Abstract

   The incidence of kidney stones is increasing worldwide. However, the
   underlying mechanism of the process of kidney stone formation and the
   kidney damage caused are not well understood. Here, it is observed that
   Lgals3, a β‐galactoside‐binding protein, is significantly increased in
   tissues with calcium oxalate (CaOx) stones, and in both in vivo and in
   vitro models. Lgals3 expression is positively correlated with the
   deposition of CaOx crystals. Knockout of Lgals3 markedly reduces the
   deposition of CaOx crystal and renal fibrosis in vivo. Furthermore,
   Lgals3 deficiency decrease the glycolytic rate and lactate production
   during the process of CaOx deposition and inhibited histone lactylation
   of H3K18la. Mechanistic studies shows that Lgals3 directly interacted
   with the key glycolysis protein pyruvate kinase M2 (PKM2) and promoted
   its expression by modulating E3 ligase Trim21, preventing the
   ubiquitination of PKM2. Furthermore, H3K18 lactylation promoted CaOx
   crystal deposition and kidney injury in vivo and in vitro. Lgals3
   deficiency inhibites the transcription, activation, and expression of
   FGFR4 through inhibition of H3K18la. These findings suggest that Lgals3
   may play a key role in CaOx stone formation and kidney injury by
   interacting with PKM2 and promoting both H3K18la‐mediated gene
   transcription and activation.

   Keywords: CaOx crystal, epigenetics, histone lactylation, kidney
   injury, Lgals3
     __________________________________________________________________

   This study indicates that Lgals3 plays a potential novel therapeutic
   target for the prevention of Calcium oxalate (CaOx) stone formation and
   subsequent renal fibrosis. Elevated Lgals3 interacted with PKM2 and
   promote the expression of FGFR4 via histone H3K18 lactylation, thereby
   facilitating CaOx crystal deposition and the development of renal
   fibrosis.

   graphic file with name ADVS-12-2413937-g008.jpg

1. Introduction

   Kidney stones are among the most prevalent urinary system diseases,
   with their incidence rising globally.^[ [44]^1 ^] While most patients
   opt for stone removal through surgery, studies have shown that this
   method has up to a 35% recurrence rate.^[ [45]^2 ^] Calcium oxalate
   (CaOx) stones account for approximately 75% of patients with kidney
   stones^[ [46]^3 ^] CaOx stone formation is primarily caused by an
   imbalance of crystallization, which involves a complex series of
   processes that begins with nucleation.^[ [47]^4 ^] The deposition of
   crystals in the kidney induces the production of reactive oxygen
   species and the aggregation of inflammatory factors, leading to the
   damage of renal tubular epithelial cells.^[ [48]^5 ^] The damaged cells
   exacerbate the aggregation of crystals, ultimately resulting in the
   formation of kidney stones, decline of renal function, and eventually
   renal fibrosis.^[ [49]^6 ^]

   Galactose lectin 3 (Lgals3) is a β‐galactoside‐binding lectin that
   binds to β‐galactosides via its carbohydrate‐recognition structural
   domain.^[ [50]^7 ^] Epidemiological research indicates that higher
   levels of plasma or serum Lgals3 are linked to reduced renal function
   and a heightened risk of developing chronic kidney disease (CKD).^[
   [51]^8 ^] In the presence of tissue damage, evidence suggests that
   Lgals3 can modulate a variety of glycosylated matrix proteins, such as
   laminin, fibronectin, and integrins, to promote cell adhesion and
   proliferation, leading to tissue fibrosis.^[ [52]^9 ^] In addition, a
   study found that Lgalse3 in a unilateral ureteral obstruction model
   promoted the M2 macrophage polarization that led to renal fibrosis.^[
   [53]^10 ^] However, the underlying mechanism of Lgals3 in CaOx kidney
   formation is unclear.

   The kidneys are rich in mitochondria that produce large quantities of
   ATP to provide energy for cellular processes, including renal transport
   and reabsorption.^[ [54]^11 , [55]^12 ^] Research has demonstrated the
   significance of metabolic reprogramming in acute kidney injury (AKI)
   and chronic kidney disease (CKD).^[ [56]^13 ^] Under renal ischemic and
   hypoxic conditions, fatty acid oxidation is inhibited, resulting in a
   reduction of ATP biosynthesis. In addition, the glycolytic pathway
   displays progressive upregulation in response to sustained renal
   injury.^[ [57]^14 ^] This metabolic shift exhibits a double‐edged role.
   Primarily, it provides the kidneys with essential ATP to maintain renal
   function. However, it can lead to renal lactic acid accumulation and
   lipid deposition, contributing to the progression of renal fibrosis.^[
   [58]^15 ^] Traditionally, lactic acid was considered a metabolic waste
   product of glycolysis during hypoxia. However, Zhao et al found that
   lactic acid plays an important role in posttranslational modifications
   by lactylating histone and non‐histone proteins.^[ [59]^16 ^] Histone
   lactylation controls downstream genes' transcription by modulating open
   regions of chromatin, similar to histone methylation and acetylation.^[
   [60]^17 , [61]^18 , [62]^19 ^] Previous studies that evaluated
   sepsis‐associated AKI models found that histone H3K18 lactylation was
   elevated and promoted the transcription of RhoA, leading to
   inflammation, cell apoptosis, and kidney dysfunction.^[ [63]^20 ^]
   However, the role of lactylation in CaOx crystal formation and renal
   fibrosis remains to be explored.

   In the present study, RNA‐seq, immunoprecipitation mass spectrum, and
   CUT&Tag were utilized. The results revealed that Lgals3‐mediated
   lactate generation promoted lactylation at H3K18, which contributed to
   FGFR4 expression, and promoted CaOx kidney stone formation and renal
   fibrosis. These findings suggest that Lgals3 and metabolic‐related
   lactylation may be potential therapeutic targets for patients with
   kidney stones and offer novel insights into the mechanism to prevent
   the development of stone formation.

2. Results

2.1. Lgals3 is Markedly Upregulated in Both Mouse and Human Kidney Stone
Specimens

   Kidney tissues from CaOx kidney stone patients and control kidney
   tissues from renal carcinoma patients were collected for RNA‐seq
   (Figure [64]S1A, Supporting Information). In total, 2728 differentially
   expressed genes (DEGs) were identified, with 1768 genes showing
   upregulation and 960 genes showing downregulation (Figure [65]S1B,
   Supporting Information). The Kyoto Encyclopedia of Genes and Genomes
   (KEGG) and Gene Ontology (GO) enrichment analysis based on the DEGs
   found that the upregulated genes were closely associated with
   extracellular matrix (ECM) and ECM‐receptor interaction (Figure
   [66]S1C,D, Supporting Information). Then, we established an CaOx stone
   mouse model (Figure [67]S1E, Supporting Information). HE, Von‐kossa
   staining and Western blot analysis successfully validated the
   establishment of the model (Figure [68]S1F—H, Supporting Information).
   We collected the kidney tissues and performed RNA‐seq and
   four‐dimensional independent data acquisition (4D‐DIA) proteomic
   analyses (Figure [69]S1I, Supporting Information).

   To explore the role of Lgals family in the process of kidney injury
   caused by CaOx crystal, individual members of the Lgals family were
   characterized and quantified based on the RNA‐seq and 4D‐DIA proteomic
   analyses. The results found that Lgals3 exhibited the most pronounced
   upregulation in the kidney from CaOx stone mice (Figure [70]1A–D).
   Immunofluorescence staining and Western‐blot analysis further confirmed
   increased Lgals3 levels in vivo and in vitro (Figure [71]1E–I).
   Meanwhile, we co‐stained the Lgals3 with proximal tubules and found
   that Lgals3 was mainly increased in proximal tubules (Figure [72]S2A,
   Supporting Information).

Figure 1.

