitor of DUSP26) potentiated cisplatin-induced apoptosis as shown by TUNEL staining (Fig. 4E, F).
In contrast, stable overexpression of DUSP26 in BUMPT cells via lentiviral transduction conferred significant protection against cisplatin-induced injury. Immunoblot analysis revealed lower c-Cas3 levels in DUSP26-overexpressing cells after 24 hours of cisplatin treatment than in cells transduced with control lentivirus (Fig. 4G, H), which was corroborated by fewer TUNEL-positive cells (Fig. 4I, J). DUSP26 overexpression similarly attenuated caspase 3 cleavage/activation following H/R injury (Fig. 4K, L).
Domain mapping of full-length DUSP26 (211 amino acids) confirmed the presence of a 131-amino acid dual-specificity phosphatase (DSP) catalytic domain, which contains the conserved DSP motif “VHCAVGVSRS” and the MKP motif “AYLM” (Fig. 4M). We generated a phosphatase-inactive mutant by substituting the catalytic cysteine at position 152 with serine (C152S) (Supplementary figure 3A). Importantly, this point mutation attenuated the protective effect of DUSP26, indicating that the phosphatase activity of DUSP26 is critical for its cytoprotective function (Fig. 4N, O). Collectively, these results highlight a protective role of DUSP26 in kidney proximal tubular cells via its phosphatase activity.

5. Pharmacologic inhibition of DUSP26 sensitizes the kidney to AKI.
NSC87877 has been identified as a small-molecule inhibitor of DUSP2627. Given the cytoprotective role of DUSP26 in BUMPT cells in vitro, we next investigated whether pharmacologic inhibition of DUSP26 would exacerbate kidney injury in vivo. Mice were pretreated with the DUSP26 inhibitor NSC87877 (NSC) prior to cisplatin administration. At 48 hours of cisplatin treatment, NSC-pretreated mice exhibited significantly higher levels of serum creatinine and blood urea nitrogen (BUN) compared to vehicle-treated controls, indicating worsened AKI (Fig. 5A). Renal histology also revealed more severe tubular damage in NSC-treated mice, characterized by tubular dilation, brush border loss, epithelial cell sloughing, and cast formation (Fig. 5B), which was confirmed by a significantly higher tubular injury score (Fig. 5C). Consistent with these findings, immunofluorescence analysis demonstrated increased expression of kidney injury molecule-1 (KIM-1), a proximal tubule injury marker, in the kidneys of NSC-treated mice (Fig. 5D). TUNEL staining further revealed an increased number of apoptotic tubular cells in these tissues (Fig. 5E). Quantitative analysis confirmed a significantly higher number of TUNEL-positive cells in the NSC-pretreated group (Supplementary figure 4), suggesting that pharmacologic suppression of DUSP26 amplifies tubular cell death during toxic injury.
To determine whether these effects extend to ischemic injury, we subjected mice to bilateral renal ischemia-reperfusion injury (IRI), with or without NSC pretreatment. NSC administration significantly aggravated renal dysfunction, as evidenced by higher serum creatinine and BUN levels compared to vehicle-treated IRI mice (Fig. 5F). Histopathological analysis confirmed exacerbated tubular damage in the NSC-treated group (Fig. 5G), with increased injury scores (Fig. 5H). Furthermore, Western blot analysis of whole kidney lysates confirmed that NSC-pretreated mice had significantly higher protein levels of both KIM-1 and the apoptotic marker cleaved-Caspase 3 (c-Cas3) (Fig. 5I). Consistent with this finding, these mice also showed an increased number of TUNEL-positive apoptotic cells (Fig. 5J), which was confirmed by quantitative analysis (Supplementary figure 4). Together, these results indicate that DUSP26 inhibition sensitizes the kidney to both toxic and ischemic injury, supporting a protective role of DUSP26 in maintaining tubular epithelial integrity in AKI.

6. Proximal tubule-specific knock-in of DUSP26 attenuates AKI.
To validate the protective role of DUSP26 in vivo using a genetic approach, we generated proximal tubule-specific DUSP26 knock-in mice by crossing conditional Rosa26loxP-STOP-loxP-Dusp26 mice with Pepck-Cre mice, yielding Rosa26loxP-STOP-loxP-Dusp26; Pepck-Cre animals (referred to as PT-Dusp26-KI), while Cre-negative littermates served as controls (PT-Dusp26-WT) (Fig. 6A, B). We validated the knock-in efficacy. Immunofluorescence staining confirmed strong DUSP26 expression co-localizing with the proximal tubule marker LTL in PT-Dusp26-KI mice, which was absent in PT-Dusp26-WT controls (Fig. 6C). This was further confirmed by Western blot, which showed markedly elevated DUSP26 protein levels in the kidneys of PT-Dusp26-KI mice (Fig. 6D). Baseline renal function did not differ significantly between these two groups. However, following cisplatin treatment, PT-Dusp26-KI mice displayed markedly lower serum creatinine and BUN levels at 24 hours, indicating better renal function (Fig. 6E). Histological analysis revealed significantly less tubular damage in PT-Dusp26-KI mice compared with WT, as evidenced by H&E staining and quantification of injury scores (Fig. 6F, G). Immunoblotting further demonstrated less expression of cleaved caspase-3 and neutrophil gelatinase-associated lipocalin (NGAL), two markers of apoptosis and kidney injury, respectively, in PT-Dusp26-KI mice (Fig. 6H). Consistently, the knock-in group had less TUNEL staining (Fig. 6I). To determine whether this protective effect extends to ischemic injury, PT-Dusp26-KI and PT-Dusp26-WT mice were subjected to bilateral renal ischemia-reperfusion. Similar to the cisplatin model, PT-Dusp26-KI mice exhibited significantly better renal function and histology, as reflected by lower serum creatinine/BUN levels and less tubular injury (Fig. 6J-L). These findings confirm that proximal tubule-specific overexpression of DUSP26 attenuates kidney injury in both toxic and ischemic models of AKI, reinforcing its role as a tubular protective factor.

7. DUSP26 suppresses pro-apoptotic gene expression via interaction with p53.
To investigate the mechanisms by which DUSP26 protects kidney tubular epithelial cells, we analyzed transcriptomic changes associated with DUSP26 expression in human ischemic AKI from the GEO database (GSE21374, GSE30718, and GSE43974). AKI patients were stratified into high- and low-DUSP26 expression groups, revealing differential expression of p53 and related pro-apoptotic genes, with higher BAX, BAK1, CASP3, and APAF1 expression in the DUSP26-low group (Fig. 7A). Co-expression analysis confirmed a strong negative correlation between DUSP26 and these p53-regulated apoptotic genes across datasets (Fig. 7B). To experimentally validate these associations in vivo, we measured pro-apoptotic gene expression in mice treated with the DUSP26 inhibitor NSC87877. In both cisplatin-induced AKI and ischemia-reperfusion injury (IRI) models, NSC significantly increased the expression of Bax, Puma, and Noxa mRNA in kidney tissues (Fig. 7C, D). Given that p53 is a key transcriptional regulator of these pro-apoptotic genes, we examined whether DUSP26 modulates p53 directly. DUSP26 knockdown increased total p53 protein levels in cisplatin-treated BUMPT cells, whereas DUSP26 overexpression suppressed p53 accumulation (Fig. 7E). Co-immunoprecipitation (Co-IP) assays further detected the interaction of DUSP26 with p53 following cisplatin exposure (Fig. 7F), suggesting a direct regulatory relationship. To explore whether this interaction involves post-translational modifications, we performed phosphoproteomic profiling in BUMPT cells with DUSP26 overexpression or knockdown. Among differentially regulated phospho-sites, p53 phosphorylation at serine 312 emerged as the top-ranked site affected by DUSP26 expression, based on Andromeda scoring (Fig. 7G). This residue, conserved across species (corresponding to Ser315 in human p53), may be involved in regulating p53 transcriptional activity and protein stability28-30. Together, these findings demonstrate that DUSP26 negatively regulates pro-apoptotic gene expression by interacting with and modulating the phosphorylation status of p53, particularly at Ser312. These findings support a DUSP26–p53 axis that contributes to tubular cell survival during AKI.

