Abstract

   Histone lactylation is a metabolic stress‐related histone modification.
   However, the role of histone lactylation in the development of
   sepsis‐associated acute kidney injury (SA‐AKI) remains unclear. Here,
   histone H3K18 lactylation (H3K18la) is elevated in SA‐AKI, which is
   reported in this study. Furthermore, this lactate‐dependent histone
   modification is enriched at the promoter of Ras homolog gene family
   member A (RhoA) and positively correlated with the transcription.
   Correction of abnormal lactate levels resulted in a reversal of
   abnormal histone lactylation at the promoter of RhoA. Examination of
   related mechanism revealed that histone lactylation promoted the
   RhoA/Rho‐associated protein kinase (ROCK) /Ezrin signaling, the
   activation of nuclear factor‐κB (NF‐κB), inflammation, cell apoptosis,
   and aggravated renal dysfunction. In addition, Ezrin can undergo
   lactylation modification. Multiple lactylation sites are identified in
   Ezrin and confirmed that lactylation mainly occurred at the K263 site.
   The role of histone lactylation is revealed in SA‐AKI and reportes a
   novel post‐translational modification in Ezrin. Its potential role in
   regulating inflammatory metabolic adaptation of renal proximal tubule
   epithelial cells is also elucidated. The results provide novel insights
   into the epigenetic regulation of the onset of SA‐AKI.

   Keywords: ezrin, glycolysis, lactylation, RhoA, sepsis‐associated acute
   kidney injury
     __________________________________________________________________

   H3K18la is elevated and positively correlated with the RhoA
   transcription in SA‐AKI. Mechanistically, histone lactylation promotes
   the RhoA/ROCK/Ezrin signaling pathway, NF‐κB activation, inflammation,
   and cell apoptosis and aggravates renal dysfunction. In addition,
   exogenous lactate promotes Ezrin lactylation, which mainly occurs at
   the K263 site.

   graphic file with name ADVS-11-2307216-g003.jpg

1. Introduction

   Sepsis‐associated acute kidney injury (SA‐AKI) is commonly
   characterized as sepsis or septic shock affecting the kidneys, leading
   to a gradual deterioration in kidney function, as per the criteria
   outlined by the Global Renal Disease Prognosis Organization (KDIGO) for
   acute kidney injury (AKI). This definition also involves the exclusion
   of alternative causes of kidney injury.^[ [46]^1 , [47]^2 ^]
   Extrapolating from incidence rates in the United States, Adhikari
   et al. determined that as many as 190 000 cases of sepsis are reported
   worldwide each year.^[ [48]^3 ^] Compared with non‐septic AKI, SA‐AKI
   is strongly associated with adverse clinical outcomes.^[ [49]^4 ,
   [50]^5 , [51]^6 ^]

   Renal tubular epithelial cells (RTECs) are the most metabolically
   active cells in the kidney and are sensitive to sepsis‐related damage.
   In both the cecal ligation and puncture (CLP) model and human SA‐AKI,
   RTECs may undergo an initial shift to glycolysis, converting pyruvate
   to lactic acid. This represents an inefficient mechanism for producing
   ATP. Importantly, serum lactate has been recognized as a biomarker of
   sepsis prognosis, and elevated serum lactate levels are positively
   associated with sepsis‐related mortality.^[ [52]^7 ^] Lactate, a
   derivative of aerobic glycolysis, is known to directly enhance the
   release of inflammatory factors, such as tumor necrosis factor α
   (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β),^[ [53]^8 ^]
   and a large number of inflammatory factors that lead to worsening of
   renal function, apoptotic cell death, and sublethal damage (loss of
   cell polarity, reduced expression of tight junction protein, and
   bioenergy interference).^[ [54]^9 ^] In contrast, inhibiting aerobic
   glycolysis and inducing oxidative phosphorylation (OXPHOS) can reduce
   susceptibility to AKI and significantly improve survival, improving
   inflammation‐related conditions such as sepsis and multiple
   sclerosis.^[ [55]^10 , [56]^11 ^] Therefore, reprogramming glycolysis
   in the proximal tubule epithelium may be a promising strategy for
   maintaining or restoring kidney function. This modulation may regulate
   the potential role of the inflammatory metabolic adaptation of the
   proximal tubule epithelium, thereby offering a potential avenue for
   improving inflammation and apoptosis.

   Long‐established protein post‐translational modifications (PTMs)
   include acetylation, ubiquitination, and methylation of lysine residues
   and phosphorylation and glycosylation of serine or threonine
   residues.^[ [57]^12 ^] These modifications form a part of the hierarchy
   of epigenetic regulatory mechanisms. Glycolysis‐derived lactate has
   been identified as a substrate for histone lactylation and activates
   downstream gene transcription directly.^[ [58]^13 , [59]^14 ^]
   Interestingly, histone lactylation plays an important role as an
   epigenetic regulator mechanism in pathogenesis. For example, histone
   lactylation regulates the M1/2 polarization of macrophages and promotes
   the transformation of macrophages from an inflammatory state to a
   repaired state.^[ [60]^15 ^] Histone lactylation promotes the
   expression of m6A reader protein YTHDF2 to promote the development of
   tumors.^[ [61]^16 ^] Glis1 promotes somatic reprogramming through an
   epigenome‐metabolome‐epigenome signaling cascade.^[ [62]^17 ^] However,
   the role of histone lactylation in the development of SA‐AKI remains
   unclear. Because of the metabolic reprogramming of RTECs during the
   early stage of SA‐AKI, histone lactylation in RTECs is likely to be
   abnormal, and it is necessary to explore the potential function of
   histone lactylation in the development of SA‐AKI. Moreover, PTMs can
   widely regulate the function of proteins, shape cell phenotypes, and
   play a vital role in the endogenous regulation of Ezrin. Examination of
   PTMs of Ezrin would help enrich the understanding of the Ezrin
   regulation mechanism.

   Therefore, in this study, we report for the first time the potential
   role of H3K18la and lactylation on the FREM domain of Ezrin in SA‐AKI,
   identifying Ezrin as a lactate substrate. We provide evidence that
   SA‐AKI promotes an increase in lactate and H3K18la and activates
   RhoA/ROCK1/Ezrin signaling and Ezrin K263 lactylation, leading to
   downstream inflammation and apoptosis. Inhibition of GLUT1 and Ezrin
   K263 site mutations (K263R) alleviated inflammation and apoptosis,
   providing a new mechanism and potential target for delaying renal
   dysfunction.