   Figure 1
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   Expression and distribution of Lgals3 in both mouse and human CaOx
   stone specimens. A) heatmap for differentially expressed Lgals family
   member in the kidney tissues from NC mice and CaOx stone mice based on
   the RNA‐seq. B) heatmap for differentially expressed Lgals family
   member in the kidney tissues from NC mice and CaOx stone mice based on
   the DIA proteomic analysis. C) The expression of Lgals3 based on the
   result of RNA‐seq. D) The expression of Lgals3 based on the DIA
   proteomic analysis. E‐F) The immunofluorescence images and
   quantification of Lgals3 and α‐SMA levels in the kidney tissues from NC
   mice and CaOx stone mice (scale bar = 100 µm in first colcumn and 40 µm
   in second colcums, n = 5 mice per group). G) Immunoblots of the protein
   expression levels of Lgals3 in kidney tissues from NC mice and CaOx
   stone mice (n = 5 mice per group). H) Immunoblots of the protein
   expression levels of Lgals3 in HK‐2 cells treated with COM for 48h. I)
   Immunofluorescence images showing the Lgals3 level in HK‐2 cell with
   COM treated for 48h. J) Immunoblots of the protein expression levels of
   Lgals3 in kidney tissues from CaOx kidney stone patients (n = 10 per
   group). K) mRNA levels of Lgals1‐Lgals7 in kidney tissues from CaOx
   kidney stone patients (n = 10 per group). L,M) Immunohistochemical
   staining and quantification of Lgals3 in kidney tissues from CaOx
   kidney stone patients. (scale bar = 50 µm, n = 10 per group). N) The
   correlation between Lgals3 expression and kidney function (eGFR) in the
   clinical cohort. *P<0.01; **P<0.05.

   Then, a single‐nucleus atlas of the human nephrolithiasis database
   ([74]GSE231568) was used to test the expression of Lgals3. As is shown
   in the Figure [75]S2B (Supporting Information), the cell was divided
   into 9 different types such as proximal tubule cell, mastcell and
   endothelial. Then, we evaluated the Lgals3 expression and the results
   showed that Lgals3 was significantly increased in the kidney of
   nephrolithiasis patients (Figure [76]S2C—E, Supporting Information). In
   addition, the upregulation of Lgals3 was further confirmed in the
   kidneys of nephrolithiasis patients by western blot and qPCR
   (Figure [77]1J–K).To assess the clinical relevance of Lgals3 in CaOx
   stone, 10 kidney tissues from patients with CaOx kidney stone patients
   and 10 kidney tissues from radical nephrectomy for renal cell carcinoma
   were analyzed, showing a significant increase in Lgals3 expression in
   patients with kidney stone, which correlated closely with kidney
   function (Figure [78]1L–N).

2.2. Lgals3 Deficiency Inhibits Kidney Injury and Renal Fibrosis Caused by
CaOx Crystal

   To explore the role of Lgals3 in kidney injury caused by CaOx crystal,
   Lgals3 knockout (Lgals3^−/−) mice were generated (Figure [79]S3A,B,
   Supporting Information). qPCR, Western blot and immunohistochemistry
   staining were used to verify knockout efficiency (Figure [80]S3C—E,
   Supporting Information). Subsequently, a CaOx kidney stone model was
   established (Figure [81]2A). CaOx crystal deposition markedly elevated
   blood urea nitrogen (BUN) and creatinine levels, whereas Lgals3
   knockout ameliorated kidney function (Figure [82]2B,C). Histological
   assessment of kidney pathology by hematoxylin and eosin (HE) and Von
   Kossa staining showed that tubular injury and CaOx crystal deposition
   were significantly increased in the model group, whereas knockout of
   Lgals3 alleviated kidney injury and CaOx crystal formation
   (Figure [83]2D). Meanwhile, renal fibrotic structural alterations were
   evaluated, and Lgals3 knockout reduced collagen fiber deposition
   (Figure [84]2E,F). In addition, Inflammatory responses play a crucial
   role in the formation of calcium oxalate kidney stones. We found that
   CaOx crystals leads to an increase in the expression of inflammatory
   cytokines such as IL‐1,IL‐6 and TNF‐α (Figure [85]S3F, Supporting
   Information). Conversely, the knockout of Lgals3 results in a
   significant decrease in the expression of these inflammatory factors.

Figure 2.

   Figure 2
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   Lgals3 deficiency inhibits kidney injury and renal fibrosis caused by
   CaOx crystal. A) The schematic of the experimental design. B‐C) The BUN
   and Scr level in blood from WT mice and Lgals3 knockout (Lgals3^−/−)
   mice (n = 5 mice per group). D) Representative images and
   quantification of HE and Von‐kossa staining in kidney tissues from WT
   mice and Lgals3 knockout (Lgals3^−/−) mice (scale bar = 50 µm, n = 5
   mice per group). E) Representative images and quantification of Masson
   staining and the immunohistochemical staining of α‐SMA and Collagen1 in
   kidney tissues from WT mice and Lgals3 knockout (Lgals3^−/−) mice
   (scale bar = 50 µm, n = 5 mice per group). F) Immunoblots of the
   protein expression levels and quantification of Fibronectin, α‐SMA and
   Collagen1 in kidney tissues from WT mice and Lgals3 knockout
   (Lgals3^−/−) mice (n = 5 mice per group). **P<0.01, compared to the NC
   group; ^# P<0.05, compared with WT‐Stone mice.

   To identify the role of Lgals3 in vitro, a Lgals3‐knockdown HK‐2 cell
   line was established and western blot analysis confirmed that Lgals3
   knockdown was successful (Figure [87]S4A, Supporting Information). The
   HK‐2 cells were treated with COM for 48h to established the cell model.
   Immunofluorescence staining found that Lgals3 knockdown alleviated
   α‐SMA expression in COM‐treated HK‐2 cells (Figure [88]S4C, Supporting
   Information). Western blot analysis showed a significant decrease in
   fibrosis‐related protein expression under the stimulation of COM in
   vitro (Figure [89]S4D, Supporting Information). In addition, the
   immunofluorescence staining of ROS showed that COM significantly
   increased the level of ROS, while knockdown of Lgals3 decreased the
   level of ROS (Figure [90]S4E, Supporting Information). These results
   suggest that deficiency of Lgals3 inhibits CaOx crystal deposition and
   renal fibrosis in vivo and in vitro.

2.3. Lgals3 Overexpression Promotes Kidney Injury and Renal Fibrosis Induced
by CaOx Crystal

   To investigate whether Lgals3 promoted kidney injury and renal fibrosis
   caused by CaOx crystal, a kidney‐specific overexpression system that
   overexpressed Lgals3 was utilized in mice (Figure [91]S5A, Supporting
   Information). After four weeks of injection, the effectiveness of
   Lgals3 overexpression was confirmed by qPCR and Western blot (Figure
   [92]S5B,C, Supporting Information). Subsequently, a CaOx kidney stone
   model was established (Figure [93]3A). The serum BUN and Creatinine
   results showed that Lgals3 overexpression migrates the kidney injury
   (Figure [94]3B,C). HE and Von Kossa staining found that Lgals3
   overexpression aggravated kidney injury and CaOx crystal deposition
   (Figure [95]3D). In addition, pathology staining and Western blot
   analysis found that overexpression of Lgals3 significantly increased
   renal fibrosis and inflammation response caused by CaOx crystal
   deposition (Figure [96]3E,F; Figure [97]S5D, Supporting Information).

Figure 3.

   Figure 3
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   Lgals3 overexpression promotes kidney injury and renal fibrosis induced
   by CaOx crystal. A) The schematic of the experimental design. B,C) The
   BUN and Scr level in blood from AAV9‐NC mice and AAV9‐Lgals3 mice (n =
   5 mice per group). D) Representative images and quantification of HE
   and Von‐kossa staining in kidney tissues from AAV9‐NC mice and
   AAV9‐Lgals3 mice (scale bar = 50 um, n = 5 mice per group). E)
   Representative images and quantification of Masson staining and the
   immunohistochemical staining of α‐SMA and Collagen1 in kidney tissues
   from AAV9‐NC mice and AAV9‐Lgals3 mice (scale bar = 50 um, n = 5 mice
   per group). F) Immunoblots of the protein expression levels and
   quantification of Fibronectin, α‐SMA and Collagen1 in kidney tissues
   from AAV9‐NC mice and AAV9‐Lgals3 mice (n = 5 mice per group).
   **P<0.01, compared to the NC group; ^# P<0.05, compared with
   AAV9‐Ctrl‐Stone mice.

   An adenoviral vector encoding the Lgals3 gene was utilized in vitro to
   investigate the effects of Lgals3 overexpression. The overexpression
   efficiency was confirmed by Western blot (Figure [99]S4B, Supporting
   Information). Western blot analysis and immunofluorescence staining of
   ROS found that overexpression of Lgals3 significantly increased the
   expression of fibrosis‐related protein and ROS expression (Figure
   [100]S4F,G, Supporting Information). Taken together, these results
   indicate that Lgals3 overexpression can promote CaOx crystal deposition
   and renal fibrosis.