8. DUSP26 regulates tubular cell apoptosis in AKI by dephosphorylating p53 at serine 312.
We then determined whether DUSP26 directly regulates p53 phosphorylation at serine 312. As shown in Fig. 8A, DUSP26 knockdown significantly enhanced, while DUSP26 overexpression suppressed, p53 phosphorylation at Ser312 (p-p53S312) following cisplatin treatment. Similar findings were observed in vivo: PT-Dusp26-KI mice had less p-p53S312 in kidney tissues than PT-Dusp26-WT littermates after cisplatin treatment (Fig. 8B). To confirm the specificity of this in vivo regulation, we comprehensively examined other key N-terminal phosphorylation sites. Crucially, in the same kidney samples, DUSP26 overexpression had no significant effect on the phosphorylation of p53 at Ser20 or Ser46. While a modest decrease was seen at Ser15, the profound inhibition of Ser312 contrasted with the lack of effect at other major N-terminal sites, demonstrating that the regulatory effect of DUSP26 is highly specific to the Ser312 site (Supplementary figure 7). To functionally validate the role of Ser312 phosphorylation, we reconstituted p53-knockout (p53-KO) BUMPT cells with either wild-type (WT) p53 or a Ser312-to-alanine (S312A) mutant. The constructs were confirmed by Sanger sequencing (Supplementary figure 3B). Upon cisplatin treatment, S312A-transfected cells exhibited markedly less cell death and cleaved caspase-3 than WT-p53 cells (Fig. 8C-E). Moreover, qPCR analysis showed that transcription of Bax and Puma, canonical p53 target genes, was significantly attenuated in S312A-expressing cells (Fig. 8F). We further evaluated this mechanism under H/R conditions. DUSP26 knockdown in BUMPT cells enhanced p-p53S312 after 8 hours of H/R, whereas DUSP26 overexpression suppressed this phosphorylation (Fig. 8G, H). Consistently, Bax and Puma expression during H/R was lower in S312A p53 cells than in WT-p53 cells (Fig. 8I). We also performed chromatin immunoprecipitation (ChIP) to assess p53’s binding to the promoters of its target genes (Fig. 8J). ChIP-qPCR analysis revealed that while wild-type p53 robustly bound to the p53 response elements in both the Bax and Puma promoters, the binding of the p53-S312A mutant was significantly impaired (Fig. 8K). This indicates that Ser312 phosphorylation is critical for p53’s ability to occupy the promoters of its pro-apoptotic targets.

9. Phosphorylation of p53 at Ser312 is required for its pro-apoptotic function and AKI in vivo.
We further verified the role of p53 Ser312 phosphorylation in vivo using a genetic rescue model. To this end, we re-expressed either wild-type p53 (p53-WT), its non-phosphorylatable p53-S312A mutant, or an empty vector (EV) in p53-knockout (KO) mice via a kidney proximal tubule-specific Ksp-cadherin promoter-driven adeno-associated virus (AAV) system (Fig. 9A, B). Immunofluorescence analysis confirmed efficient and specific AAV transduction in renal tubular epithelial cells (Fig. 9C). We validated the model’s response to cisplatin injury. As expected, a robust p-p53(S312) signal was detected by Western blot in kidneys re-expressing WT p53. In stark contrast, this signal was absent in mice with S312A p53 mutant (Fig. 9D), confirming the non-phosphorylatable nature of the mutant. Importantly, re-expression of WT p53 in p53-KO mice restored their susceptibility to cisplatin, leading to severe renal dysfunction (significantly elevated BUN and sCr), whereas the mice expressing the S312A mutant were strikingly protected (Fig. 9E). Histological analysis with H&E staining (Fig. 9F, G) and TUNEL staining (Fig. 9H, I) mirrored these functional data: the WT p53 rescue group exhibited extensive tubular injury and widespread apoptosis, whereas the S312A p53 group showed markedly reduced tubular damage and significantly fewer apoptotic cells. At the molecular level, the expression of kidney injury (NGAL) and apoptotic (cleaved caspase-3) markers were significantly increased in the WT p53 kidney, but this induction was largely blunted in the S312A group (Fig. 9J, K). Furthermore, the p53 target genes Bax and Puma were dramatically upregulated in the WT p53 kidneys, while their transcription was significantly attenuated in mice kidneys expressing the S312A p53 mutant (Fig. 9L). Taken together, these data provide direct evidence that the phosphorylation of p53 at Ser312 is required for its full pro-apoptotic transcriptional activity and the subsequent development of acute kidney injury.

Methods
Ethics statement
All human studies complied with relevant ethical regulations and were approved by the Institutional Ethics Committee of the Second Xiangya Hospital, Central South University (Approval/Protocol No. 2020TYFC2005004). Written informed consent was obtained for the use and publication of de-identified clinical information; for individuals under 18 years of age, consent was obtained from a parent/legal guardian in accordance with institutional guidelines. No identifiable individual-level information is included. No compensation was provided to participants.
All animal procedures complied with relevant ethical regulations and the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Second Xiangya Hospital, Central South University (Approval No. 2022418). All mice were housed under specific-pathogen-free conditions in a temperature-controlled room (21–26 °C) with a 12 h dark/12 h light cycle and 40–60% humidity. Prior to tissue and blood collection, mice were euthanized by placement in a 5% isoflurane-saturated plexiglass chamber for 5 min, followed by cervical dislocation after confirming deep anesthesia by the absence of an active paw-withdrawal reflex.

Sex as a biological variable
Human renal tissue specimens were obtained from male and female patients, and sex was abstracted from the clinical record. Sex-stratified analyses were not performed due to limited sample size and the use of archival specimens, which precluded adequately powered assessment of sex-specific effects. For all mouse experiments, only male mice were used to minimize potential confounding effects of estrogen on acute kidney injury susceptibility.