2. Results

2.1. Histone Lactylation Increases Rapidly in Renal Tissue and Tubular
Epithelial Cells After SA‐AKI

   Although the pathophysiological mechanisms of SA‐AKI, especially the
   inflammatory response, microvascular dysfunction, and metabolic
   recombination, have been studied, the role of histone lactylation as
   epigenetic modification in the development of SA‐AKI remains unclear.
   Given the metabolic reprogramming from OXPHOS to glycolysis in RTECs
   during SA‐AKI,^[ [63]^18 , [64]^19 ^] we found that GLUT1 protein
   expression levels were significantly increased after stimulation of
   HK‐2 cells with LPS (Figure [65]S1A,B, Supporting Information).
   Therefore, we first examined lactate levels in the peripheral blood of
   CLP mice. Colorimetric measurements showed that serum lactate levels
   were significantly higher in mice in the CLP group compared to those in
   matched Sham mice (Figure [66] 1A). The results were consistent with
   previously reported clinical observations indicating that patients with
   SA‐AKI have higher lactate concentrations than healthy controls.^[
   [67]^20 , [68]^21 , [69]^22 ^] Similarly, lactate concentration in the
   supernatant and cell lysate of HK‐2 cells was higher in the
   LPS‐stimulated group than in the vehicle group (Figure [70]1B,C). Since
   lactate acts as a precursor that can stimulate histone lactylation,^[
   [71]^23 ^] we hypothesized that histone lactylation might change in the
   context of SA‐AKI. Western blot analysis showed increased levels of Pan
   Kla, H3K9la, and H3K18la in total kidney proteins extracted from mice
   in the CLP group compared to that in the Sham group (Figure [72]1D,E).
   Consistent with observations in mice, levels of Pan Kla and H3K18la
   were significantly increased in HK‐2 cells and TCMK‐1 cells
   (Figure [73]1F,G). To further evaluate the lactylation‐related changes
   in RTECs in vitro after LPS stimulation at different time and
   concentration gradients, western blot analysis was performed. The
   results showed that the lactylation level increased as early as 3 h
   after LPS stimulation in the early stage, with HK‐2 cells reaching a
   peak at 12 h and TCMK‐1 cells reaching a peak at 6 h. Lactylation
   decreased to baseline levels within 48 h of LPS stimulation. In
   contrast, the level of acetylation did not change significantly
   (Figure [74]1H). Subsequently, we examined the lactylation of H3K18la.
   The results showed that H3K18la levels gradually increased in HK‐2
   cells and began to decline at 24 h. It remained elevated compared to
   baseline and the difference was statistically significant. In
   comparison, TCMK‐1 cells maintained a high level of H3K18la until 48 h
   (Figure [75]1I). In response to stimulation with different
   concentrations of LPS, the levels of global lactylation and H3K18la
   levels showed a similar trend, and with the increase of LPS
   concentration, the global lactylation and H3K18la levels were gradually
   upregulated. However, the levels of global acetylation and H3K18ac were
   not significantly changed (Figure [76]1J,K). These results all support
   increased lactate and histone lactylation in the context of SA‐AKI.

Figure 1.

   Figure 1
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   Increased histone lactylation in SA‐AKI mice and RTECs induced by LPS.
   A) Lactate levels in the serum of CLP model and Sham group mice (n  = 6
   mice per group). B) The relative levels of lactate in the supernatant
   of HK‐2 cells treated with LPS, n  =  3 independent experiments. C)
   Changes in intracellular lactate levels in HK‐2 cells after LPS
   treatment, n  =  3 independent experiments. D) Western blotting
   analysis of Pan Kla in the total proteins extracted from the kidney
   tissues of CLP and Sham group mice (n  = 6 mice per group); β‐actin
   served as an internal control; normalized corresponding densitometric
   analyses are shown as a histogram. E) Western blotting analysis of
   site‐specific histone lactylation in the total proteins extracted from
   the kidney tissue of CLP and Sham group mice (n  = 6 mice per group);
   Histone H3 served as an internal control; normalized corresponding
   densitometric analyses are shown as a histogram. F) Western blotting
   analysis of Pan Kla in HK‐2 cells and TCMK‐1 cells; Histone H3 served
   as an internal control. G) Western blotting analysis of site‐specific
   histone lactylation in HK‐2 cells and TCMK‐1 cells. Histone H3 served
   as an internal control. H) The Pan Kla and Pan Kac western blotting
   analysis of HK‐2 cells and TCMI‐1 cells was performed at 0, 3, 6, 12,
   24, and 48 h after LPS treatment. I) Time gradient‐based western
   blotting analysis of H3K18la and H3K18ac in HK‐2 cells and TCMK‐1 cells
   after LPS treatment. J) The Pan Kla and Pan Kac western blotting
   analysis of HK‐2 cells and TCMI‐1 cells treated with LPS at 0, 0.3, 1,
   3, 10, and 30 µg mL^−1. K) Concentration gradient‐based western
   blotting analysis of H3K18la and H3K18ac in HK‐2 cells and TCMK‐1 cells
   after LPS treatment. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not
   significant.

2.2. Inhibition of Histone Lactylation Alleviates LPS‐Induced RTEC Damage

   Lactate production depends on a balance between glycolysis and
   mitochondrial metabolism. We first tested whether inhibition of
   glycolysis‐related enzyme activity reduced histone lactylation levels
   in RTECs (Figure [78] 2A). In this paper, we applied two inhibitors: 1)
   Glucose transporter GLUT1 inhibitor, BAY‐876; 2) Glycolysis inhibitors,
   the non‐metabolizable glucose analogs 2‐deoxyd‐glucose (2‐DG) and
   Oxamic acid sodium. Notably, both classes of inhibitors achieved
   significant reductions in lactic acid within RTECs (Figure [79]S2A,B,
   Supporting Information) and reductions in global lactylation and
   H3K18la levels (Figure [80]2B,C; Figure [81]S2C,D, Supporting
   Information). Immunofluorescence also confirmed changes in H3K18la
   levels in HK‐2 cells (Figure [82]2D). And we introduced siRNAs specific
   to GLUT1 and LDHA/LDHB.^[ [83]^16 ^] to further validate the role of
   histone lactylation in SA‐AKI. We observed that silencing GLUT1 or
   LDHA/LDHB with LPS treatment significantly impaired histone
   lactylation, increased cell apoptosis, and exacerbated damage.
   Reintroducing lactate into GLUT1 or LDHA/LDHB‐deficient cells with LPS
   treatment successfully elevated histone lactylation levels and
   partially restored the extent of cell damage (Figure [84]S2E,F,
   Supporting Information). Conversely, mitochondrial electron transport
   chain Complex I inhibitors (Rotenone) mediated cellular glycolysis and
   increased intracellular lactate levels, as well as lactate and H3K18la
   levels (Figure [85]S2G,H, Supporting Information).

Figure 2.

   Figure 2
   [86]Open in a new tab

   Inhibition of GLUT1 can decrease the level of histone lactylation and
   inhibit LPS‐induced RTECs injury. A) Schematic diagram of histone
   lactylation inhibition. B) Pan Kla and Pan Kac levels were detected in
   HK‐2 cells and TCMK‐1 cells cultured in different concentrations of
   BAY‐876 and 10 µg/mL of LPS for 24 h by western blotting analysis. C)
   H3K18la and H3K18ac levels were detected in HK‐2 cells and TCMK‐1 cells
   cultured in different concentrations of BAY‐876 and 10 µg/mL of LPS for
   24 h by western blotting analysis. D) Immunofluorescence staining for
   H3K18la (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI; blue) in HK‐2
   cells (scale bar = 20 µm). E) Flow cytometry and corresponding
   quantitative analysis of the extent of HK‐2 cell apoptosis by Annexin
   V/PI staining, n  =  3 independent experiments. F,G,H) Detection of SOD
   activities (F), MDA levels (G), and ROS levels (H) in HK‐2 cells after
   different treatments, n  =  3 independent experiments. (I) Western
   blotting analysis of BCL2 and BAX protein levels in HK‐2
   cells incubated with 10 µg/mL LPS ± 1 µM BAY‐876 for 24 h, n  =  3
   independent experiments. (J) Detection of IL (interleukin)−1, IL
   (interleukin)−6, and TNF‐α (tumor necrosis factor‐α) mRNA expression
   levels by real‐time qPCR, n  =  3 independent experiments. ^* p <0.05,
   ^** p <0.01, and ^*** p <0.001; ns, not significant.