2.4. Lgals3 Regulates Glycolysis During the Process of Kidney Injury Caused
by CaOx Crystal

   To explore the underlying mechanism of Lgals3 in kidney injury caused
   by CaOx crystal, RNA‐seq and 4D‐DIA proteomic analysis was performed on
   Lgals3^−/‐ and WT mice that with CaOx crystal deposition. RNA‐seq
   results identified a total of 1932 DEGs, with 1005 exhibiting
   significant upregulation and 927 exhibiting significant downregulation
   (Figure [101]4A). The KEGG analysis showed that inflammation‐related
   and glycolysis‐related pathways were significantly enriched based on
   the DEGs, and the gene set enrichment analysis (GSEA) analysis showed
   that Lgals3 knockout inhibited glycolysis‐related genes at the RNA
   level (Figure [102]4B,C). Moreover, 4D‐DIA proteomic results showed a
   total of 437 DEPs, with an upregulation of 222 and a downregulation of
   215 proteins (Figure [103]4D). The KEGG and GSEA enrichment analysis
   also showed that glycolysis‐related pathways were significantly
   enriched, and were subsequently inhibited after Lgals3 knockout
   (Figure [104]4E,F). To validate these results, 2‐NDBG glucose uptake
   detection and lactate production assays were performed. The results
   indicated that the knockdown of Lgals3 reduced both glucose uptake and
   lactate production (Figure [105]4G,I). Conversely, Lgals3
   overexpression promoted glucose uptake and lactate production
   (Figure [106]4K–M). Meanwhile, Seahorse glycolytic rate analysis found
   that COM‐treated cells exhibited an increase in basic and compensatory
   glycolysis, whereas Lgals3 knockdown decreased the basic and
   compensatory glycolysis (Figure [107]4J). Conversely, overexpression of
   Lgals3 promoted glycolytic activity (Figure [108]4K). In addition, HK‐2
   cells exposed to COM showed that knockdown Lgals3 showed no
   significantly difference in basal respiration oxygen consumption rate,
   maximal respiration oxygen consummation rate, indicated that Lgals3
   knockdown do not affect oxidative phosphorylation (Figure [109]S6,
   Supporting Information).

Figure 4.

   Figure 4
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   Lgals3 regulates glycolysis during the process of kidney injury caused
   by CaOx crystal. A) Volcano plot for differentially expressed genes
   (DEGs) in the kidney tissues from WT mice and Lgals3 knockout
   (Lgals3^−/−) mice that with CaOx crystal deposition. (n = 5 mice per
   group). B) Bubble chart showing the KEGG pathway enrichment analysis of
   the DEGs. C) Gene Set Enrichment Analysis (GSEA) of WT and Lgals3^−/−
   mice from RNA‐seq data. D) Volcano plot for differentially expressed
   proteins (DEPs) in the kidney tissues from WT mice and Lgals3 knockout
   (Lgals3^−/−) mice (n = 5 mice per group). E) Bubble chart showing the
   KEGG pathway enrichment analysis of the DEPs. F) Gene Set Enrichment
   Analysis (GSEA) of WT and Lgals3^−/− mice from DIA proteomic data. G)
   The flow cytometry was used to monitor glucose uptake in Sh‐ctrl HK‐2
   and Sh‐Lgals3 HK‐2 cells incubated with 2‐NDBG. H,I) The glucose uptake
   and lactate production in Sh‐ctrl HK‐2 and Sh‐Lgals3 HK‐2 cells. J)
   Seahorse metabolic analysis to test the glycolysis in Sh‐ctrl HK‐2 and
   Sh‐Lgals3 HK‐2 cells. **P<0.01, compared to the Sh‐Ctrl group group; ^#
   P<0.05, compared with COM + Sh‐Ctrl group. K) The flow cytometry was
   used to monitor glucose uptake in Lv‐Vector HK‐2 and Lv‐Lgals3 HK‐2
   cells incubated with 2‐NDBG. L,M) The glucose uptake and lactate
   production in Lv‐Vector HK‐2 and Lv‐Lgals3 HK‐2 cells. N) Seahorse
   metabolic analysis to test the glycolysis in Lv‐Vector HK‐2 and
   Lv‐Lgals3 HK‐2 cells. **P<0.01, compared to the Lv‐Vector group; ^#
   P<0.05, compared with COM +Lv‐Vector group.

2.5. Lgals3 Interacts with PKM2 and Regulates PKM2 Level to Promote Lactate
Generation

   To determine the direct target of Lgals3 that regulates glycolytic
   function, whole‐cell lysates were subjected to immunoprecipitation (IP)
   using Flag antibodies. Protein bands were visualized utilizing silver
   staining and the products were analyzed using mass spectrometry (IP‐MS;
   Figure [111]5A–C). A total of 171 interacting proteins were identified
   which were cross‐referenced with the DEPs obtained in the 4D‐DIA
   proteomics sequencing, revealing 5 differentially expressed proteins
   (Slc16A7, PKM2, Nut, ABCC1, and Abhd5) that bind to Lgals3
   (Figure [112]5D). Among the Lgals3‐interacting proteins, PKM2 was of
   particular interest as it represented one of the key regulatory enzymes
   of the glycolytic pathway (Figure [113]5E). Molecular docking
   simulations of Lgals3 with PKM2 revealed that Lgals3 could binds to
   PKM2 (Figure [114]5F). Co‐IP assays were conducted in HK‐2 cells to
   further verify Lgals3 and PKM2 binding (Figure [115]5G). In addition,
   Co‐IP assays with epitope‐tagged proteins were conducted in HK‐2 and
   293T cells. Flag‐Lgals3 and His‐PKM2 were shown to co‐precipitate in
   293T and HK‐2 cells (Figure [116]5H,I). Moreover, Lgals3 and PKM2
   exhibited co‐localization in the kidney and HK‐2 cells
   (Figure [117]5J,K).

Figure 5.

   Figure 5
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   Lgals3 interacts with PKM2 and regulates PKM2 level to promote lactate
   generation. A) The schedule of IP‐MS. B) Silver staining showing IP
   products. C) The top8 proteins that binding to Lgals3. D) The overlap
   protein from the IP‐MS database and DIA protemic analysis. E) The
   peptide of PKM2 from IP‐MS. F) The molecular docking of Lgals3 and
   PKM2. G) Cell lysates of HK‐2 cells were immunoprecipitated with Lgals3
   or PKM2 antibodies, and immunoblot assays were performed. H) 293T cells
   were transfected with plasmids encoding Flag‐Lgals3 or His‐PKM2. Cell
   lysates were immunoprecipitated with Flag and HA antibodies and
   immunoblot assays were performed. I) HK‐2 cells were transfected with
   plasmids encoding Flag‐Lgals3 or His‐PKM2. Cell lysates were
   immunoprecipitated with Flag and HA antibodies and immunoblot assays
   were performed. J) Representative images of the immunofluorescence
   staining of Lgals3 (green) and PKM2 (red) in kidney tissues. K)
   Co‐localization of Lgals3 (green) and PKM2 (red) in the HK‐2 cells.

   The role of PKM2 in renal fibrosis caused by CaOx crystal was explored
   further. To investigate the functional role of PKM2, an siRNA‐mediated
   knockdown of PKM2 was employed, and the results demonstrated that
   inhibition of PKM2 alleviated renal fibrosis in vitro (Figure
   [119]S7A,B, Supporting Information). In addition, the western blot
   analysis confirmed that the expression of PKM2 was decreased after the
   knockout of Lgals3 in vivo and in vitro (Figure [120]S7C,D, Supporting
   Information). Then, a rescue assay was performed and found that Lgals3
   knockdown decreased the expression of fibrosis‐related proteins.
   However, the overexpression of PKM2 restored the expression of these
   proteins in vitro (Figure [121]S7E,F, Supporting Information).