Human kidney specimens and clinicopathological assessment
Human renal tissue specimens were obtained from patients diagnosed with acute tubular necrosis (ATN) by clinically indicated renal biopsy (ATN group, n = 8; male and female), together with de-identified clinicopathological information (Supplementary Table 1). Control renal tissue specimens (n = 7; male and female) were derived from the unaffected poles of nephrectomy samples from patients undergoing surgery for localized renal tumors. All analyses were performed on archival formalin-fixed, paraffin-embedded sections; no additional tissue was collected for research purposes beyond standard-of-care procedures, and no participants were prospectively recruited for this study. Because inclusion was based on clinical indication and tissue availability, selection/indication bias is possible (e.g., biopsy-confirmed ATN may not represent all AKI phenotypes). In addition, nephrectomy-adjacent “control” tissue may not fully represent healthy kidney, which may limit generalizability.
To assess DUSP26 expression in proximal tubules, paraffin sections were subjected to immunofluorescence co-staining for DUSP26 and Lotus tetragonolobus lectin (LTL; a proximal tubule marker) as described below. Quantification was performed using Image-Pro Plus 6.0 by measuring DUSP26 integrated optical density (IOD) and normalizing to area (IOD/area, arbitrary units). Briefly, LTL-positive regions were identified on the LTL channel using a consistent thresholding strategy across all samples to generate an LTL (+) mask. This mask was applied to the DUSP26 channel to quantify DUSP26 IOD/area within proximal tubules (LTL (+)). LTL-negative regions (LTL (−)) were defined as tissue areas outside the LTL (+) mask within the same field and quantified in an identical manner. For each specimen, values from five non-overlapping cortical fields were averaged to yield a single biological value per individual. Comparisons between Control and ATN groups were performed separately for LTL (−) and LTL (+) compartments using two-sided unpaired Student’s t-tests. Correlations between LTL (+) tubular DUSP26 IOD/area and clinical/pathological indices (ASTN score and eGFR) were assessed using two-sided Pearson correlation, with corresponding P values reported.

Mouse strains and sources
Male C57BL/6J mice were purchased from SJA Laboratory Animal Corporation (Hunan, China). Proximal tubule–specific DUSP26 knock-in mice (PT-Du26-KI; male) were generated by crossing conditional Rosa26-LSL-DUSP26 knock-in mice (Shanghai Model Organisms Center, Inc.) with Pepck-Cre transgenic mice kindly provided by Dr. Volker Haase (Vanderbilt University School of Medicine, Nashville, TN, USA). Male p53-knockout (p53-KO) mice on a C57BL/6J background (generated by Cyagen Bioscience) were used for in vivo p53 rescue experiments.

Generation of proximal tubule-specific DUSP26 knock-in mice
Following previously established methods52, we generated proximal tubular epithelial cell-specific DUSP26 knock-in (PT-Du26-KI) mice. Briefly, conditional DUSP26 knock-in mice, in which a loxP-STOP-loxP (LSL) cassette followed by the DUSP26 cDNA was inserted into the Rosa26 locus (designated as Dusp26KI/KI), were obtained from Shanghai Model Organisms Center, Inc. These mice were crossed with Pepck-Cre transgenic mice, which express Cre recombinase under the control of the Pepck promoter, leading to Cre activity specifically in proximal tubular epithelial cells. Since the Pepck-Cre transgene is X-linked, male offspring with the genotype Dusp26KI/KI; XCreY were designated as PT-Du26-KI mice. Their Cre-negative male littermates, Dusp26KI/KI; XY, served as wild-type controls (PT-Du26-WT). The breeding strategy is illustrated in Supplementary figure 1.

AAV-mediated in vivo p53 Rescue Model
p53-knockout (p53-KO) mice (on a C57BL/6J background) were generated by deleting exon 3 through exon 9 using CRISPR/Cas9 technology, and were obtained from Cyagen Bioscience. Adeno-associated virus (AAV) vectors were custom-packaged by Genechem (Shanghai, China). The AAV vector backbone used was GV908, which employs the kidney tubular epithelial cell-specific Ksp-cadherin promoter (1.3k) to drive transgene expression. The following constructs were generated: AAV-p53-WT: Carrying the full-length murine Trp53 cDNA; AAV-p53-S312A: Carrying the Trp53 (NM_011640.4 ) cDNA with a site-directed S312A mutation. The empty vector control (CON753). All vectors co-expressed EGFP via an FT2A element. Vectors were packaged as AAV9 serotype.
Male p53-KO mice were randomly allocated into three groups. Male mice were exclusively used in this study to minimize the potential confounding effects of estrogen, which is known to confer protection against AKI. For injection, the required volume of each virus was calculated to achieve the target dose of 8×1011 vg/mouse. The calculated viral volume was then diluted with sterile PBS to a final injection volume of 100 µL (within the 80-120 µL range). Prior to injection, both the viral aliquots and the mice were pre-warmed. Mice received a single, slow tail vein injection of the corresponding AAV-p53-WT, AAV-p53-S312A, or AAV-EV. Twenty-one days post-injection, successful and specific AAV transduction in renal tubular epithelial cells was confirmed by EGFP fluorescence (Fig. 9C). To induce AKI, mice were administered a single intraperitoneal (i.p.) injection of cisplatin (30 mg/kg). Mice were euthanized 48 hours after cisplatin administration, and blood and kidney tissues were harvested for subsequent analysis.

Acute kidney injury (AKI) models
For bilateral renal ischemia–reperfusion injury (bIRI), 8–10 weeks old (22–25 g) mice were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneal), placed on a temperature-controlled surgical platform, and core temperature was maintained at 37 °C throughout the procedure. Following a midline laparotomy, both renal pedicles were exposed and occluded with microvascular clamps for 30 min. Reperfusion was initiated by clamp removal and confirmed visually by restoration of kidney color. Blood and kidneys were harvested 48 h after reperfusion unless otherwise specified. For cisplatin-induced AKI, mice received a single intraperitoneal injection of cisplatin (30 mg/kg) and were euthanized 48 h later; control mice received an equivalent volume of saline. For pharmacological intervention, the DUSP26 inhibitor NSC87877 was dissolved in PEG300/Tween-80/saline to a working concentration of 10 mg/mL. Mice received NSC87877 (50 mg/kg) or vehicle by intraperitoneal injection 1 h prior to bIRI surgery or cisplatin administration and then once daily thereafter. Serum creatinine and blood urea nitrogen (BUN) were measured using commercial kits (Roche Diagnostics, Germany) according to manufacturer instructions. Blood was collected via retro-orbital bleeding and centrifuged at 3,000 × g for 10 min to obtain serum.

Hepatic ischemia–reperfusion injury (HIRI) model
HIRI was performed in anesthetized mice using intraperitoneal injection of pentobarbital sodium (60 mg/kg). Body temperature was maintained using a heating pad, and monitored via a rectal probe throughout the procedure. After a midline laparotomy and gentle exteriorization of the intestine, the portal triad branch supplying the left lateral liver lobe was occluded for 1.5 hours using a microvascular clamp to induce regional hepatic ischemia, which was visually confirmed by blanching of the liver. The exposed abdominal organs were kept moist using sterile saline-soaked gauze. Reperfusion was initiated by clamp removal. Mice were euthanized 24 hours after reperfusion under deep anesthesia. Blood was collected for plasma ALT and AST assays, and liver tissues were harvested, rinsed in saline, and divided: one portion was snap-frozen and stored at -80°C for molecular analyses, while the other was fixed and paraffin-embedded for histological studies. Histological liver injury was assessed using the Suzuki criteria, based on scoring sinusoidal congestion, hepatocyte ballooning, and hepatocellular necrosis.