   Next, we evaluated whether Histone lactylation inhibition mitigated
   LPS‐induced RTEC damage in vitro. Flow cytometry (FCM) showed that
   BAY‐876 reduced LPS‐induced apoptosis (P<0.01, Figure [87]2E). Upon
   measuring the expression of oxidative stress markers in HK‐2 cell
   supernatant, we observed that LPS up‐regulated MDA and down‐regulated
   SOD compared to the corresponding levels observed in the control group.
   In contrast, the BAY+LPS group inhibited LPS‐induced oxidative stress
   factors (p <0.05, Figure [88]2F,G). LPS then induced ROS expression,
   while BAY‐876 prevented LPS‐mediated oxidative stress (p <0.01,
   Figure [89]2H). Similarly, Western blot showed that compared with the
   control group, the level of apoptosis marker BCL2‐associated X (BAX)
   was increased, whereas the addition of BAY‐876 inhibited LPS‐induced
   kidney injury and apoptosis (Figure [90]2I). The expression of
   inflammatory factors in HK‐2 cells was also confirmed by Real‐time qPCR
   (Figure [91]2J). Thus, BAY‐876 alleviated LPS‐induced inflammation,
   oxidative stress, and apoptosis of renal tubular epithelial cells.

2.3. Identification of Potential Downstream Targets of H3K18la Regulation

   Histone modification can affect the transcription level of target
   genes.^[ [92]^24 , [93]^25 , [94]^26 ^] Zhang et al. further
   demonstrated that lactate‐derived histone lactylation (for example, at
   the H3K18la site) can directly stimulate gene transcription.^[ [95]^23
   ^] In order to reveal the regulatory role of histone lactylation in
   gene expression, we first performed ATAC‐seq analysis and CUT&Tag
   analysis with the whole kidney tissue of the SA‐AKI mouse model,
   followed by sequencing using anti‐H3K18la antibodies. The results
   showed that compared with the sham group, chromatin accessibility was
   higher in the CLP group. In CUT&Tag deep Tools analysis, the peak of
   H3K18la was enriched in the CLP group (Figure [96]S3A, Supporting
   Information). To explore potential candidate genes regulated by H3K18la
   in renal tubular epithelial cells (MRPTEpiC) of SA‐AKI mice, we
   isolated and identified primary MRPTEpiC (Figure [97] 3A;
   Figure [98]S3B–D, Supporting Information) for three independent
   replicate of CUT&Tag experiments (grouped as MRPTEpiC_Sham,
   MRPTEpiC_CLP, and MRPTEpiC_BAY+CLP). The results showed that compared
   to MRPTEpiC_Sham, MRPTEpiC_CLP exhibits enrichment of H3K18la peaks,
   with 10 978 (25.08%) H3K18la binding peaks located within promoter
   region (≤3Kb) (Figure [99]3B). After the addition of the BAY‐876
   inhibitor, the enrichment of H3K18la peaks decreased compared to
   MRPTEpiC_CLP (Figure [100]3C). To investigate the epigenetic regulatory
   role of H3K18la in MRPTEpiC, Kyoto Encyclopedia of Genes and Genomes
   (KEGG) analysis was performed on the target genes of H3K18la binding
   peaks. The results indicate that, compared to MRPTEpiC_Sham, the
   H3K18la‐specific genes in the MRPTEpiC_CLP group are significantly
   enriched in the regulation of actin cytoskeleton. Additionally,
   pathways involved in metabolism and inflammation of epithelial cell, as
   well as cell apoptosis, are also enriched (Figure [101]3D).
   Furthermore, these enrichments can be suppressed by BAY‐876
   (Figure [102]3E).

Figure 3.

   Figure 3
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   Histone lactyation activated transcription of RhoA. A) Schematic
   diagram of the extraction and purification of RTECs from kidney tissue.
   Created with BioRender.com with permission. B) Pie plot of the genomic
   distribution of H3K18la peaks in MRPTEpiC_CLP group. C) Heatmaps of
   H318la CUT&Tag signals were visualized by deepTools in MRPTEpiC_Sham,
   MRPTEpiC_CLP, and MRPTEpiC_BAY+CLP groups. The findings are ordered by
   signal strength. D) Bubble chart showing the KEGG pathway enrichment
   analysis of MRPTEpiC_CLP group upregulated genes with increased H3K18la
   modification. E) The KEGG pathway enrichment analysis of
   MRPTEpiC_BAY+CLP group downregulated genes with decreased H3K18la
   modification. F) The flow chart depecting selection of RhoA as the
   downstream target of H3K18la by bioinformatics analysis. G) IGV tracks
   for RhoA from MRPTEpiC CUT&Tag analysis. H,I) Western blot was
   performed to test RhoA expression in HK‐2 and TCMK‐1 cells after
   treatment with different concentrations of BAY‐876 and 10 µg/mL of LPS.
   n  =  3 independent experiments. ^* p <0.05, ^** p <0.01, and ^***
   p <0.001; ns, not significant.

   Next, we performed the CUT&Tag and ATAC‐seq and combined these results
   with those of the transcriptomic sequencing analyses of mouse kidney
   tissues with or without BAY‐876. RhoA, Chac1, Akt1s1, Zfp36l1, and
   Scn1b were selected as candidate genes (Figure 3F; Figure [104]S3E,
   Supporting Information). Among these candidate genes, RhoA, a member of
   the small GTPase family, is critical in the regulation of cellular
   barrier function in a variety of inflammatory diseases such as
   sepsis,^[ [105]^27 , [106]^28 ^] and also plays a crucial role in cell
   morphology, motility, adhesion, and proliferation.^[ [107]^29 ,
   [108]^30 ^] Hence, we aimed to focus on RhoA. To verify whether
   transcription of RhoA is positively correlated to H3K18la, we initially
   compared the signals of MRPTEpiC CUT&Tag peaks in the RhoA region. We
   found a significant enrichment of the H3K18la signal at the RhoA
   promoter in the genomic position of the CLP group compared to that in
   the Sham group (Figure [109]3G). This marked enrichment was reduced by
   BAY‐876 (Figure [110]3G). In addition, as expected, BAY‐876 treatment
   led to a significant decrease in protein levels of RhoA expression
   (Figure [111]3H,I).