2.6. Lgals3 Inhibits PKM2 Ubiquitination and Degradation by Interact with
Trim21

   Next, we explored how Lgals3 regulates PKM2. Using western blot and
   qPCR assays, we found that Lgals3 treatment increases the PKM2 protein
   level in HK‐2 and 293T cells, but had no impact on mRNA level (Figure
   [122]6A,B). Therefore, we hypothesized that Lgals3 may regulate PKM2
   protein levels via posttranslational modifications. To confirm this
   hypothesis, control HK‐2 cells and overexpressing Lgals3 HK‐2 cells
   were treated with cycloheximide, an inhibitor of protein synthesis. The
   results showed that Lgals3 overexpression prolonged the half‐life of
   PKM2 (Figure [123]6C,D). Subsequently, MG132, a proteasome inhibitor,
   was administered, which restored PKM2 protein levels in Lgals3
   knockdown HK‐2 cells (Figure [124]6E). However, the lysosomal inhibitor
   chiloroquine (CQ) and NH[4]CL do not restored PKM2 protein levels,
   indicating that Lgals3 regulates the expression of PKM2 through the
   ubiquitination pathway (Figure [125]6F). Further examination of
   ubiquitination levels was conducted in both in vivo and in vitro
   models. The findings demonstrated a significant decrease in PKM2
   ubiquitination levels in COM‐treated HK‐2 cells compared to the
   controls (Figure [126]6G). Similarly, in the CaOx stones mice model,
   the ubiquitination level of PKM2 was significantly decreased, which was
   restored by Lgals3 knockout (Figure [127]6H). In addition,
   overexpression or knockdown Lgals3 in HK‐2 cells decreased or increased
   PKM2 ubiquitination, respectively (Figure [128]6I,J). Hence, the above
   results indicated that Lgals3 regulates the expression of PKM2 through
   the ubiquitination pathway.

Figure 6.

   Figure 6
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   Lgals3 inhibits PKM2 Ubiquitination and degradation. A) Immunoblots of
   the protein expression levels of PKM2 in HK‐2 and 293T cells
   transfected with the indicated doses of plasmids encoding Flag‐Lgals3.
   **P<0.01, ***P<0.005, compared to the NC group. B) The mRNA level of
   PKM2 in HK‐2 and 293T cells transfected with the indicated doses of
   plasmids encoding Flag‐Lgals3. C) Cycloheximide (CHX) was used to block
   protein synthesis. Immunoblots of the protein expression levels and
   quantification of PKM2 in HK‐2 cells. D) Cycloheximide (CHX) was used
   to block protein synthesis. Immunoblots of the protein expression
   levels and quantification of PKM2 in 293T cells. E) Immunoblots of the
   protein expression levels and quantification of PKM2 in MG132 treated
   HK‐2 cells (n = 3 per group). F) Immunoblots of the protein expression
   levels and quantification of PKM2 in CQ and NH[4]CL treated HK‐2 cells
   (n = 3 per group). **P<0.01, compared to the Sh‐Ctrl group; ^# P<0.05,
   compared with Sh‐Lgals3 group. G) Detection of endogenous
   ubiquitination levels of PKM2 in COM treated HK‐2 cells. H) Detection
   of endogenous ubiquitination levels of PKM2 in kidney tissues of CaOx
   mice. I,J) Cells were pretreated with MG132 (5 um) for 8h. Detection of
   the ubiquitinated levels of PKM2 in Lgals3‐overexpression or
   Lgals3‐knockdown HK‐2 cells.

   Next, IP‐MS data collected in this study was subsequently searched to
   identify potential E3 ligases that interacted with Lgals3 and modulated
   PKM2 stability. Trim21 was identified as the sole E3 ligase that
   interacted with Lgals3 (Figure [130]5A,B). Co‐IP assays verified that
   Trim21 directly interacted with both Lgals3 and PKM2 (Figure
   [131]7A–D). Subcellular co‐localization of these proteins in HK‐2 cells
   also confirmed their relationship (Figure [132]7N). We found that
   overexpression of Trim21 decreased the protein level of PKM2 in a
   dose‐dependent manner, while had no impact on its mRNA level
   (Figure [133]7E,F). In contrast, knockdown of Trim21 significantly
   increased the protein level of PKM2, but had no effect on its mRNA
   level (Figure [134]7G). In addition, control HK‐2 cells and knockdown
   Trim21 HK‐2 cells were treated with cycloheximide and the results
   showed that Lgals3 overexpression prolonged the half‐life of PKM2
   (Figure [135]7H,I). Furthermore, the decrease of PKM2 protein level
   induced by Trim21 overexpression could be reversed by MG132, but CQ and
   NH[4]CL did not have the same effect (Figure [136]7J,K).

Figure 7.

   Figure 7
   [137]Open in a new tab

   Lgals3 inhibits PKM2 Ubiquitination and degradation by interact with
   Trim21. A‐D) Cell lysates of HK‐2 cells were immunoprecipitated with
   Flag, Myc and His antibodies, and immunoblot assays were performed. E)
   Immunoblots of the protein expression levels of PKM2 in HK‐2 and 293T
   cells transfected with the indicated doses of plasmids encoding
   Myc‐Trim21. F) The mRNA level of PKM2 in HK‐2 and 293T cells
   transfected with the indicated doses of plasmids encoding Myc‐Trim21.
   G) Immunoblots of the protein expression levels and quantification of
   PKM2 in Trim21 knockdown HK‐2 cells (n = 3 per group). H) Cycloheximide
   (CHX) was used to block protein synthesis. Immunoblots of the protein
   expression levels and quantification of PKM2 in HK‐2 cells. I)
   Cycloheximide (CHX) was used to block protein synthesis. Immunoblots of
   the protein expression levels and quantification of PKM2 in 293T cells.
   J) Immunoblots of the protein expression levels and quantification of
   PKM2 in MG132 treated HK‐2 cells (n = 3 per group). **P<0.01, compared
   to the Myc‐Ctrl group; ^# P<0.05, compared with Myc‐Trim21 group. K)
   Immunoblots of the protein expression levels and quantification of PKM2
   in CQ and NH[4]CL treated HK‐2 cells (n = 3 per group). L‐M) HK‐2 and
   293T cells were pretreated with MG132 (5 um) for 8h. Detection of the
   ubiquitinated levels of PKM2 in Trim21‐overexpression or
   Trim21‐knockdown cells. N) Co‐localization of Lgals3, PKM2 and Trim21
   in HK‐2 cells.

   Then, we assessed the effect of Trim21 on the ubiquitination level of
   PKM2 and the results showed that overexpression of Trim21 enhanced PKM2
   ubiquitination, whereas Trim21 knockdown inhibited this process in HK‐2
   and 293T cells (Figure [138]7L,M). These results indicate that Lgals3
   inhibits PKM2 Ubiquitination and degradation by interact with Trim21.

2.7. Histone Lactylation is Enhanced during CaOx Crystal Formation

   Lactylation is a recently discovered epigenetic modification that
   regulates gene expression. In the current study, global lactylation
   levels were determined in HK‐2 cells under the stimulation of COM. The
   results showed that global lactylation levels were elevated, and the
   predominant band observed was 17 kDa, which corresponded to histone H3
   (Figure [139]8A). Next, various lactylation sites on histone H3 and
   histone H4 were examined, and the level of H3K18 lactylation (H3K18la)
   increased significantly after COM treatment (Figure [140]8B,C; Figure
   [141]S8A,B, Supporting Information). Immunofluorescence analysis also
   indicated that COM‐stimulated HK‐2 cells exhibited increased levels of
   H3K18la compared to the controls (Figure [142]8D). Immunofluorescence
   was utilized to examine global lactylation and H3K18la levels in CaOx
   stone mouse model kidney tissues. The analysis confirmed that both
   global lactylation and H3K18la levels were significantly increased in
   vivo (Figure [143]8E,F).

Figure 8.

   Figure 8
   [144]Open in a new tab

   Histone lactylation promotes CaOx crystal formation and renal fibrosis.
   A) Immunoblots of the protein expression levels and quantification of
   Lacty‐lysine in HK‐2 cells with COM treated for 48h (n = 3 per group).
   B,C) Immunoblots of the protein expression levels and quantification of
   H3K9la, H3K14la, H3K18la and H3K56la in HK‐2 cells with COM treated for
   48h (n = 3 per group). D) Immunofluorescence images showing the H3K18la
   level in HK‐2 cell with COM treated for 48h (n = 3 per group). E)
   Representative images and quantification of the immunofluorescence
   staining of Lacty‐lysine and α‐SMA in kidney tissues from NC mice and
   CaOx mice (scale bar = 50 um, n = 5 mice per group). F) Representative
   images and quantification of the immunofluorescence staining of H3K18la
   and α‐SMA in kidney tissues from NC mice and CaOx mice (scale bar = 50
   um, n = 5 mice per group). **P<0.01, compared to the NC group. G) The
   schematic of the experimental design. H) Representative images and
   quantification of the von‐kossa staining and the immunofluorescence
   staining of α‐SMA in kidney tissues from mice treated with Lac (scale
   bar = 50 um, n = 5 mice per group). **P<0.01, compared to the Stone
   group. I) Immunoblots of the protein expression levels and
   quantification of H3K18la in HK‐2 cells with Lac treated for 48h (n = 3
   per group). J) Immunofluorescence images showing the α‐SMA level in
   HK‐2 cell with Lac treated for 48h (n = 3 per group). **P<0.01,
   compared to the COM group.