Cell culture and in vitro injury models
Mouse proximal tubular epithelial cells (BUMPT; originally established by Dr. Lieberthal, Boston University) were maintained at 37 °C in a humidified incubator with 5% CO₂ and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in standard tissue culture dishes. For cisplatin injury, BUMPT cells were treated with cisplatin (20 μM; Sigma-Aldrich) for 12, 24, or 36 h. For hypoxia/reoxygenation (H/R), cells were cultured in serum- and glucose-free DMEM under 1% O₂ for 8 h in a hypoxia chamber, followed by reoxygenation in complete DMEM under normoxia for 2, 4, 6, 8, or 10 h.

siRNA knockdown and lentiviral overexpression
For transient silencing, BUMPT cells were transfected with Dusp26-targeting siRNAs (RiboBio, Guangzhou, China): si-m-Dusp26-1 (GTGGTCATAGTCTCACTGA) and si-m-Dusp26-2 (GGAAGCAGCTGAGCTGTAA), using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. A scrambled siRNA served as the negative control. For transfection, cells were seeded to reach ~70–80% confluence at the time of transfection, and siRNAs were used at a final concentration of 50 nM. Cells were incubated for 24 h prior to downstream treatments or analyses. For stable overexpression, BUMPT cells were transduced with lentivirus encoding mouse DUSP26 (pLenti-EF1-mCherry-P2A-Puro-CMV-DUSP26-WPRE; MOI = 40) or control lentivirus (pLenti-EF1-mCherry-P2A-Puro-CMV-MCS-WPRE). Media were replaced 12 h post-infection, and mCherry fluorescence was assessed at 72 h to confirm transduction efficiency. For selection of transduced cells, puromycin was applied at 2.0 µg/mL for 48–72 h as the primary selection condition.

DUSP26 catalytic mutant and p53 rescue constructs in vitro
A catalytically inactive DUSP26 mutant (C152S) was generated by substituting cysteine 152 with serine. Plasmids encoding wild-type DUSP26 (pcDNA3.1(+)-DUSP26-3×FLAG; OBiO plasmid H37372) and DUSP26(C152S) (pcDNA3.1(+)-DUSP26(C152S)-3×FLAG; OBiO plasmid H37373), as well as the corresponding empty vector control, were constructed in the pcDNA3.1(+)-MCS-3×FLAG backbone (H6919). A CRISPR/Cas9-based p53-knockout (p53-KO) BUMPT cell line was generated using guide RNAs targeting mouse Trp53 (GenBank NM_011640.4). sgRNA protospacer sequences (5′–3′) was PCA16734, ATAAGCCTGAAAATGTCTCC. The sgRNA/Cas9 expression vector used was GV465 (hU6-sgRNA–CBA promoter–3×FLAG-SpCas9–T2A–puro; GeneChem, Shanghai, China). Single-cell clones were expanded, and p53 depletion was confirmed by western blot.
For rescue experiments, p53-KO cells were transfected with plasmids encoding wild-type p53 (pcDNA3.1(+)-Trp53-3×FLAG, LOBiO, H38331; GenBank NM_011640.3), p53-S312A (pcDNA3.1(+)-Trp53(S312A)-3×FLAG, OBiO, H38332), or the matched empty vector (pcDNA3.1(+)-MCS-3×FLAG, OBiO, H6919) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The S312A substitution was introduced by site-directed mutagenesis, and the insert sequence was verified by Sanger sequencing performed by OBiO (sequencing primers: CMV-F, 5′-CGCAAATGGGCGGTAGGCGTG-3′; pcDNA-SEQR, 5′-TTATTAGGAAAAGACAGTGGG-3′).

Tissue processing and hematoxylin and eosin (H&E) staining
Kidney tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned at 4 µm. H&E staining was performed using standard procedures. Tubular injury was scored semi-quantitatively based on tubular dilation, brush border loss, tubular epithelial cell swelling/necrosis, and intraluminal debris or cast formation using the following scale: 0 (no injury), 1 (<25% of tubules injured), 2 (25–50%), 3 (50–75%), and 4 (>75%). Ten randomly selected cortical fields per animal were scored and averaged to yield a single value per animal. All scoring was performed by a pathologist blinded to the experimental groups.

Immunofluorescence staining
Kidney sections were dewaxed, rehydrated, and subjected to antigen retrieval by immersion in citrate buffer (pH 6.0) followed by microwave heating using a stepped program (8 min at medium power, 8 min standby, and 7 min at medium–low power), then cooled to room temperature. Samples were blocked for 1 h at room temperature in blocking buffer 5% BSA in PBS with 0.1% Triton X-100, and incubated overnight at 4 °C with primary antibodies against DUSP26 (1:100; Invitrogen, PA5-22013), KIM-1 (1:200; R&D Systems, AF1817), and Lotus tetragonolobus lectin (LTL; 1:100; Vector Laboratories, RFL-1321). After washing, samples were incubated for 1 h at room temperature with species-appropriate secondary antibodies (Abcam, 1:400). Nuclei were counterstained with Hoechst 33342 (Beyotime, C1029) according to the manufacturer’s instructions. Images were acquired on a Zeiss Axio Imager Z2 fluorescence microscope using ZEN (version 3.7; Carl Zeiss, Germany) acquisition software, with identical settings maintained across experimental groups within each experiment. Whole-slide images were acquired using CaseViewer (v2.4.0; 3DHISTECH, Hungary). Quantitative analysis, including fluorescence intensity and positive area percentage, was performed using Image-Pro Plus (v6.0; Media Cybernetics, USA). Specifically, the integrated optical density (IOD) was measured in five randomly selected high-power fields per section to ensure unbiased quantification.

TUNEL assay
Apoptotic cells were detected using a TUNEL assay kit (Roche, Germany) according to the manufacturer’s instructions. Sections were deparaffinized, rehydrated, permeabilized, and incubated with terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP. Nuclei were counterstained with Hoechst 33342 (Beyotime, C1029). TUNEL-positive cells were quantified in 10 randomly selected fields per section.

Immunoblotting
Total protein was extracted from tissue or cultured cells using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (sigma, P8340). Protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4°C with primary antibodies (all diluted at 1:1000) against DUSP26 (Invitrogen, PA5-22013), p53 (Cell Signaling Technology, 2524S), p-p53 (S15) (Cell Signaling Technology, 9284S), p-p53 (S20) (Cell Signaling Technology, 9287T), p-p53 (S46) (Proteintech, 28960), cleaved caspase-3 (Cell Signaling Technology, 9664S), NGAL (R&D Systems, AF1857), KIM-1 (R&D Systems, AF1817) , GAPDH (Proteintech, 10494), β-actin (Proteintech, 66009), DNMT1 (Cell Signaling Technology, 5032), DNMT3a (abcam, ab188470), DNMT3b (abcam, ab2851) and FLAG (Sigma-Aldrich, 102698217), as appropriate. After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:5000; Proteintech). Chemiluminescent western blot signals were captured using Tanon Image Analysis Software (v4.2; Tanon, China). Quantification was performed using ImageJ (v1.54g; NIH, USA). Uncropped and unprocessed scans of blots/gels are provided in the Source Data file.