2.4. LPS‐Mediated RhoA‐ROCK Pathway Induced Ezrin Phosphorylation and
Sublocalization

   To investigate the potential involvement of RhoA/ROCK signaling in
   LPS‐stimulated HK‐2 cells, we used western blotting to examine the
   expression and phosphorylation levels of myosin phosphatase target
   subunit 1 (MYPT1), the downstream target of RhoA and ROCK. The results
   of time response showed that LPS‐induced RhoA and ROCK1 protein levels
   increased 3 h after LPS stimulation, reached a peak at 6 h, and
   returned to baseline 48 h later. The dose‐response results showed a
   dose‐dependent increase (Figure [112] 4A,B). Notably, ROCK is involved
   in Ezrin activation.^[ [113]^31 , [114]^32 , [115]^33 ^] Therefore, we
   first verified the knockdown and overexpression efficiency of RhoA and
   Ezrin. Western blot analysis showed that transfection of RhoA shRNA and
   Ezrin siRNA significantly reduced RhoA and Ezrin protein expression
   levels. Anterograde RhoA plasmid and Ezrin lentivirus significantly
   increased the respective expression levels of the two proteins
   (Figure [116]4C,D). HK‐2 cells were transfected with RhoA shRNA or
   pretreated with ROCK1 inhibitor Y‐27632 before LPS stimulation. Western
   blot results showed that the protein expression level of p‐ezrin
   increased significantly in LPS‐treated cells. Up‐regulation of p‐Ezrin
   was significantly inhibited by transfection with RhoA shRNA or
   pretreatment with Y‐27632 (Figure [117]4E). In addition,
   immunofluorescence analysis showed the intracellular sublocalization of
   Ezrin. Resting cells fluoresce weakly with p‐Ezrin and are mainly
   located in the cytoplasm, where p‐Ezrin appears to be transferred from
   the cytoplasm to the membrane in response to LPS, as shown by the
   yellow arrow. This was further demonstrated by co‐localization with
   F‐actin cytoskeletal recombination. This migration was significantly
   restored by transfection with RhoA shRNA (Figure [118]4F).

Figure 4.

   Figure 4
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   RhoA/ROCK signaling pathway mediated LPS‐induced Ezrin phosphorylation
   and translocation. A) HK‐2 cells were treated with LPS (10 µg mL^−1)
   for 0, 3, 6, 12, 24, and 24 h, respectively. RhoA, phosphorylated
   MYPT1, MYPT1, phosphorylated Ezrin, and Ezrin protein level was
   evaluated by western blotting. B) HK‐2 cells were treated with LPS for
   24 h at concentrations of 0, 0.3, 1, 3, and 10 µg mL^−1, respectively.
   RhoA, MYPT1, Ezrin, phosphorylated MYPT1, and Ezrin protein level was
   evaluated by western blotting. C) Western blot showed that RhoA was
   knocked down after shRNA transfection and overexpressed after being
   transfected with a plasmid carrying RhoA gene in HK‐2 cells. D) Western
   blot showed that Ezrin was knocked down after siRNA transfection and
   overexpressed after transfected with a lentiviral vector carrying the
   Ezrin‐encoding gene in HK‐2 cells. n  =  3 independent experiments. ^*
   p <0.05, ^** p <0.01, and ^*** p <0.001; ns, not significant. E) Total
   Ezrin and phosphorylated Ezrin protein levels were detected in HK‐2
   cells challenged with RhoA shRNA or Y‐27632 ± 10 µg mL^−1 LPS for 24 h
   by western blotting. ^** p <0.01 versus Con, ^# p <0.05 versus LPS. F)
   The localization of Ezrin was analyzed by immunofluorescence staining
   (scale bar = 20 µm). p‐Ezrin was stained with Alexa Fluor
   594‐conjugated IgG (red), F‐actin was stained with phalloidin (green),
   and nuclei were stained with DAPI (blue). n  =  3 independent
   experiments.

2.5. RhoA‐ROCK1‐Ezrin Pathway Induces NF‐κB Activation, and Ezrin Inhibition
Reduces the Membrane Expression of GLUT1

   We examined potential interactions between Ezrin and myeloid
   differentiation factor 88 (MyD88)/IL‐1R‐associated kinase 1 (IRAK‐1).
   The interaction was significantly enhanced after LPS stimulation via
   COIP experiments, indicating that Ezrin binds to MyD88/IRAK1 in an LPS
   stimulation‐dependent manner (Figure [120] 5A). To explore further
   Ezrin‐mediated downstream signaling, HK‐2 cells were transfected with
   siNC or siEzrin in the presence or absence of LPS. Western blotting
   analysis was used to measure the protein expression and phosphorylation
   levels of NF‐κB inhibitor alpha (IκBα) and NF‐κB, as well as changes in
   apoptotic and renal injury biomarkers (KIM‐1 and NGAL). LPS treatment
   significantly increased the activation of NF‐κB in HK‐2 cells
   transfected with siNC. The expression level of B‐cell lymphoma protein
   2 (BCL2) decreased, whereas the expression level of BAX, KIM‐1, and
   NGAL protein increased. Ezrin knockdown partially suppressed the
   increase of LPS‐induced NF‐κB activation, apoptosis, and the elevation
   of kidney injury markers (Figure [121]5B). Rescue tests further
   demonstrated that knockdown of RhoA and overexpression of Ezrin could
   restore downstream NF‐κB activation (Figure [122]5C). The effects of
   Ezrin knockdown on the proinflammatory cytokines production in HK‐2
   cells with or without LPS were measured by real‐time qPCR. As
   anticipated, LPS mediated a significant increase in mRNA expression
   levels of IL‐1β, IL‐6, and TNF‐α. Knockdown of Ezrin inhibited this
   upregulation (Figure [123]5D).

Figure 5.

   Figure 5
   [124]Open in a new tab

   Ezrin is a key downstream gene of RhoA, regulating RTEC damage and
   translocation of GLUT1 in the plasma membrane. A) The association
   between Ezrin and MyD88/IRAK1 in HK2 cells in the presence or absence
   of LPS (10 µg mL^−1) was detected by co‐immunoprecipitation. B) HK‐2
   cells transfected with siNC or siEzrin and incubated with ±10 µg mL^−1
   LPS for 24 h. Western blot was performed to analyze p‐IKBα, IKBα,
   p‐NF‐κB, NF‐κB, BCL2, BAX, KIM‐1, and NGAL protein levels. C) The
   rescue experiment was performed to confirm the relationship between
   ROCK and Ezrin in vitro. D) Detection of IL‐1, IL‐6, and TNF‐α mRNA
   expression levels by real‐time qPCR, n  =  3 independent experiments.
   E) Western blot analysis was performed to detect the plasma membrane
   (PM)/ WCL whole‐cell lysate (WCL) expression level of GLUT1 in HK‐2
   cells; the internal reference for cell membrane protein was Na+/K+
   ATPase. n  =  3 independent experiments. ^* p <0.05, ^** p <0.01, and
   ^*** p <0.001; ns, not significant. F) The localization of GLUT1 was
   analyzed by immunofluorescence staining (scale bar = 20 µm). Ezrin was
   stained with Alexa Fluor 594‐conjugated IgG (red), GLUT1 was stained
   with Alexa Fluor 488‐conjugated IgG (green), and nuclei were stained
   with DAPI (blue), n  =  3 independent experiments.

   Ezrin positively correlates with the expression of HK2, a key enzyme
   involved in glycolysis. We aimed to examine whether Ezrin was related
   to the sublocalization and expression of GLUT1. Total proteins were
   extracted from HK‐2 cells, and membrane proteins were separated using
   the Membrane/Cytosol/Nuclear Protein Extraction Kit (Beijing Solaibio
   Technology Co., Ltd., EX1400). Western blot analysis demonstrated that
   in HK‐2 cells, the knockdown of Ezrin resulted in a decrease in the
   ratio of cell membrane protein GLUT1 to total cellular protein GLUT1
   (Figure [125]5E). This phenomenon was also confirmed by
   immunofluorescence (Figure [126]5F). The results indicate that LPS
   promotes GLUT1 membrane localization, while knockdown of Ezrin
   significantly reduces the protein levels of GLUT1 in the PM/WCL.