2.8. Histone Lactylation Promotes CaOx Crystal Formation and Renal Fibrosis

   To assess the effects of histone lactylation on CaOx crystal formation
   and renal fibrosis, mice were intraperitoneally injected with sodium
   lactate to induce histone lactylation (Figure [145]8G). As shown in
   Figure [146]8H, lactate supplementation increased CaOx crystal
   deposition and renal fibrosis, evidenced by Von Kossa and
   immunofluorescence staining. In vitro, the supplementation of lactate
   contributed to increased H3K18la levels and α‐SMA expression under COM
   treatment (Figure [147]8I,J). Additionally, mice were injected
   intraperitoneally with FX‐11, a specific inhibitor of LDHA, to suppress
   histone lactylation (Figure [148]9A). The pathological staining showed
   that inhibition of histone lactylation alleviated CaOx crystal
   deposition and renal fibrosis (Figure [149]9B). Consistent with the
   results of the mouse model, the addition of FX‐11 decreased the H3K18la
   levels and α‐SMA expression after COM treatment (Figure [150]9C,D).

Figure 9.

   Figure 9
   [151]Open in a new tab

   Inhibition of Lgals3 reduces H3K18la during the formation of CaOx
   crystal. A) The schematic of the experimental design. B) Representative
   images and quantification of the von‐kossa staining and the
   immunofluorescence staining of α‐SMA in kidney tissues from mice
   treated with FX‐11 (scale bar = 50 um, n = 5 mice per group). **P<0.01,
   compared to the Stone group. C) Immunoblots of the protein expression
   levels and quantification of H3K18la in HK‐2 cells with FX‐11 treated
   for 48h (n = 3 per group). D) Immunofluorescence images showing the
   α‐SMA level in HK‐2 cell with FX‐11 treated for 48h (n = 3 per group).
   E) Immunoblots of the protein expression levels and quantification of
   Lacty‐lysine in Lgals3 knockdown HK‐2 cells with COM treated for 48h (n
   = 3 per group). F) Immunoblots of the protein expression levels and
   quantification of H3K9la, H3K14la, H3K18la and H3K56la in Lgals3
   knockdown HK‐2 cells with COM treated for 48h (n = 3 per group). G)
   Immunofluorescence images showing the H3K18la level in Lgals3 knockdown
   HK‐2 cell with COM treated for 48h (n = 3 per group). **P<0.01,
   compared to the COM group. H) Representative images and quantification
   of the immunofluorescence staining of Lacty‐lysine and α‐SMA in kidney
   tissues from WT mice and Lgals3 knockout mice (scale bar = 50 um, n = 5
   mice per group). I) Representative images and quantification of the
   immunofluorescence staining of H3K18la and α‐SMA in kidney tissues from
   WT mice and Lgals3 knockout mice (scale bar = 50 um, n = 5 mice per
   group). **P<0.01, compared to the WT+Stone group. J) Immunoblots of the
   protein expression levels and quantification of Lacty‐lysine and
   H3K18la in kidney tissues from WT mice and Lgals3 knockout (Lgals3^−/−)
   mice (n = 5 mice per group). **P<0.01, compared to the NC group; ^#
   P<0.05, compared with WT‐Stone mice.

2.9. Inhibition of Lgals3 Reduces H3K18la During CaOx Crystal Formation

   Furthermore, this study investigated whether Lgals3 deficiency reduced
   H3K18la during CaOx deposition. Western blot and immunofluorescence
   staining showed that Lgals3 knockdown in vitro decreased the levels of
   global lactylation and H3K18la levels (Figure [152]9E–G). Similarly,
   Lgals3^−/− mice exhibited significant decreases in global lactylation
   and H3K18la levels compared with the WT mice through Western blot and
   immunofluorescence staining (Figure [153]9H–J). Meanwhile, we also
   tested the level of acetylation of histone H3 and histone H4 and the
   results showed that Lgals3 deficiency have no impact on the expression
   of acetylation of histone H3 and histone H4 (Figure [154]S9, Supporting
   Information)

2.10. Lgals3‐Mediated H3K18la Targets Multiple Fibrosis‐Related Genes

   Previous studies have shown that histone lactylation may mediate gene
   expression. This study employed cleavage under targets and tagmentation
   (CUT&Tag) targeting H3K18la to characterize genome‐wide changes of
   histone modifications. Analysis of the CUT&Tag signal enrichment data
   revealed a distinct heatmap pattern demonstrating that Lgals3 knockdown
   significantly decreased the H3K18la enrichment signal around the
   transcription start sites (Figure [155]10A,B). Meanwhile, global
   mapping analysis showed that the H3K18la sites were primarily enriched
   at the promoter regions (Figure [156]10C). A volcano plot analysis
   identified a total of 48661 genetic loci bound by H3K18la, of which 907
   were downregulated and 287 were upregulated in the Lgals3 knockdown
   cells compared with the WT HK‐2 cells (Figure [157]10D). The GO
   enrichment analysis of the target genes associated with the
   H3K18la‐binding peaks showed significant enrichment in processes
   related to tight junctions and focal adhesions (Figure [158]10E).

Figure 10.

   Figure 10
   [159]Open in a new tab

   Lgals3‐mediated H3K18la targets multiple fibrosis‐related genes. A,B)
   The binding density of H3K18la in the transcriptional start site was
   visualized. C) Genome‐wide distribution of differentiated
   H4K12la‐binding peaks in Sh‐Ctrl HK‐2 cell and Sh‐Lgals3 HK‐2 cells. D)
   Volcano plot for different genetic loci bound by H3K18la in CUT‐tag. E)
   Bubble chart showing the KEGG pathway enrichment analysis of the
   H3K18la binding DEGs. F) Venn diagram showed genes downregulated in WT
   and Lgals3 knockout mice and the downregulated target genes bound by
   H3K18la. G) Integrative Genomics Viewer analysis representing H3K18la
   peaks at the FGFR4 locus. H) Chip‐qPCR assays of H3K18la in the FGFR4
   in HK‐2 cells. I) Immunoblots of the protein expression levels and
   quantification of FGFR4 in kidney tissues from WT mice and Lgals3
   knockout (Lgals3^−/−) mice (n = 5 mice per group). **P<0.01, compared
   to the NC group; ^# P<0.05, compared with WT‐Stone mice. J) Immunoblots
   of the protein expression levels and quantification of FGFR4 in Lgals3
   knockdown HK‐2 cells with COM treated for 48h (n = 3 per group).
   **P<0.01, compared to the Sh‐Ctrl group; ^# P<0.05, compared with
   COM+Sh‐Ctrl group. K) Immunofluorescence images showing the α‐SMA level
   in Lgals3 overexpression HK‐2 cells with BLU9931 treated for 48h (n = 3
   per group). L) Immunoblots of the protein expression levels and
   quantification of Collagen1 and α‐SMA in Lgals3 overexpression HK‐2
   cells with BLU9931 treated for 48h (n = 3 per group). **P<0.01,
   compared to the Lv‐Vector group; ^# P<0.05, compared with Lv‐Lgals3
   group.

   To investigate the target gene of H3K18la as a result of Lgals3
   deficiency, integrative CUT&Tag and RNA‐seq analysis were utilized.
   Four overlapping genes were identified: FGFR4, NOX4, Wnt3, and CDH4
   (Figure [160]10F), among which, FGFR4 exhibited the strongest
   association with fibrosis. Integrative Genomics Viewer analysis of the
   H3K18la CUT&Tag analysis indicated that H3K18la binds to the FGFR4
   promoter region (Figure [161]10G). In addition, Chip‐qPCR analysis
   confirmed that H3K18la was significantly enriched on the FGFR4
   promoter, whereas Lgals3 knockdown decreased this enrichment
   (Figure [162]10H). Concomitantly, qRT‐PCR and Western blot analysis
   found that the mRNA and protein level of FGFR4 was significantly
   decreased in Lgals3 deficient mice and in Lgals3 knockdown cells
   (Figure [163]10I,J).