Quantitative real-time PCR (qPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Japan). qPCR was performed using SYBR Green Master Mix (Roche, Germany) on a LightCycler 96 Application Software (v1.1; Roche, Germany) and QuantStudio Real-Time PCR Software (v1.3; Applied Biosystems, USA). Gene expression was normalized to ACTIN, and relative expression levels were calculated using the 2−ΔΔCt method. Primer sequences are listed in Supplementary Table 2.

Chromatin immunoprecipitation (ChIP) and ChIP–qPCR
ChIP was performed using the High-Sensitivity ChIP Kit (ab185913; Abcam) according to the manufacturer’s protocol with the following conditions. BUMPT cells (1 × 106 per reaction) were cross-linked with 1% formaldehyde for 10 min at room temperature and quenched with 1.25 M glycine. Chromatin was sonicated (15 s on/30 s off, 20 cycles, 170–190 W) to yield DNA fragments of 100–700 bp (peak ~300 bp), verified by agarose gel electrophoresis. For each reaction, 2 μg chromatin was incubated in antibody-coated strip wells (100 μL total volume) containing antibody buffer, blocker, and enrichment enhancer. Antibodies were added on a per-well mass basis following the kit guidance (final amount 0.8 µg antibody per well for antibodies of interest; 0.8 µg per well for RNA polymerase II as the positive control; and 0.8 µg per well for non-immune IgG as the negative control).
For p53 ChIP, antibodies against p53 ( Cell Signaling Technology, 2524) were used; normal mouse IgG served as negative control, and RNA polymerase II antibody provided in the kit served as positive control. After immunoprecipitation, wells were washed and DNA was released. Crosslinks were reversed with RNase A (10 mg/mL, 42 °C, 30 min) and Proteinase K (10 mg/mL, 60 °C, 45 min), followed by heating at 95 °C for 15 min. DNA was purified and eluted in 20 μL. ChIP DNA was quantified by qPCR using SYBR Green Master Mix (Applied Biosystems) on a QuantStudio 6 Flex system (Thermo Fisher Scientific). Primers targeting p53 response element–containing regions in Bax and Puma promoters are listed in Supplementary Table 2.
To assess DNMT occupancy at the Dusp26 promoter, BUMPT cells were treated with cisplatin (20 μM) for 24 h and subjected to ChIP–qPCR using anti-DNMT1 (CST, 5032), anti-DNMT3A (CST, 9768), anti-DNMT3B (CST, 44145), or species-matched IgG. The same primer set spanning the CpG-rich region of the Dusp26 promoter was used across DNMT ChIP assays (Supplementary Table 2). Fold enrichment over IgG was calculated as 2(Ct(IgG) − Ct(IP)).

Methylation-specific PCR (MSP)
MSP primers for methylated (M) and unmethylated (U) alleles were designed using MethPrimer. Genomic DNA was isolated from cells after the indicated treatments and subjected to bisulfite conversion using the DNA Bisulfite Conversion Kit (DP215; TIANGEN, Beijing, China) according to the manufacturer’s instructions, which converts unmethylated cytosines to uracils while preserving methylated cytosines. Bisulfite-converted DNA was eluted in 20 μL elution buffer. MSP was performed using the MSP Kit (EM101; TIANGEN) in 20 μL reactions containing bisulfite-converted DNA (<500 ng), 1 μL each primer (10 μM), 1.6 μL dNTPs (2.5 mM), 1 U MSP DNA Polymerase, 2 μL 10× MSP PCR Buffer, and nuclease-free water to volume. Separate PCR reactions were set up for M-primers and U-primers using the same bisulfite-converted DNA template. Primer sequences were shown in Supplementary Table 2. PCR products (10 μL) were resolved by agarose gel electrophoresis and assessed for the presence of methylated or unmethylated amplicons.

Targeted bisulfite sequencing (TBS)
Genomic DNA was extracted from mouse kidneys collected after ischemia–reperfusion injury (Control, n = 3; AKI, n = 3). For each sample, 100 ng genomic DNA was bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions. Three bisulfite-PCR amplicons spanning the Dusp26 promoter (mm39; chr8: 31,579,529–31,579,791 (Dusp26_1, 263 bp, −25 bp to TSS), 31,579,670–31,579,930 (Dusp26_2, 261 bp, +116 bp), and 31,579,843–31,580,126 (Dusp26_3, 284 bp, +289 bp)) were amplified using ZymoTaq PreMix (Zymo Research) with the following primer pairs (Supplementary Table 2). Amplicons were purified and used for library construction with the VAHTS Universal Plus DNA Library Prep Kit (Vazyme), followed by paired-end sequencing on an Illumina NovaSeq 6000 using NovaSeq Control Software (Illumina; version 1.7.5). Raw reads were quality-filtered and adapter-trimmed using fastp v0.20.0, aligned to the mm39 reference genome, and CpG methylation calls were extracted using Bismark v0.22.3. CpG sites with ≥10× coverage were retained for downstream analysis. Differential methylation between groups was assessed using DMLtest (Wald test) implemented in the DSS (v2.43.2), and sites with p < 0.05 were considered significant.

Generation and purification of phospho-specific anti–p-p53 (Ser312) antibody
A phospho-specific antibody recognizing p53 phosphorylated at Ser312 was generated by Hangzhou HuaAn Biotechnology Co., Ltd. using the synthetic phosphopeptide TSA(pSer)PPQKKKPLDC. The peptide was conjugated to KLH via Sulfo-SMCC crosslinking and used to immunize three New Zealand White rabbits (RB8154, RB8155 and RB8156) with a primary injection followed by boosters on days 14, 21 and 35 (Freund’s complete adjuvant for priming and incomplete Freund’s adjuvant for boosters). Serum collected after the final boost was evaluated by indirect ELISA against phosphorylated and non-phosphorylated peptides. Phospho-specific IgG was purified using a dual-affinity strategy: enrichment on a phosphopeptide column followed by depletion on a non-phosphorylated peptide column. Purified IgG was dialyzed into PBS (pH 7.4), sterile-filtered (0.22 μm), and quantified by absorbance at 280 nm (DeNovix DS-11). The final concentration of the RB8154 preparation was 0.54 mg/mL.