2.6. The Reduction of H3K18la has Anti‐Inflammatory Effects and Contributes
to the Improvement of Renal Function After SA‐AKI

   Clinical evidence suggests that high serum lactate levels are
   associated with activity and mortality in patients with sepsis.^[
   [127]^34 , [128]^35 , [129]^36 ^] We established a mouse model of
   SA‐AKI using cecal puncture ligation and observed a significant
   increase in serum lactate levels and serum creatinine and urate
   nitrogen levels (Figures [130]1A and [131]6A). In order to investigate
   the changes in histone lactylation at different time points of CLP, we
   performed western blot analysis of total protein collected from kidney
   tissue of mice with sepsis at different time points. The results showed
   that lactylation level was significantly up‐regulated 24 h after CLP
   and remained high until 72 h. The lactylation trend of H3K18la was
   consistent with the change in total lactylation level (Figure [132]
   6B). Similarly, immunohistochemistry at 24 h after CLP showed a
   significant increase in renal H3K18la levels, and the images showed
   that lactylation was mainly observed in the nucleus of renal proximal
   convoluted tubule epithelial cells (Figure [133]6C).

Figure 6.

   Figure 6
   [134]Open in a new tab

   GLUT1 inhibition reduces H3K18la and improves renal function impairment
   after SA‐AKI. A) Mouse CLP model diagram (Created with BioRender.com
   with permission) and mouse serum CREA and UREA levels in the Sham, CLP,
   and CLP+BAY‐876 groups. B) Western blotting analysis of Pan Kla and
   H3K18la in the total proteins extracted from the kidney tissues of the
   Sham group and 6 h, 24 h, 72 h after CLP (n  = 4 mice per group). C)
   Hematoxylin‐eosin staining (H&E) staining showing the degree of
   inflammatory cell infiltration. Immunohistochemical (IHC) staining
   showed the protein expression levels and sublocalization of Pan Kla,
   H3K18la, and RhoA in mouse kidneys (scale bar = 50 µm, n  = 6 mice per
   group). D) Western blotting analysis of Pan Kla and H3K18la in the
   total proteins extracted from the kidney tissues of the Sham group and
   CLP group ± BAY‐876 (n  = 4 mice per group). (E) Kidney tissue protein
   from Sham group and CLP group ±10 µg mL^−1 LPS for 24 h was evaluated
   by western blotting for the RhoA, p‐IKBα, IKBα, p‐NF‐κB, and NF‐κB
   protein levels. F) Serum cytokine concentrations were measured using a
   Mouse Inflammation Panel (13‐plex) with a V‐bottom Plate and
   demonstrated IL‐6 and TNF‐α levels (n  = 5 mice per group). G) Western
   blotting analysis of H3K18la in the total kidney tissue protein of the
   CLP group and CLP+LAC group (n  = 3 mice per group). ^* p <0.05 and ^**
   p <0.01 versus LPS group; ns, not significant versus LPS group.

   To evaluate the effect of histone lactylation on renal function in
   SA‐AKI models, BAY‐876 was intraperitoneally injected 24 h before
   surgery to control histone lactylation in proximal tubule epithelial
   cells. BAY‐876 treatment inhibited renal global lactylation and H3K18la
   levels, decreased RhoA protein expression and downstream IKBα and NF‐κB
   phosphorylation levels, and inhibited the expression of inflammatory
   factors IL‐6 and TNF‐α (Figure [135]6D,E,F). Hematoxylin and eosin
   staining and immunohistochemical staining confirmed that H3K18la and
   inflammatory cell infiltration were decreased in proximal convoluted
   renal tubule epithelial cells compared to those in the CLP group
   (Figure [136]6C). To determine whether increased lactate levels could
   modulate H3K18la levels during sepsis, we increased serum lactate
   levels via peritoneal injection of lactate 6 h after induced sepsis and
   examined changes in H3K18la levels. In contrast to BAY‐876, the H3K18la
   levels were significantly increased after lactic acid treatment
   (Figure [137]6G).

   Taken together, these results suggest that during SA‐AKI, increased
   H3K18la levels increase NF‐κB pathway activation and aggravate kidney
   injury, whereas a reduction in H3K18la has anti‐inflammatory effects
   with mitigation of kidney injury, contributing to the improvement of
   renal function after SA‐AKI.

2.7. Lactate Promotes Ezrin K263 Lactylation, and K263R Mutation Reverses the
Regulation of Ezrin‐Mediated Kidney Injury by Lactate

   CUT&Tag sequencing using anti‐H3K18la antibodies was performed in whole
   kidney tissue of SA‐AKI mouse models. DeepTools analysis showed that
   H3K18la peaks were significantly enriched in the CLP+lactate (CLP+LAC)
   group compared to those in the CLP group (Figure [138]S4A, Supporting
   Information). Genome Explorer tracked CUT&Tag signals at the
   representative target locus Ezrin, and these peaks identified elevated
   H3K18la levels in the Ezrin gene promoter region. Moreover, no
   significant change was observed in the chromatin accessibility of the
   Ezrin gene in ATAC‐seq (Figure [139]S4B, Supporting Information). To
   determine whether Ezrin can be lactated in cells, we transfected
   Ezrin‐overexpressing lentivirus into HK‐2 cells and added 30 mm lactate
   to the medium. Immunoprecipitation tests were then performed, Ezrin was
   pulled down with anti‐Ezrin antibodies, and Ezrin lactatation levels
   were detected with anti‐acetylation antibodies. We observed that Kla
   was indeed present on Ezrin, and the presence was significantly
   enhanced after lactic acid treatment (Figure [140] 7A). These results
   indicate that exogenous lactate supplementation promoted an increase in
   Ezrin lactylation levels.

Figure 7.

   Figure 7
   [141]Open in a new tab

   Lactate promotes K263 lactylation of Ezrin. A) Lactylation of Ezrin in
   HK‐2 cells was confirmed by the IP method. HK‐2 cells were treated with
   ddH2O or 30 mM L‐lactic acid for 24 h, followed by western blotting. B)
   Schematic diagram showing 4D labeled unlactated quantitative
   proteomics. Created with BioRender.com with permission. C) Illustration
   of possible lactylation sites of Ezrin in the CLP kidney tissue
   analyzed via immunoprecipitation (IP)‐mass spectrometry analysis. The
   six possible lactylation sites of Ezrin observed via IP‐mass
   spectrometry are shown. D) Molecular docking in the molecular operating
   environment (MOE) was used to predict the binding affinity between the
   Ezrin FREM domain and the L‐lactic acid molecule. E) Flag‐Ezrin (WT),
   Flag‐Ezrin (K3R), Flag‐Ezrin (K143R), Flag‐Ezrin (K162R), Flag‐Ezrin
   (K263R), and Flag‐Ezrin (K523R) were transfected into 293T cells for
   48 h via overexpression plasmid. Proteins obtained from the Flag‐Ezrin
   overexpressed 293T cells were pulled down by flag antibody and detected
   with anti‐lactyllysine antibody. F) A ribbon diagram of the crystal
   structure of human Ezrin protein ([142]P26040) was obtained from
   AlphaFold Protein Structure Database
   ([143]https://alphafold.ebi.ac.uk). We used Chimera1.17,1 software to
   visualize the protein structure and label the K263 site. G) The
   sequences around Ezrin K263 from different mammal species were
   conserved. Ezrin K263 conservative lysine residues are marked in red.
   H) Ezrin‐WT or Ezrin‐K263R site mutation in overexpressed HK‐2 cells
   was constructed via the lentiviral vector. Then, the cells were
   incubated with 10 µg/mL LPS ± 30 mm lactate for 24 h. Cell proteins
   were analyzed by WB for p‐IKBα, IKBα, p‐NF‐κB, NF‐κB, KIM‐1, and NGAL
   protein levels. n  =  3 independent experiments. ^* p <0.05 and ^**
   p <0.01; ns, not significant.