   To determine whether activation of FGFR4 is required for the
   pro‐fibrotic effects of Lgals3, an FGFR4 inhibitor, BLU9931, was used
   in vitro after COM treatment. The increase in α‐SMA expression induced
   by Lgals3 overexpression was significantly ameliorated after the
   treatment of BLU9931 (Figure [164]10K). In addition, Western blot
   analysis corroborated the findings of the immunofluorescence staining
   (Figure [165]10L).

2.11. Pharmacological Inhibition of Lgals3 Ameliorates Kidney Injury and
Renal Fibrosis Caused by CaOx Crystal

   To affirm the therapeutic potential of Lgals3, wild type CaOx stone
   mice were treated with MCP, an inhibitor of Lgals3 (Figure [166]11A).
   The BUN and Scr results showed that MCP administration alleviated
   kidney injury caused by CaOx crystal (Figure [167]11B). Histological
   results showed that MCP significantly alleviated kidney injury, CaOx
   crystal deposition and renal fibrosis (Figure [168]11C,D). In addition,
   western blot analysis affirmed that downregulation of PKM2, FGFR4,
   α‐SMA and Collagen1 after MCP injection. Furthermore, MCP decreased the
   global and histone H3K18 lactylation level (Figure [169]11E,F).

Figure 11.

   Figure 11
   [170]Open in a new tab

   Pharmacological inhibition of Lgals3 ameliorates CaOx crystal formation
   and renal fibrosis. A) The schematic of the experimental design. B) The
   BUN and Scr level in blood from Caox stone mice and CaOx stone+MCP mice
   (n = 5 mice per group). C,D) Representative images and quantification
   of HE, Von‐kossa and α‐SMA staining in kidney tissues from Caox stone
   mice and CaOx stone+MCP mice (scale bar = 50 um, n = 5 mice per group).
   E,F) Immunoblots of the protein expression levels and quantification of
   PKM2, Lacty‐lysine, H3K18la, FGFR4, α‐SMA and Collagen1in kidney
   tissues from Caox stone mice and CaOx stone+MCP mice (n = 5 mice per
   group). **P<0.01, compared to the NC group; ^# P<0.05, compared with
   Stone group.

   Meanwhile, to verify the clinical potential of targeting Lgals3 for
   treatment of kidney stones, we established another CaOx crystal mouse
   model according our previous studies by injecting glycolic acid for
   consecutively 12 days and inhibited Lgals3 through MCP and GB1107
   (Figure [171]S10A, Supporting Information). The results demonstrated
   that MCP and GB1107 also inhibited the activation of H3K18la in the
   kidneys of the acute calcium oxalate crystal deposition mice and
   alleviated both crystal deposition and kidney damage (Figure
   [172]S10B—E, Supporting Information).

2.12. Lgals3 May be Key Factors Involved in CaOx Stone Patients

   To determine whether serum Lgals3 levels are associated with kidney
   injury and fibrosis in human patients, we measured serum Lgals3 levels
   in 10 healthy participants and 10 nephrolithiasis patients (Figure
   [173]12A,B). The level of Lgals3 was elevated in nephrolithiasis
   patients compared with healthy participants (Figure [174]12C).
   Furthermore, the HK318la level was also significantly increased in
   nephrolithiasis patients (Figure [175]12D,E). These results provide
   strong evidence that Lgals3 play a key role in the kidney injury caused
   by CaOx crystal.

Figure 12.

   Figure 12
   [176]Open in a new tab

   Lgals3 may be key factors involved in CaOx stone patients. A–C) Graphic
   presentation shows serum Lgals3 levels in a cohort of patients with
   nephrolithiasis (n = 10) and healthy participants (n = 10). D,E) The
   H3K18la level in the kidney tissues from CaOx stone patients. **P<0.01
   compared to the NC group.

3. Discussion

   Previous studies have found that Lgals3 plays a crucial role in
   inflammation, immunity, injury, and fibrosis.^[ [177]^21 , [178]^22 ,
   [179]^23 ^] The result of the present study revealed that Lgals3
   exhibited several novel functions and mechanisms in the formation of
   kidney stones and the development of renal fibrosis (Figure [180]13 ).
   It was found that Lgals3 was highly expressed in CaOx crystal
   deposition and stimulated the activation of glycolysis during crystal
   deposition and renal fibrosis. Knockout or pharmacological inhibition
   of Lgals3 demonstrated a significant reduction of crystal deposition
   and renal fibrosis. In addition, IP‐MS analysis identified PKM2, a key
   molecule in the regulation of glycolytic function, as the direct
   binding target of Lgals3. Furthermore, this study integrated analyses
   of CUT&Tag and RNA‐seq and demonstrated that Lgals3‐mediated histone
   lactylation promoted FGFR4 expression during the formation of CaOx
   stones and renal fibrosis.^[ [181]^24 ^] Lgals3 is considered a
   disease‐associated biomarker and it is significantly increased in cases
   of acute myocardial infarction or AKI.^[ [182]^25 , [183]^26 ^]
   Furthermore, recent investigations have shown that it has significant
   potential as a therapeutic target for various inflammatory and fibrotic
   illnesses, including lung or kidney fibrosis.^[ [184]^27 ^] In this
   study, it was found that Lgals3 expression was increased in both mouse
   and human CaOx crystal kidney tissues. This study utilized Lgals3^−/‐
   mice and demonstrated that Lgals3 deficiency alleviated CaOx crystal
   deposition and renal fibrosis. The deposition of CaOx crystals and the
   development of renal fibrosis are complex processes regulated by
   numerous genes and signaling pathways. RNA‐seq and 4D‐DIA proteomics
   were performed to detect alterations in mRNA and protein expression in
   Lgals3‐deficient cells under COM stimulation. The KEGG analysis showed
   that Lgals3 deficiency primarily enriched metabolic‐related pathways,
   specifically glycolysis. When cells are exposed to various stimuli, the
   mitochondrial energy metabolism undergoes alterations, leading to
   significant activation of glycolysis, which in turn increases the
   overall energy supply.^[ [185]^28 ^] Yang et al have suggested that the
   knockout of phosphofructokinase 1 (PFKP), an enzyme in glycolysis, in
   renal tubular epithelial cells inhibited the TGF‐beta pathway, thereby
   alleviating renal fibrosis.^[ [186]^29 ^] In addition, a recent study
   reported that Lgals3 was overexpressed in several metabolic conditions,
   such as diabetes, obesity, and atherosclerosis.^[ [187]^30 ^] In the
   current study, it was found that glycolysis was activated after COM
   stimulation. Further analysis using the Seahorse glycolytic rate
   confirmed that the knockdown of Lgals3 inhibited the glycolytic flux
   and reduced glucose uptake and lactate production, suggesting that
   Lgals3 plays a key role in regulating glycolytic metabolism.

Figure 13.

   Figure 13
   [188]Open in a new tab

   Schematic representation of the mechanism of Lgals3 in CaOx stone
   formation. Elevated Lgals3 interacted with PKM2 and promoted the
   expression of FGFR4 via H3K18la, thereby facilitating CaOx crystal
   deposition and the development of renal fibrosis.

   Pyruvate kinase M2 (PKM2) is a crucial enzyme in the glycolysis pathway
   that controls the rate at which phosphopyruvate is converted into
   pyruvate and ATP by phosphorylation.^[ [189]^31 ^] Xie et al found that
   PKM2 regulated mitochondrial homeostasis in cisplatin‐induced acute
   kidney injury by binding to MYH9 to promote dynamin‐related protein
   1‐mediated mitochondrial fragmentation, alleviating renal tubular
   injury and cell death.^[ [190]^32 ^] In the present study, it was found
   that the expression of PKM2 was significantly increased in both in vivo
   and in vitro models, and the knockdown of PKM2 attenuated CaOx
   crystal‐induced renal fibrosis. In addition, further analysis showed
   that Lgals3 promoted PKM2 protein levels by inhibiting ubiquitination
   and degradation by modulating Trim21. The ubiquitin‐proteasome system
   (UPS) selectively degrades the majority of cellular proteins through an
   ATP‐driven process.^[ [191]^33 ^] UPS protein degradation has been
   implicated in various kidney diseases, such as AKI, diabetic
   nephropathy, and CKD.^[ [192]^34 ^] Data from this study suggest that
   Lgals3 directly interacted with the E3‐ubiquitin ligase Trim21 and
   prevented PKM2 from undergoing Trim21‐mediated ubiquitination and
   degradation. These results highlight the importance of the
   Lgals3‐Trim21‐PKM2 axis in the pathogenesis of renal fibrosis and
   kidney stone formation.