Mass spectrometry (proteomics) and data processing
To identify DUSP26-regulated phosphorylation events under cisplatin stress, BUMPT cells with DUSP26 knockdown or overexpression (and matched controls) were treated with cisplatin (20 μM) for 24 h. For discovery analysis, cells from three biological replicates per group were pooled into one analytical sample, yielding four samples. Proteins were reduced with DTT and alkylated with iodoacetamide, digested with trypsin (Promega; enzyme-to-protein ratio 1:50) at 37 °C for 20 h, and peptides were desalted, lyophilized, and reconstituted in 0.1% formic acid. Peptides were analyzed on an Easy-nLC 1000 system coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific) operated in positive ion mode. MS1 scans were acquired over m/z 300–1800 at a resolution of 70,000 (at m/z 200) with an AGC target of 1×106 and a maximum injection time of 50 ms. The top 20 precursors were selected for HCD fragmentation with an isolation window of 2 m/z; MS2 spectra were acquired at a resolution of 17,500 with NCE 27 eV, dynamic exclusion of 30 s, and an underfill ratio of 0.1%. Raw files were processed using MaxQuant (v1.6.14) against the UniProt Mus musculus database (uniprot_Mus_musculus_87808_20240110). Carbamidomethylation (C) was set as a fixed modification; oxidation (M), phosphorylation (STY), GlyGly (K), acetylation (K), and protein N-terminal acetylation were set as variable modifications. Up to two missed cleavages were allowed. Peptide mass tolerance was 20 ppm and fragment mass tolerance was 0.1 Da. Protein- and peptide-level FDR were controlled at 1%. iBAQ was enabled. Phosphorylation sites with localization probability >0.75 were considered Class I sites.

Bioinformatic and machine learning analysis (GEO)
We analyzed three GEO datasets (GSE21374, GSE30718, and GSE43974) derived from patients with early-stage AKI after kidney transplantation without evidence of rejection. In this context, the donor kidney inevitably undergoes ischemia-reperfusion injury (IRI), making these datasets a valuable model for studying human IRI-induced AKI. Batch effects across datasets were corrected using the sva (v3.52.0) and limma (v3.60.0) packages in R (v4.4.0). Differentially expressed genes (DEGs) between AKI and control samples were identified with adjusted P values < 0.05 and |log2 fold change| > 1. To prioritize candidate genes, we applied two machine learning approaches: least absolute shrinkage and selection operator (LASSO) regression and support vector machine-recursive feature elimination (SVM-RFE). LASSO regression was performed using the glmnet (v4.1-8) package to identify variables with minimal binomial deviance. SVM-RFE was conducted using the e1071 (v1.7-14) and caret (v6.0-94) packages with 10-fold cross-validation. The diagnostic performance was assessed by receiver operating characteristic (ROC) curve analysis using the pROC (v1.18.5) package. Heatmaps and volcano plots were generated using the pheatmap (v1.0.12) and ggplot2 (v3.5.1) packages.