   PTMs plays a central role in the functional regulation of proteins, and
   lactic acid can directly modify proteins.^[ [144]^23 ^] The regulatory
   mechanism by which the lactated modification of Ezrin influences its
   function remains elusive. Next, we explored the underlying mechanism
   further. The kidney tissues of mice in the Sham group and CLP group
   were subjected to 4D label‐free quantitative proteomics
   with lactylation modification‐specific proteomics analysis
   (Figure [145]7B). No significant difference was observed in Ezrin
   protein levels in the proteomics analysis. However, liquid
   chromatography‐mass spectrometry (LC‐MS) analysis showed that there may
   be significant differences in lactylation sites K3, K143, K162, K263,
   K450, and K523 in Ezrin (Figure [146]7C). We used molecular docking in
   the molecular operating environment (MOE) to predict the binding
   affinity between L‐lactic acid molecules and the Ezrin FERM domain,
   predicting the site where the amino acid will be lactated
   (Figure [147]7D). It is generally believed that the interaction of the
   lysine residue K253/254/262/263 in the ezrin FERM domain with
   phosphatidylinositol, 4, 5‐diphosphate (PIP2) is essential for
   activation.^[ [148]^37 , [149]^38 ^] In this study, we mutated lysine
   to arginine for de‐lactate modification. By overexpressing
   Flag‐Ezrin‐WT plasmid into 293T cells, we constructed 293T cells
   mutated at K3R, K143R, K162R, and K523R due to poor species‐specific
   specificity of K450 sites. The results showed that the level of Flag in
   Ezrin overexpressed cells was significantly up‐regulated. Next, we
   pulled down Ezrin with Flag antibodies and detected altered lactate
   levels of Ezrin point mutations using anti‐lactylation antibodies. We
   found that mutations at K263R significantly reduced Ezrin lactylation,
   whereas mutations at other sites had no significant effect on
   lactylation levels (Figure [150]7E). The tertiary structure of Ezrin
   indicated that K263 was located in the FERM domain of the Ezrin protein
   (Figure [151]7F) and was conserved in a variety of mammalian species
   (Figure [152]7G). These results indicate that K263 is the leading
   lactylation site of Ezrin, and Anti‐Lactyla‐Ezrin (K263) antibody
   validation was performed (Figure [153]S4C–F, Supporting Information).

   Next, we attempted to investigate the regulatory role of Ezrin‐K263
   lactylation on the NF‐κB pathway by overexpressing Ezrin‐WT and
   Ezrin‐K263R mutations in HK‐2 cells. The addition of lactate increased
   NF‐κB activation levels and cell damage in HK‐2 cells to those observed
   in the Ezrin‐WT+LPS group. However, this effect was significantly
   diminished in K263R mutated cells. Compared with the Ezrin‐WT+LPS+LAC
   group, the Ezrin‐K263R+LPS+LAC group also showed lower levels of NF‐kB
   phosphorylation and alleviated cell damage (Figure [154]7H). These
   results support that lactate promoted LPS‐induced renal injury
   exacerbation via lactylation of Ezrin at K263, which may be a potential
   lactylation modification target.

3. Discussion

   Despite significant advances in understanding the pathophysiology and
   detection markers of SA‐AKI, it remains a common and hazardous
   complication of critical illness.^[ [155]^39 ^] The pathophysiology of
   SA‐AKI involves damage and dysfunction in many cell types, including
   macrophages, vascular endothelial cells (ECs), and RTECs, as well as
   their crosstalk and associations.^[ [156]^40 ^] Accumulating evidence
   suggests that the pathogenesis of SA‐AKI is multifactorial and complex,
   involving interactions between inflammation, microcirculatory
   dysfunction, and metabolic reprogramming. However, the mechanisms in
   terms of epigenetic regulation are not fully understood.^[ [157]^41 ^]
   This study investigated the changes in lactylation modifications in
   SA‐AKI and explored their relationship with glycolysis inhibitors.
   Despite the stability of global histone acetylation and H3K18ac, global
   histone lactylation and H3K18la showed a significant increase in
   SA‐AKI, which was markedly reduced upon the addition of glycolysis
   inhibitors. In previous studies, hypoxia‐induced lactate production in
   MCF‐7 cells increased the level of histone lactylation but did not
   elevate the level of histone acetylation.^[ [158]^23 ^] Moreover,
   hypoxia did not significantly alter the acetylation levels of H3K23ac
   and H3K18ac before embryo implantation.^[ [159]^42 ^] H3K18la and
   H3K18ac levels do not follow the same temporal dynamics. In liver
   stellate cell activation and hepatic fibrosis, despite an increase in
   lactate and acetyl‐CoA levels induced by the upregulation of glycolysis
   through HK2 expression, the impact was limited to histone lactylation
   and did not induce histone acetylation.^[ [160]^43 ^] Therefore, these
   studies suggest functional differences in the patterns of histone
   lactylation and acetylation.

   RhoA is a universally expressed cytoplasmic protein belonging to the
   small GTPase family that acts as a molecular switch, is activated in
   response to the binding of chemokines, cytokines, and growth factors,
   and can regulate the activation of cytoskeletal proteins and other
   factors via a ROCK signaling cascade.^[ [161]^44 ^] The RhoA/ROCK
   pathway can promote the progression of various diseases, such as
   inflammatory bowel disease,^[ [162]^29 ^] those involving microglia
   activation,^[ [163]^45 , [164]^46 ^] and acute lung injury.^[ [165]^47
   , [166]^48 ^] In a rat model of unilateral ureteral obstruction (UUO),
   extracellular vesicles produced by bone marrow mesenchymal stem cells
   mitigated renal fibrosis by inhibiting the RhoA/ROCK pathway.^[
   [167]^49 ^] The role of RhoA in SA‐AKI remains to be explored. This
   study demonstrated that the expression level of RhoA protein and
   phosphorylation level of ROCK1 downstream target protein MYPT1
   increased in SA‐AKI, and the inhibition of GLUT1 decreased the
   expression of RhoA protein.