   Lactate was traditionally considered a metabolic waste product produced
   during glycolysis.^[ [193]^35 ^] However, previous studies have found
   that lactate functions as a precursor which mediate the modification of
   histone and non‐histone gene expression.^[ [194]^36 ^] For instance,
   previous research suggests that H4K12 lactylation was enriched in the
   promoter of the NF‐KB signaling pathway, which subsequently activated
   transcription in kidney tubular cells and aggravated kidney
   inflammation and fibrosis.^[ [195]^37 ^] Observations in the present
   study found increased global lactylation and histone lactylation
   levels, particularly H3K18la, during the process of CaOx crystal
   deposition. Conversely, Lgals3 deficiency inhibited H3K18la activity. A
   combination of H3K18la‐targeted CUT&Tag and RNA‐seq analyses identified
   FGFR4 as the key downstream target gene of H3K18la. Additionally,
   H3K18la was significantly enriched in the promoter region of FGFR4,
   suggesting that this histone modification may play a role in activating
   its transcription. FGFR4 is a member of the fibroblast growth factor
   receptor family and plays a key role in cell survival, proliferation,
   and migration.^[ [196]^38 ^] Previous studies found that FGF23 binds to
   FGFR4 and increases kidney inflammation. The current study found that
   FGFR4 was significantly increased and is essential for Lgals3‐mediated
   CaOx crystal deposition and the development of renal fibrosis.

   In conclusion, this study demonstrated the significant role of Lgals3
   in the process of CaOx crystal deposition and stone formation. Elevated
   Lgals3 interacted with PKM2 and promoted the expression of FGFR4 via
   H3K18la, thereby facilitating CaOx crystal deposition and the
   development of renal fibrosis (Figure [197]11). These findings provide
   a potential novel therapeutic target for the prevention of CaOx stone
   formation and subsequent renal fibrosis.

4. Experimental Section

Clinical Kidney Tissues

   The non‐function human kidney samples were collected from patients
   diagnosed with kidney stone and control samples from patients diagnosed
   with renal cell carcinoma in Renmin Hospital of Wuhan University
   between 2022‐1‐1 and 2024‐1‐1, after obtaining approval from the
   hospital's Ethics Committee (WDRY2023‐KS033). Normal samples were
   obtained from healthy kidneys of individuals who underwent tumor
   nephrectomies without other kidney diseases. The clinical information
   of the patients was presented in Table [198]S1 (Supporting
   Information).

Animal Studies

   All experimental procedures received approval from the ethics committee
   at Renmin Hospital, Wuhan University (WDRM‐2023070). Lgals3 knockout
   mice (Lgals3^−/− mice) were obtained from the Cyagen (Suzou, China).
   The male C57/BL6 mice were obtained from the Center of Experimental
   Animals at Wuhan university. All mice were kept in 20–22 with a 12‐hour
   light‐dark cycle. The control group (C57BL/6J mice) and experimental
   group (Lgals3^−/− mice) mice used in the study were littermates.

   Two different types of CaOx crystal mice models, including calcium and
   1,25(OH)[2]D[3]‐induced CaOx crystal formation and Glyoxylate acid
   (Gly)‐induced CaOx crystal formation were used in this study.

   For calcium and 1,25(OH)[2]D[3]‐induced CaOx crystal mice model. The
   mice were given free access to food containing 0.73% calcium and 600IU
   1,25(OH)[2]D[3] and drinking water containing calcium (2g L^−1) for a
   period of 3 months to established the CaOx stone model.

   For Gly‐induced CaOx crystal mice model. The mice were received an
   intraperitoneal injection of Gly (120mg k^−1g d^−1) for 12 consecutive
   days to established the CaOx stone model.

Renal Function

   Prior to sacrifice, blood samples were harvested from the orbital sinus
   of the mice. The collected samples were then analyzed utilizing an
   automated biochemical analyzer (Beckman, USA) to assess serum Cr, and
   BUN levels.

AAV9 Virus Injection

   The AAV9 virus were provided by Jiman Biotechnologies. The AAV9
   carrying Lgals3 gene with KSP promoter (AAV9‐Lgals3) or the control
   virus (AAV9‐vector) were injected into 5 different sites of the renal
   cortex according to the manufacture instruction. One month later,
   kidney were collected to tested the overexpression efficiency.

RNA‐Sequencing (RNA‐seq)

   This study used the kidney tissues to obtain total RNA for RNA‐Seq
   analysis. The RNA extraction process involved the use of Trizol
   reagent, followed by the purification of poly(A) RNA. This purified RNA
   was then used to form a cDNA library. The sequencing of all samples was
   performed using the Illumina HiSeq X Ten platform.

4D‐DIA Proteomic Sequencing

   Kidney tissues were combined with a solution containing 8 M Urea and
   100 mM Tris‐Cl, followed by treatment with water bath sonication.
   Proteins were reduced and alkylated using TCEP and CAA at 37 °C for 1
   hour. The urea concentration was then diluted to less than 2 M with 100
   mM Tris‐HCl (pH 8.0). Trypsin was added at a 1:50 ratio for overnight
   digestion at 37 °C. The next day, the digestion process was stopped by
   adjusting the pH to 6.0 with TFA. After centrifuging at 12 000 g for 15
   minutes, the supernatant was processed for peptide purification using a
   custom‐made SDB‐RPS desalting column. The peptide eluate was vacuum
   evaporated and stored at ‐20 °C for future use. Samples were analyzed
   using the timsTOF Pro, a hybrid trapped ion mobility spectrometer
   (TIMS) quadrupole time‐of‐flight mass spectrometer by Bruker Daltonics.
   To identify proteins differentially expressed among groups, statistical
   significance was assessed using an unpaired t‐test.

Histological Analysis of Kidney Tissues and Immunohistochemical Staining

   Kidney tissues were isolated and fixed in 4% paraformaldehyde for 24
   hours before being embedded in paraffin. The sections were then stained
   using Haematoxylin‐eosin (H&E), Von Kossa, and Masson techniques.

   For immunohistochemistry, tissue sections were deparaffinized with
   xylene, ethanol, and water, respectively. Sections were blocked with
   10% normal goat serum and incubated with primary antibody against
   Lgals3 (1:200 dilution) and α‐SMA (1:400 dilution) at 4 °C overnight.
   After three times washes with TBST, the sections were incubated with
   DAB for 5 min.

Cell Culture

   Human tubular epithelial cells (HK‐2 cells) were sourced from the
   Chinese Academy of Sciences Cell Bank. The cells were maintained in
   DMEM medium supplemented with 10% FBS and 1% antibiotics (penicillin
   and streptomycin), and cultured in a 37 °C incubator with 5% CO2. To
   establish the cell model in vitro, HK‐2 cells were exposed to 100
   µg ml^−1 calcium oxalate monohydrate (COM) for 48 hours.

Cell Transfection

   The stably Lgals3 knockdown and Lgals3 overexpression cell lines were
   constructed by transferring lentivirus into HK‐2 cell according to the
   manufacturer's instructions (GeneChem, Shanghai).

   The expression plasmids encoding Flag‐Lgals3, His‐PKM2, Myc‐Trim21 and
   HA‐tagged ubiquitin were purchased from Jiman Bio. For transient
   transfection, plasmids were transfected into HK‐2 or HEK 293T cells
   with Lipofectamine™ 2000 transfection reagent as required for the
   experiment.

   The PKM2 siRNA (si‐PKM2) and negative control (si‐NC) were acquired
   from Jiman Bio. The HK‐2 cells were transfected with si‐PKM2 and si‐NC
   using Lipofectamine™ 2000 transfection reagent following the
   instructions provided by the manufacturer. The sequence of the shRNA
   and siRNA were listed in the Table [199]S2 (Supporting Information)

Immunofluorescence Staining

   The kidney sections were dewaxed, rehydrated, and rinsed with deionized
   water. Antigen retrieval was then performed by microwaving, followed by
   blocking for 1 hour. Next, the sections were incubated with primary
   antibodies, including Lgals3, α‐SMA, Collagen1, PKM2, L‐lactyllysine,
   and H3K18la. All sections were counterstained with DAPI. Fluorescence
   images were captured using a laser scanning confocal microscope.