Statistical analysis
Statistical analyses were performed using GraphPad Prism v10. Data are presented as mean ± SEM unless otherwise specified. Two-group comparisons used unpaired two-tailed Student’s t-test. One-way ANOVA with Tukey’s post hoc test was used for single-factor designs with ≥3 groups. Two-way ANOVA with Sidak’s or Tukey’s post hoc test was used for two-factor designs as specified in figure legends. No outliers were excluded. Sample sizes were determined by experimental feasibility and prior experience in the field, and exact n values are indicated in the figure legends and Source Data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "While the importance of phosphorylation events in AKI has been extensively studied, little is known about the role of dephosphorylation. DUSP6 is an atypical dual-specificity phosphatase. Although DUSP6 has been implicated in multiple diseases, the role of DUSP26 in the kidney remains unclear.\nUsing integrative bioinformatic analyses of publicly available AKI datasets, the authors identified DUSP26 as a potential important gene in AKI pathogenesis. Subsequently, they found that DUSP26 was highly expressed in proximal tubular cells in normal kidneys, but its expression was significantly reduced due to hypermethylation of the Dusp26 promoter during AKI in both patients and mice.\nThrough pharmacological inhibition and proximal tubule-specific overexpression of DUSP26, the authors demonstrated that DUSP26 exerts a strong protective effect against AKI induced by cisplatin or I/R. Mechanistically, DUSP26 inactivated p53 by dephosphorylating it at S312, thereby suppressing apoptosis in renal tubular cells.\nThis study reveals a previously unrecognized and important role of DUSP26 in attenuating kidney injury, positioning it as a promising therapeutic target for AKI. The work is very well-designed and comprehensive, spanning from patient dataset analysis, phenotypic characterization of genetic mouse models, and mechanistic elucidation.\nI have a few minor comments:\n1. TUNEL positive tubular cells in Figure 5 may need to be quantified to assess apoptosis levels. Were there any alterations in cleaved caspase 3 in the kidneys following NSC treatment?\n2. The phenotypes of the PT-specific DUSP26 overexpressing mice are significant. It would be helpful to confirm DUSP26 expression levels by Western blotting and immunofluorescence.\n3. The findings on the role of promoter methylation in Dusp26 silencing in renal tubular cells are exciting (Figure 3). In Figure 3E, treatment with 5-Aza, a DNA methyltransferase inhibitor, enhanced DUSP26 protein expression in DUMPT cells in the presence of cisplatin. It would be interesting to see whether p53 phosphorylation at S312 and cleaved casp3 were decreased following 5-Aza treatment.\n4. The authors may consider moving the routine methods (e.g., cell culture and animal models of AKI) to Supplemental Materials.", "reviewer_2": "Does your article contain western blots?\nYes - For manuscripts that include western blots, authors are required to provide the full length uncropped original western blots used in their manuscript, as part of their original submission. Full length western blots should be uploaded as a single ‘Supplemental Material’ file, should be clearly cited in the main text, and will be published if your article is accepted for publication.\nIn this manuscript, the authors demonstrate that DUSP26 directly binds and dephosphorylates p53 at Ser312, thereby attenuating p53-mediated apoptosis and reducing tubular epithelial cell death. Through an integrated approach involving clinical kidney biopsies, murine models of AKI, epigenetic analysis, and mechanistic cell-based assays, this study suggests the DUSP26-p53 axis as a regulatory node in renal injury, and addresses a clinically relevant issue. However, several important concerns must be addressed before further consideration.\nMajor Concerns\n1. Figure 2B, statistical comparisons are not provided? It is unclear whether the differences reach statistical significance. Figures 2D and 2H, the DUSP26 staining in the control group are overexposed, which affects interpretation. Additionally, the Hoechst nuclear staining is poorly visible, making it difficult to assess cellular localization. The overall immunofluorescence quality should be improved.\n2. Figure 3, although the authors employed methylation-specific PCR (MSP) to show increased promoter methylation of Dusp26, this method is semi-quantitative and lacks resolution at the single-nucleotide level. You are suggested to perform bisulfite sequencing or MeDIP-qPCR to quantify methylation at specific CpG sites accurately. In addition, the expression levels of DNA methyltransferases (e.g., DNMT1, DNMT3a, DNMT3b) in AKI models should be examined to support the epigenetic regulation mechanism.\n3. Figure 4A, the Western blot for DUSP26 is unclear and difficult to interpret. A higher-quality blot is needed.\n4. Functional validation of the p53 S312A mutant remains insufficient. The authors transfected WT and S312A p53 constructs into p53-knockout BUMPT cells and examined downstream gene expression and apoptosis. However, the effect of p53 WT and S312A overexpression on cleaved caspase-3 (c-Cas3) in Figure 8E is mild, and it is unclear how statistical significance was determined. Please clarify the statistical method and number of replicates used. Important functional aspects, such as the stability, nuclear translocation, and coactivator interactions of the S312A mutant, are not addressed. You should determine whether the S312A mutation affects p53 binding to Bax or Puma promoters, for example through ChIP assays.\n5. Figure 5E, the quality and reliability of the TUNEL staining are questionable. Representative images do not clearly show nuclear fragmentation, and the staining lacks specificity.\n6. Figure 6D. In the PT-Dusp26-WT+Cisplatin group, renal injury appears relatively mild, raising concerns about the consistency of the model.\n7. Figure 7I. The expression pattern of β-actin closely resembles that of KIM-1, suggesting possible issues with loading control or blot normalization.\n8. Figure 8. The band intensity and pattern of total p53 and phospho-p53 (p-p53 S312) vary substantially between different panels, which affect data interpretation. Please ensure consistency and normalization across replicates.\nMinor Concerns\n1. Cisplatin exposure time is inconsistent throughout the study. In some experiments, the treatment duration is 24 hours, while 20 hours in others. Please explain the rationale for choosing different time points and ensure consistency across experiments or clarify if time-course variation is intentional.\n2. Some figure legends (e.g., Figures 4 and 6) lack sufficient details to interpret results.\n3. Please clarify your raw phosphoproteomic data will be publicly available.", "reviewer_3": "Thank you for inviting me to evaluate the work conducted by Fu et al., which presents a thorough investigation finding DUSP26 as a new phosphatase that safeguards proximal tubule cells from acute kidney injury by the dephosphorylation of p53 at serine 312. In this study, Fu et al. employed an integrative transcriptomic analysis, human biopsy staining, in vitro cell models, and various mouse injury models, demonstrating that DUSP26 expression is downregulated in AKI and that the loss of DUSP26 exacerbates tubular apoptosis, whereas DUSP26 overexpression confers protection against both CIS-AKI and IR-AKI. Mechanistically, they illustrate that DUSP26 interacts with p53 and specifically dephosphorylates Ser312, attenuating p53’s pro-apoptotic transcriptional activity. The p53 S312A mutant notably replicates the protective effect of DUSP26 overexpression, while NSC87877 exacerbates harm in a liver ischemia model, indicating a more extensive organ-protective function.\nThe study examines a critical issue: how phosphatases mitigate stress-induced p53 in acute kidney injury, within a domain where p53 is extensively recognized as a principal facilitator of tubular cell apoptosis. The study is methodologically rigorous and comprehensive, encompassing human data, genetic models, CRISPR cell lines, biochemical assays (using a proprietary phospho-p53S312 antibody and phosphoproteomics), and two separate AKI models, hence providing robust support for the conclusions. The discovery that DUSP26 influences a non-canonical p53 site (Ser312) is unprecedented and may have significant ramifications for renal disease and cancer biology. Furthermore, the translational aspect of human tissue and liver injury is a strength, indicating that this pathway may be amenable to therapeutic targeting.\nThe work possesses significant scientific worth and importance to nephrology and cell death research. Nevertheless, essential clarifications and supplementary evidence are required to comprehensively substantiate the assertions (see to points below).\nMajor issues\n1. The ethic number should be provided on line 96.\n2. To further clarify the relationship between DUSP26 and kidney function, Figure 1 should include the correlation between DUSP26 and creatine, nitrogen, and eGFR.\n3. Figure 3E illustrates the 20-hour treatment of BUMPT cells with the DUSP26 phosphatase inhibitor NSC87877 (5 μM) and cisplatin (20 μM). Figure 3i illustrates the TUNEL staining of DUSP26-overexpressing cells after 24 hours of cisplatin treatment. The time point for tunel staining is distinct; how can this be elucidated?\n4. The PT-Dusp26-KI model is crucial; however, the manuscript fails to demonstrate the baseline DUSP26 levels of these animals. The authors should quantify the expression of DUSP26 in KI proximal tubules compared to WT proximal tubules, for example, through immunostaining or Western blotting of DUSP26 in untreated kidneys.\n5. A recent study conducted by Xiang et al. (2024) in the field of toxicology and applied pharmacology demonstrated that DUSP26 protects against renal IR injury by restricting TAK1-JNK/p38 activation. The present study identifies a unique mechanism (p53 S312 dephosphorylation) and does not address MAPKs. The authors should elaborate on the connections between these discoveries. For instance, is the DUSP26–p53 axis independent of DUSP26's impact on JNK/p38? The discussion should acknowledge that DUSP26 likely has multiple substrates and cite Xiang et al., as well as clarify how the p53 effect fits into this context.