   The Ezrin/radixin/moesin (ERM) protein family consists of an N‐terminal
   FERM domain (a dense clover‐shaped structure consisting of three
   structural modules F1, F2, and F3) and a C‐terminal domain containing
   an F‐actin binding site, connected by a central alpha helix
   structure.^[ [168]^50 , [169]^51 ^] The FERM domain is involved in
   binding membrane proteins and scaffold proteins.^[ [170]^52 , [171]^53
   ^] Ezrin is a member of the ERM protein family whose n‐terminal FERM
   domain interacts with the C‐terminal domain when dormant, whereas
   PtdIns (4,5) P[2] binds to the F3 lobe of the FERM domain.
   Subsequently, phosphorylation of the C‐terminal domain Thr567 residues
   of RhoA downstream effector ROCK leads to the transition from the
   inactive to the active form, and Ezrin is recruited from the cytoplasm
   to the membrane.^[ [172]^54 , [173]^55 ^] Y‐27632 is a ROCK inhibitor
   that blocks Ezrin phosphorylation and binds to the actin
   cytoskeleton.^[ [174]^56 ^] Based on the evidence indicating that
   Y‐27632 blocks Ezrin phosphorylation, this study further explored
   whether Ezrin bonded with MYD‐88 and IRAK1 in a LPS‐dependent manner to
   induce downstream NF‐κB activation and release of inflammatory factors,
   leading to the aggravation of kidney injury. We found that Ezrin may be
   partially involved in regulating glycolytic reprogramming and GLUT1
   sublocalization and expression.

   PTMs of proteins include acetylation, methylation, phosphorylation,
   ubiquitination, and glycosylation.^[ [175]^12 ^] Histone acylation is a
   crucial component of epigenetic regulation of gene transcription, and
   acetylation has been the first observed and most studied in renal
   diseases, including ischemia‐reperfusion injury‐induced AKI‐CKD
   transition model and unilateral ureteral obstruction‐induced renal
   fibrosis model.^[ [176]^57 , [177]^58 ^] Non‐histone acylation
   regulates protein functions such as protein activity, stability, and
   interactions. For example, p53 deacetylation can reduce acute kidney
   injury caused by sepsis by promoting autophagy,^[ [178]^59 ^] and
   SIRT1‐mediated HMGB1 deacetylation can inhibit sepsis‐related acute
   kidney injury.^[ [179]^60 ^] Recently, novel lysine acylation
   reactions, including propionylation, butyrylation, malonylation,
   succinylation, crotonylation, 2‐hydroxy‐butyrylation,
   β‐hydroxy‐butyrylation, glutarylation, and lactylation, have been
   extensively examined. As a potential modification substrate, lactate
   regulates epigenetic regulation of histones by introducing lactyl
   functional groups to histones. including H3K9,^[ [180]^61 ^] H3K14,^[
   [181]^62 ^] H3K18,^[ [182]^17 , [183]^63 ^] h3k23,^[ [184]^42 ^]
   H3K56,^[ [185]^61 ^] and H4K12,^[ [186]^62 , [187]^64 , [188]^65 ^]
   thereby regulating the transcription of specific genes. Subsequently,
   Non‐histone lactylation modifications have been reported, and they can
   also regulate the transcription of genes, such as those encoding
   HMGB,^[ [189]^66 ^] PKM2,^[ [190]^64 ^] HIF1α,^[ [191]^67 ^] Snail1,^[
   [192]^14 ^] YY1,^[ [193]^68 ^] and METTL3.^[ [194]^69 ^] H3K18la is
   involved in the expression of homeostatic genes related to damage
   repair, such as Arg‐1, playing a crucial role in the mechanisms of
   homeostasis and healing in vivo.^[ [195]^23 ^] In a single‐center,
   small‐sample clinical study on sepsis, researchers examined peripheral
   blood mononuclear cells from healthy volunteers and 35 critically ill
   patients (septic and nonseptic). The study revealed that H3K18la may be
   associated with the severity of critical illness and infection.
   Furthermore, H3K18la showed a positive correlation with the production
   of inflammatory factors.^[ [196]^70 ^] In a study by Pan et al.,
   enhanced H4K12 lactylation in AD model mice and microglia exacerbated
   the cognitive impairment phenotype via the glycolysis/H4K12la/PKM2
   axis.^[ [197]^64 ^] In contrast, H3K18la and H4K12la exhibited
   different localization and biological functions with distinct
   downstream targets in the pathogenesis of Alzheimer's disease. Lin
   et al. found that aging microglia undergo a metabolic shift to aerobic
   glycolysis regulated by the enhanced H3K18la/NFκB axis to modulate
   age‐related inflammation by regulating SASP components IL‐6 and IL‐8.^[
   [198]^71 ^]

   However, the changes and functions of these novel acetylations in the
   kidneys have not been extensively examined, especially since the role
   of lactylation modification in the kidneys has not been reported to
   date. We conducted the first exploration of the potential roles of
   histone lactylation in the development of SA‐AKI and generated the
   initial dataset of lactylation modifications on key enzymes in
   glycolysis and the tricarboxylic acid cycle in renal tissues of SA‐AKI
   (SFig 5). This dataset provides a foundation for further analysis of
   the physiological mechanisms underlying SA‐AKI. We observed an
   elevation in histone lactylation levels in SA‐AKI, at least partly
   mediated by histone lactylation regulating gene expression to modulate
   cellular metabolism, demonstrating the correction of abnormal lactate
   levels to effectively protect against renal function damage. Elevated
   histone lactylation promoted RhoA expression and induced
   phosphorylation and sublocation of RhoA/ROCK pathway. Notably, we
   demonstrated a significant increase in Ezrin lactylation after CLP We
   also identified multiple lactylation sites, with K263 defined as a
   potential lactylation target. Under in vitro conditions, an increase in
   lactate levels in LPS‐stimulated renal tubular epithelial cells,
   accumulation of endogenous lactate, and supplementation of exogenous
   lactate all led to an increase in Ezrin levels in renal tubular
   epithelial cells. This partially elucidates the association between
   high blood lactate levels and the high incidence and adverse prognosis
   of SA‐AKI. The increase in Ezrin may further promote inflammatory
   responses, primarily attributed to the interaction of Ezrin with MYD88
   and IRAK1, thereby activating the NF‐KB pathway. Additionally, mutation
   of the K263 site (K263R) was found to reverse the regulatory effects of
   lactate on Ezrin‐mediated renal injury. The findings of this study
   enhance our comprehension of Ezrin's regulatory mechanisms and further
   elucidate the physiological significance of lactate modification in
   renal diseases.

   Finally, in this study, SA‐AKI promoted the increase in lactic acid and
   H3K18la, activated the expression of RhoA protein, and mediated
   downstream inflammation and apoptosis, contributing to kidney injury.
   Conversely, inhibition of GLUT1 alleviated the increase in H3K18la,
   providing a new mechanistic foundation for delaying renal dysfunction.
   Our data suggest that glycolysis‐derived lactate and H3K18la may be
   potential targets for improving SA‐AKI renal function via inhibition of
   RhoA/ROCK/Ezrin signaling and Ezrin K263 lactylation mediated by
   reducing NF‐κB activation.

4. Experimental Section

Antibodies and Reagents

   LPS (L2630, E. coli 0111: B4) was purchased from Sigma–Aldrich (MO,
   United States). BAY‐876 (HY‐100017), 2‐DG (HY‐13966), L‐(+)‐lactic acid
   (Lactate, HY‐B2227), and Y‐27632 (HY‐10071) were obtained from
   MedChemExpress (Shanghai, China). Oxamic acid sodium (O7100) and
   Rotenone (C1866) were obtained from Lablead (Beijing, China).