   The HK‐2 cells were seeded onto dish and fixed with 3.7%
   paraformaldehyde for 15 min. Then cells were permeabilized, washed and
   blocked with 5% goat serum for 2 h. Cells were incubated with
   antibodies against Lgals3, α‐SMA, Collagen1, PKM2, Trim21 and H3K18la
   at 4 °C overnight. Fluorochrome‐conjugated secondary antibodies were
   dropped onto slices in the dark. The cell nuclei were counterstained
   with DAPI. Images were acquired using a confocal microscopy.

Immunoblot Analysis

   Cells or tissues were lysed with RIPA supplemented with protease and
   phosphatase inhibitor. Subsequently, the proteins were deposited onto a
   polyvinylidene fluoride (PVDF) membrane (Millipore) blocked using 5%
   milk. The membranes were exposed to primary antibodies for 12h and
   subsequently exposed to secondary antibodies. The ECL kit was utilized
   to detect the target protein band. The primary antibodies were as
   followers: anti‐Lgals3 (CST, cat#87 985, 1:1000), anti‐Fibronectin
   (Abcam, cat#ab2413, 1:1000), anti‐α‐SMA (ProteinTech Group,
   cat#14395‐1‐AP, 1:2000), anti‐Collagen1 (Abcam, cat#ab138492, 1:1000),
   anti‐PKM2 (CST, cat#4053, 1:1000), anti‐lacty‐lysine (PTM BioLab, cat#
   1401RM, 1:1000), anti‐H3K9la (PTM BioLab, cat# 1419RM, 1:1000);
   anti‐H3K14la (PTM BioLab, cat# 1414RM, 1:1000); anti‐H3K18la (PTM
   BioLab, cat# 1406RM, 1:1000); anti‐H3K56la (PTM BioLab, cat# 1421RM,
   1:1000); anti‐H3 (CST, cat#4499, 1:2000); anti‐H4K5la (PTM BioLab, cat#
   1407RM, 1:1000); anti‐H4K8la (PTM BioLab, cat# 1415RM, 1:1000);
   anti‐H4K12la (PTM BioLab, cat# 1411RM, 1:1000); anti‐FGFR4 (abclonal,
   cat#A7555, 1:1000) and anti‐GAPDH (CST, cat#2118, 1:1000).

Quantitative Real‐Time PCR (RT‐qPCR)

   RNA was extracted from cultured cells following the manufacturer's
   protocol. One microgram of RNA was reverse transcribed into cDNA using
   a gDNA eraser. Subsequently, 2 µL of cDNA was combined with qPCR SYBR
   Master Mix, forward and reverse primers, and milliQ water to reach a
   final volume of 20 µL. Real‐time PCR was conducted using SYBR Green
   Master Mix on an Applied Biosystems StepOne Real‐Time PCR System,
   following a standard cycling protocol. The different primers were
   listed in Table [200]S3 (Supporting Information).

Measurement of Extracellular Acidification Rate (ECAR)

   The extracellular acidification rate (ECAR) was measured using the
   Seahorse XFe 96 Extracellular Flux Analyzer (Agilent Biotechnologies).
   Briefly, cells were seeded into 24‐well plates and washed with Seahorse
   detection buffer. The analyzer then administered Rot/AA and 2‐DG
   (2‐deoxy‐glucose) to determine the glycolytic proton efflux rate, basal
   glycolysis rate, and compensatory glycolysis rate.

Glycolytic Process Evaluation

   Cells were incubated with
   2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐deoxy‐D‐glucose
   (2‐NBDG) at a concentration of 10 µM for 1 hour and subsequently washed
   with PBS. Fluorescence intensity was then measured using flow cytometry
   with excitation wavelengths of 488 nm and 542 nm.

Measurement of Lactate Levels

   Lactate levels were evaluated in cell culture medium from HK‐2 cells by
   using the colorimetric lactate assay kit following the manufacturer's
   instructions.

Measurement of ROS

   ROS generation was assessed using the ROS detection kit (Thermo Fisher,
   USA) following the manufacturer's specifications. Semi‐quantification
   of ROS levels was evaluated using ImageJ software.

Co‐IP and Ubiquitination Assays

   The cells were lysed with IP lysis buffer after required specific
   transfection time. The samples were incubated with 2–5 µg antibodies
   (IgG/PKM2/FLAG/His/Myc) overnight at 4 °C. Protein A/G beads were then
   added and incubated at 4 °C overnight. The beads were washed three
   times with elution buffer and resuspended with SDS loading buffer
   before being heated at 95 °C for 10 min, and then Western blot analysis
   was performed.

   For ubiquitination analysis, Lysates were precipitated with anti‐His or
   PKM2 antibody, and the IP procedure was repeated as described above.
   Immunoblotting was performed with anti‐HA or Ub antibody to measure
   ubiquitination levels.

IP‐MS

   The beads samples were subjected to incubation in a reaction solution
   consisting of 1% sodium deoxycholate, 100 mM Tris‐HCl (pH 8.5), 10 mM
   tris(2‐carboxyethyl) phosphine, and 40 mM chloroacetamide. The
   incubation was carried out at a temperature of 95 °C for a duration of
   10 minutes. The eluates were diluted with an equivalent volume of water
   and then underwent trypsin digestion overnight. Trypsin was added at a
   ratio of 1:50 for digestion at 37 °C. Following centrifugation at a
   force of 12 000 times the acceleration due to gravity for a duration of
   15 minutes, the peptide was purified using desalting columns that were
   produced in‐house using SDB. The eluate was subjected to vacuum
   evaporation and thereafter kept at a temperature of ‐20 °C for future
   utilization. The samples were analyzed using an UltiMate 3000 RSLCnano
   system that was connected to a Q Exactive HF mass spectrometer via a
   Nanospray Flex ion source.

CUT‐Tag

   The CUT&Tag assay was performed using the Hyperactive In‐Situ ChIP
   Library Prep Kit for Illumina, following the manufacturer's
   instructions. In brief, both wild‐type (WT) HK2 cells and
   Lgals3‐knockdown HK‐2 cells were targeted using ConA beads. The cell
   membranes were permeabilized with the nonionic detergent digitonin.
   Subsequently, the cells were incubated with H3K18la antibody
   (PTM‐1427RM, PTM BIO, Hangzhou, China), a secondary antibody, and
   Hyperactive pA‐Tn5 Transposase. The resulting purified PCR products
   were evaluated with the Agilent 2100 Bioanalyzer. Finally, these
   libraries were sequenced on the Illumina NovaSeq6000 platform,
   producing 150 bp paired‐end reads for further analysis.

Chip‐qPCR

   Cells were initially crosslinked by incubating with 2 mM disuccinimidyl
   glutarate for 45 minutes, followed by 1% formaldehyde for 10 minutes.
   The chromatin was then immunoprecipitated using the specified
   antibodies at 4 °C overnight with continuous rotation. Subsequently,
   the DNA was captured, eluted, reverse‐crosslinked, and purified.
   Finally, real‐time PCR amplification was performed using specific
   primers listed in Table [201]S3 (Supporting Information).

Statistical Analysis

   Statistical analysis was performed using GraphPad Prism 8.0. For
   comparisons between two groups, an independent‐sample Student's t‐test
   was employed. To compare three or more experimental groups, one‐way and
   two‐way analyses of variance (ANOVA) were utilized. A p‐value of less
   than 0.05 was deemed statistically significant.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   Z.Y., Y.S., S.Y. were co‐first authors. X.Z., and F.C. contributed
   equally to this work. Z.Y. performed conceptualisation, validation.
   Y.S. wrote – review & edited the original draft. S.Y. performed
   investigation. L.L. performed software. B.L. performed formal analysis.
   T.Y. performed data curation. Y.X. performed supervision. L.C.
   performed data curation, investigation. W.Y. performed data curation.
   W.L., F.C., and X.Z. performed supervision, funding acquisition.

Supporting information

   Supporting Information
   [202]ADVS-12-2413937-s001.docx^ (9.2MB, docx)

Acknowledgements