\n6. The mechanistic focus is on Ser312, which is non-canonical in AKI contexts. The authors demonstrate that p53-driven apoptosis is diminished by preventing Ser312 phosphorylation (S312A). To ensure comprehensiveness, it is necessary to determine whether DUSP26 directly influences the stability of p53 or other p53 phosphorylation sites. The proteomics identified Ser312 as the most significant hit (Fig. 7G); however, it is uncertain whether canonical sites (e.g. Ser15, Ser20) were also examined. Additionally, it would be beneficial to establish a connection between the Ser312 modification and existing literature. For instance, prior mouse studies demonstrated that the phosphorylation of Ser312 compromises the function of the tumor suppressor p53, which is consistent with our discovery that its dephosphorylation enhances cell survival. The importance of Ser312 would be emphasized by including a concise citation to that effect.\n7. The mechanistic insight and compelling observation that DUSP26 downregulation is mediated by promoter hypermethylation (Fig. 3) are noteworthy. Nevertheless, the authors' previous study, which was published in Kidney International (2017), indicated that the treatment with the DNA methyltransferase inhibitor 5-Aza actually exacerbated tubular cell mortality in cisplatin-induced AKI. This evident discrepancy is worthy of discussion. The authors should emphasize in the Discussion that 5-Aza may promote cell death because of its extensive epigenetic effects or potential off-target consequences, despite the fact that it restores DUSP26 expression in injured tubular cells.\n8. The representative immunofluorescence image in Fig. 1H, which depicts DUSP26 expression in the control group, is somewhat less clear and well-defined than the corresponding AKI image. The authors may wish to select a higher-resolution image that more clearly illustrates tubular DUSP26 expression in control tissue in order to enhance visual consistency and clarity.\nAdditionally, there are a few minor issues that require attention.\n1.In Fig. 6A (schematic of knock-in strategy), it would help to label the DUSP26 cDNA cassette in the Rosa26 locus for clarity.\n2. All figure legends should specify n and statistical tests (most do). For example, Fig. 1H–J should note how many human samples were quantified (n=8,7 are in Supp Table). Fig. 8C–E (cell images and blots) should state the number of replicates.\n3. In Figs. 7–9, ensure that asterisks (*) are defined (the legends mostly do). For clarity, abbreviations (e.g. AKI, IRI, HIRI) should be spelled out once in the legend.\n4. Introduction: “Dual specificity phosphatase 26 (DUSP26) is an atypical MAPK phosphatase.” Perhaps briefly define “atypical” (lacks ERK-binding motif) or cite a ref.\n5. Methods: Indicate how ATN histology was scored. The “Suzuki criteria” for liver should be defined at first mention (though that’s in legend).\n6. Discussion: In the paragraph starting “Integrating human AKI transcriptomic datasets…”, it may read better to split it into two shorter sentences for readability.\n7. Check consistency: the paper uses “Dusp26” vs “DUSP26” (gene vs protein). Ensure italicization for the gene if referring to mouse Dusp26 gene vs protein.", "reviewer_4": "The manuscript proposes that DUSP26 protects against AKI by selectively dephosphorylating p53 at Ser312 (human Ser315) and that DUSP26 is downregulated via promoter hypermethylation. The conceptual space is narrow: the p53–AKI link is already extensively established, and DUSP26 has been reported as a p53 regulator in other cell types (e.g., neuroblastoma; PMID:20562916), including reports of Ser20/Ser37 dephosphorylation. Against this background, the evidence presented for a Ser312-focused mechanism and an epigenetic cause for DUSP26 loss is not yet sufficient.\n1. The authors should more clearly distinguish a Ser312-centered pathway from the established N-terminal mechanisms (Ser20/Ser37) within the same renal/AKI models and conditions. As presented, it remains difficult to exclude that the observed effects are mediated primarily through the known N-terminal sites rather than Ser312.\n2. There is no in-vivo evidence that Ser312 phosphorylation is required. Although renal p-p53(Ser312) is reduced in PT-Dusp26-KI mice after injury, this is correlative rather than a test of necessity. Genetic evidence—for example, a non-phosphorylatable p53-S312A knock-in would be needed to show requirement.\n3. The epigenetic evidence remains preliminary. MSP is qualitative and does not resolve methylation at individual CpG sites. Please provide quantitative, locus-resolved methylation across the DUSP26 promoter, preferably using bisulfite sequencing, and relate these measurements to DUSP26 mRNA and protein in matched samples. If a causal role is proposed, include a functional rescue using demethylation by pharmacological agents or locus-targeted editing, and, where feasible, add chromatin evidence by demonstrating DNMT occupancy and relevant histone marks with ChIP.\n4. The pharmacologic inhibitor NSC87877 is known to have off-target effects. Genetic approaches to modulate DUSP26 activity would help confirm specificity and reinforce the mechanistic conclusions.\n5. Long-term outcomes after AKI, such as fibrosis and maladaptive repair, are not assessed. Given p53’s known role in these processes, examining later time points or discussing the potential implications would enhance the translational relevance. Although DUSP26 appears to suppress apoptosis and exert protective effects in the acute phase, persistent survival of abnormal cells may increase the risk of tumorigenesis or, conversely, inhibit proper tubular regeneration, potentially promoting the transition from AKI to CKD.\n6. The human biopsy data are limited by small sample size and heterogeneous underlying diseases, which complicate interpretation of associations such as the inverse correlation between DUSP26 and ATN severity.\n7. The liver ischemia–reperfusion injury extension is interesting, but it diverts focus from the kidney-centric narrative. Why was the liver selected for investigation? What is the expression profile of DUSP26 across tissues? Consider streamlining or moving some of these data to the Supplementary Information unless they are essential to the main conclusions.\nMinor Comments:\n1. Regarding the generation of proximal tubule-specific DUSP26 knock-in mice, the manuscript refers to “DUSP26 flox/flox” mice in the Method section.\n2. In Figure 2B and 2C, the y-axis lacks clarification regarding the reference value used for normalization. Please specify what was set as \"1\" in the quantification.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewer for the thorough evaluation of our manuscript. We are particularly grateful for the reviewer’s appreciation of the novelty and significance of this study. We are very encouraged that the reviewer found our study to be “very well-designed and comprehensive” and that it reveals a “previously unrecognized and important role of DUSP26 in attenuating kidney injury.” We have carefully addressed the comments, which we believe have further improved the manuscript.\n\n[... Author responses to all points ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have added substantial amounts of results to strengthen the manuscript, and my comments have been adequately addressed. The manuscript is now acceptable for publication in Nature Communications.", "reviewer_2": "The authors have addressed my concerns.", "reviewer_3": "The author has thoroughly addressed my previous concerns, and I have carefully reviewed the questions raised by other reviewers along with the author's responses. The entire revised manuscript and figure revisions have been scrutinized, and the paper now meets publication standards. I personally recommend acceptance.", "reviewer_4": "The authors have responded to my primary concerns regarding the DUSP26-p53 S312 axis with extensive new experimentation. The question of specificity for S312 dephosphorylation is resolved by new in vivo data from the PT-DUSP26-KI mice, which demonstrate a remarkably preferential effect on S312 over other N-terminal phosphorylation sites (Supplementary Fig. 7). Similarly, the question of whether S312 phosphorylation is required for AKI pathogenesis has been decisively addressed by the genetic rescue model utilizing AAV-driven p53-S312A re-expression in p53-knockout mice (Fig. 9), providing conclusive in vivo evidence of necessity. Furthermore, the conclusion that DUSP26's protective effect is dependent on its phosphatase activity is adequately supported by multiple genetic approaches, including siRNA knockdown and the phosphatase-dead C152S mutant (Fig. 4), appropriately reinforcing the pharmacological inhibitor findings. Acknowledging the long-term role of DUSP26 as a future research direction within the Discussion's limitations section is also a reasonable clarification of the study's scope.\nHowever, a lapse in mechanistic rigor remains regarding the epigenetic regulation of DUSP26 expression. While quantitative Bisulfite Sequencing confirms the existence of hypermethylation and DNMT1 recruitment is shown by ChIP-qPCR, DNMT1 is primarily a maintenance methyltransferase. Given that DUSP26 promoter hypermethylation implies de novo methylation, the principal executors of this process are DNMT3A and DNMT3B. The mechanistic chain remains incomplete because there are no direct Chromatin Immunoprecipitation (ChIP) data demonstrating the recruitment of DNMT3A or DNMT3B to the DUSP26 promoter. Proving the direct involvement of these de novo methyltransferases is highly desirable to achieve full scientific rigor for the proposed epigenetic mechanism of gene silencing.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We appreciate Reviewer #4 for clearly identifying this mechanistic gap. To directly test the involvement of de novo DNA methyltransferases, we performed additional ChIP-qPCR assays in BUMPT cells under basal conditions and after cisplatin injury (20 μM, 24 h), using ChIP-grade antibodies against DNMT3A and DNMT3B, with IgG as negative control and the same Dusp26 promoter primer pair used for DNMT1 ChIP.\nWe observed a significant increase in DNMT3A occupancy at the CpG-rich region of the Dusp26 promoter in cisplatin-treated cells compared with controls, supporting a de novo methylation component associated with Dusp26 repression. In contrast, DNMT3B did not show significant enrichment at the Dusp26 promoter under the same conditions.\nWe believe these new data directly address the reviewer’s concern by providing the missing mechanistic evidence for recruitment of a de novo DNA methyltransferase (DNMT3A) to the Dusp26 promoter during injury-associated silencing."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have fully addressed the suggestion, and I have no further comments.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewer for confirming that the previous concerns have been fully addressed. No further changes were requested by the reviewer, and we have therefore not made additional revisions specifically in response to Reviewer #4 in this final round."}]

Decision letter