   Anti‐L‐Lactyl Lysine (PTM‐1401RM), Anti‐Lactyl‐Histone H4 (Lys5) Rabbit
   mAb (PTM‐1407RM), Anti‐Lactyl‐Histone H4 (Lys8) Rabbit mAb
   (PTM‐1415RM), Anti‐Lactyl‐Histone H3 (Lys9) Rabbit mAb (PTM‐1419RM),
   Anti‐Lactyl‐Histone H4 (Lys12) Rabbit mAb (PTM‐1411RM),
   Anti‐Lactyl‐Histone H3 (Lys18) Rabbit mAb (PTM‐1406 and PTM‐1427RM),
   Anti‐Acetyllysine Rabbit mAb (PTM‐105RM), Anti‐Acetyl‐Histone H3
   (Lys18) Rabbit mAb (PTM‐114RM), and Anti‐Lactyl‐Ezrin (K263) Rabbit pAb
   were obtained from PTM Bio Inc. The glycolysis Antibody Sampler Kit
   (#8337) and the antibodies against GLUT1 (#73 015), phospho‐Ezrin
   (Thr567)/Radixin (Thr564)/Moesin (Thr558) (#3726), NF‐κB p65 (#8242),
   IκBα (#4814), Phospho‐IκBα (Ser32/36) (#9246) were obtained from Cell
   Signaling Technologies. Anti‐RhoA (sc‐418), anti‐MYPT1 (sc‐514261),
   phospho‐MYPT1 (sc‐377542), anti‐Ezrin (sc‐58758), anti‐MyD88
   (sc‐74532), anti‐IRAK1 (sc‐5288), anti‐phospho‐NF‐κB p65 (Ser536)
   (sc‐136548), and anti‐TIM‐1 (sc‐518008) were obtained from santa cruz
   biotechnology. The antibody anti‐LCN2 (26991‐1‐AP) and anti‐DYKDDDDK
   tag (66008‐4‐Ig) were from Proteintech. Anti‐β‐Actin (A0101) was
   obtained from Lablead. HRP‐conjugated Goat Anti‐Rabbit IgG (H + L)
   secondary antibody (S0101) and Goat Anti‐Mouse IgG (H + L) secondary
   antibody (S0100) were purchased from Lablead. Anti‐Histone H3 antibody
   (ab1791), Goat Anti‐Rabbit IgG H&L (Alexa Fluor 488) (ab150077), Goat
   Anti‐Mouse IgG H&L (ab150080), and Goat Anti‐Mouse IgG H&L (ab150116)
   were obtained from ABCAM.

Cell Lines and Cell Cultures

   The HK‐2 cell line (SCSP‐511) was purchased from Cell Resource Center
   (IBMS, CAMS/PUMC, China), and the following cell lines were obtained
   from American Type Culture Collection (ATCC): TCMK‐1 (CCL‐139) and
   HEK293T(CRL‐11268). All cell lines used in this study were
   authenticated by STR profiling, and the absence of mycoplasma
   contamination was confirmed. The HK2 and HEK293T cells were maintained
   in Dulbecco's modified Eagle's medium (DMEM, Gibco, MD, United States)
   containing 10% heat‐inactivated fetal bovine serum (FBS, ThermoFisher,
   United States). TCKM‐1 cells were cultured in Minimum Essential Medium
   (MEM, Gibco, MD, United States) supplemented with 10% FBS. Cells were
   maintained in an incubator with 5% CO[2] at 37 °C.

Dose‐Dependent Assay

   A 96‐well plate containing 100 µL of cell suspension and 2×103 cells
   per well was incubated in an incubator containing 5% CO2 at 37 °C for
   24 h for preculture. The culture plate was treated for three days with
   10 L of various drug concentrations containing varying amounts. Each
   well received 10 µL of CCK‐8 reagent (TargetMol, C0005) followed by
   incubation for 2 h in the incubator. The absorbance was calculated at
   450 nm using a microplate reader.

Oxidative Stress Markers Determination

   The HK‐2 cell supernatant was collected. Markers of oxidative stress,
   including superoxide dismutase (SOD, S0101S) and malondialdehyde (MDA,
   S0131S), were measured according to the manufacturer's instructions
   using a test kit purchased from Beyotime Biotechnology (Shanghai,
   China).

ROS Assay

   The HK‐2 cells were stimulated with 10 µg mL^−1 of LPS in a six‐well
   plate for 12 h. In the BAY‐876 group, cells were pretreated with
   BAY‐876 (30 nM) 3 h before LPS administration. The probe was then
   loaded on ROS Assay Kit (Beyotime Biotechnology, Shanghai, China,
   S0033S). DCFH‐DA was diluted in a serum‐free medium at a ratio of
   1:1000 to obtain a final concentration of 10 µmol L^−1. The cell
   culture medium was removed, and 1 mL of diluted DCFH‐DA was added.
   Subsequently, incubation was performed in a cell incubator at 37°C for
   20 min. The cells were washed thrice with serum‐free cell culture
   solution to remove the DCFH‐DA that did not completely penetrate the
   cells. The cells were collected and analyzed using a fluorescent enzyme
   label. The excitation and emission wavelengths of 488 and 525 nm were
   used to detect the intensity of fluorescence.

Apoptosis Assay

   The apoptosis rate of cultured HK‐2 cells was determined using flow
   cytometry with Annexin V‐FITC/PI (Dojindo Laboratories, AD10). Cell
   preparation was conducted in accordance with the manufacturer's
   instructions. The cells were simply rinsed once with cold PBS and then
   resuspended in 100 µL of 1 binding buffer. Then, 5 µL of Annexin V was
   added to the cell suspension. The cells were then gently mixed and
   incubated for 15 min at room temperature in the dark. After adding 5 µL
   of PI and 400 µL of 1x binding buffer, the cells were analyzed by flow
   cytometry for 1 h.

Lactic Acid Level Detection

   Kidney tissue, serum, or cultured HK2 cells were homogenized with lysis
   buffer solution and subjected to ultrasound on ice at 300 W (on for 3s,
   off for 7s) for 3 min, followed by centrifugation at 4 °C at 12,000 g
   for 10 min.^[ [199]^7 ^] The supernatant was collected, and the lactic
   acid concentration was determined using the Lactic Acid assay kit
   (Nanjing Jiancheng Bioengineering Institute, China, A019‐2‐1) per the
   manufacturer's instructions.

Western Blotting

   By adding 1% protease inhibitor (Shanghai Epizyme Biomedical Technology
   Co., Ltd., GRF101) and 1% phosphatase inhibitor (GRF102) to RIPA lysate
   (Applygen Technologies Inc., C1053‐100), total cell and tissue proteins
   were extracted. A membrane/cytoplasmic/nucleoprotein extraction kit
   (Beijing Solarbio Science & Technology Co. Ltd., EX1400) was used to
   isolate GLUT1 membrane protein. BCA protein assay kits (Invitrogen,
   23 227) were used to assess membrane and total protein concentrations.
   Bands from a 10% SDS‐PAGE gel were transferred to a PVDF membrane
   (Millipore, IPVH00010). The membranes were incubated with 5% skim milk
   with 0.05% Tween 20 (TBST) at room temperature for 1 h and then treated
   with primary antibody (1:1000 dilution) overnight at 4 °C. The
   membranes were rinsed thrice in TBST and then incubated with
   horseradish peroxidase‐coupled secondary antibody (1:5000). Finally, a
   1:1 Pierce ECL western blotting substrate (Millipore, WBKLS0500) was
   used to reveal proteins. The detected signals were quantified using the
   ImageJ software and normalized based on the following internal
   references: β‐actin (for total protein), histone H3 (for histone