NAT10 exacerbates acute renal inflammation by enhancing N4-acetylcytidine modification of the CCL2/CXCL1 axis

Significance

RNA modifications are associated with human diseases. N4-acetylcytidine has been identified as a mRNA modification catalyzed by N-acetyltransferase 10 (NAT10) and regulated mRNA stability and translation efficiency. During our research, NAT10 was found to serve as an important regulator of acute renal inflammation. By enhancing the ac4C acetylation of mRNAs of a range of key chemokines, including C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand 2 (CCL2), increases their stability, which in turn promotes the infiltration of macrophages and neutrophils, creating inflammatory microenvironment and exacerbating renal damage. Cpd-155 is identified as a NAT10 inhibitor, effectively protecting mice from renal inflammation and injury. These findings support NAT10 may represent a promising therapeutic target for inflammatory kidney diseases.

Abstract

Inflammation plays an essential role in eliminating microbial pathogens and repairing tissues, while sustained inflammation accelerates kidney damage and disease progression. Therefore, understanding the mechanisms of the inflammatory response is vital for developing therapies for inflammatory kidney diseases like acute kidney injury (AKI), which currently lacks effective treatment. Here, we identified N-acetyltransferase 10 (NAT10) as an important regulator for acute inflammation. NAT10, the only known “writer” protein for N4-acetylcytidine (ac4C) acetylation, is elevated in renal tubules across various AKI models, human biopsies, and cultured tubular epithelial cells (TECs). Conditional knockout (cKO) of NAT10 in mouse kidneys attenuates renal dysfunction, inflammation, and infiltration of macrophages and neutrophils, whereas its conditional knock-in (cKI) exacerbates these effects. Mechanistically, our findings from ac4C-RIP-seq and RNA-seq analyses revealed that NAT10-mediated ac4C acetylation enhances the mRNA stability of a range of key chemokines, including C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1(CXCL1), promoting macrophage and neutrophil recruitment and accelerating renal inflammation. Additionally, CCL2 and CXCL1 neutralizing antibodies or their receptor inhibitors, abrogated renal inflammation in NAT10-overexpression TECs or NAT10-cKI mice. Importantly, inhibiting NAT10, either through Adeno-associated virus 9 (AAV9)-mediated silencing or pharmacologically with our found inhibitor Cpd-155, significantly reduces renal inflammation and injury. Thus, targeting the NAT10/CCL2/CXCL1 axis presents a promising therapeutic strategy for treating inflammatory kidney diseases.

Acute kidney injury (AKI), characterized by a sudden decrease in renal function and glomerular filtration rate, causes high morbidity and mortality, and an extended hospital stay. Annually, approximately 40 to 60 % of patients admitted to intensive care units develop AKI, leading to 1.7 million deaths (1). Moreover, patients with incomplete recovery from AKI have an increased risk of chronic kidney disease (CKD) and eventually, irreversible end-stage renal disease (ESRD) (2). However, there are no specific or effective therapies for AKI. Inflammation is essential for eliminating microbial pathogens and repairing tissues after various forms of injury. AKI is known to be associated with intrarenal and systemic inflammation (3, 4). Therefore, elucidating the improved understanding of the cellular and molecular mechanisms underlying renal inflammation is important for the development of effective therapies to prevent or treat AKI.

Posttranscriptional modification is closely associated with major human diseases including autoimmune diseases, neurodegenerative diseases, and various cancers (5). RNA modification plays an important role in the development of diseases. More than 170 RNA modifications have been identified to date. Our previous studies have reported the critical roles of methyltransferase- and demethylase-mediated N6-methyladenosine (m6A) modifications in various kidney diseases, such as AKI (6), obstructive nephropathy (7), diabetic nephropathy (8), and alcohol-induced kidney injury (9), supporting the involvement of RNA modification in the pathophysiology of kidney disease.

N4-acetylcytidine (ac4C) has been identified as a novel and highly conserved mRNA modification catalyzed by NAT10 and plays an important role in regulating mRNA stability and translation efficiency (10). NAT10 is the only known “writer” protein for ac4C modification, which is the only protein with an acetylase structural domain and RNA-binding activities domain, widely involve in DNA damage repair, cell cycle, telomerase activity, rRNA synthesis, and other pathophysiological processes (11–14). Accumulating evidence indicates that aberrant regulation of NAT10 and ac4C modifications is correlated with disease progression, including tumor proliferation, metastasis, invasion, acute myeloid leukemia, and embryonic development (15–17). NAT10 also plays an important role in the tumor microenvironment, ultimately affecting tumor immune infiltration and targeted therapy response (18). Although two recent studies have identified the pathological role of NAT10 in polycystic kidney disease and renal cell ferroptosis (19, 20), the study of ac4C modifications and NAT10 in kidney disease is still at an early stage, and the detailed functions and mechanisms of ac4C modifications and NAT10 in renal acute inflammation are largely unknown.

In this study, we aimed to evaluate the functional role of NAT10 in kidney injury and inflammation. We found that NAT10 was significantly upregulated in murine models and human biopsies of AKI and correlated with renal inflammation and injury. NAT10 conditional knockout (cKO) was alleviated, whereas NAT10 conditional knock-in promoted macrophage and neutrophil infiltration. ac4C-RIP-seq analysis showed that NAT10 mediates the ac4C modification of a range of inflammatory cytokines and chemokines. Therefore, we hypothesized that NAT10 may serve as a key regulator of inflammatory kidney diseases. Furthermore, we also evaluated the efficacy of NAT10-targeted therapy in a mouse model of AKI. These results may provide a molecular basis for targeting NAT10-mediated ac4C modifications to treat kidney inflammation and AKI.

Results

NAT10 Is Overexpressed in Humans and Mice with AKI.

To investigate whether ac4C acetylation is involved in AKI, we first performed single-cell sequencing analysis from published studies. By analysis of Lake et al. (21) (GSE183276), which examined patients with AKI, alongside the study by Li et al. (22) (GSE274819) involving I/R 24 h AKI mice, we found NAT10 increased in renal tubular cells in both human and mouse samples (Fig. 1 A–C and SI Appendix, Fig. S1 A–C). We quantified ac4C levels in total RNA extracted from human kidney tissues of patients with AKI and controls using dot blot assay. Our findings showed that ac4C levels significantly increased in patients with AKI (Fig. 1D). Next, to understand the expression and localization of NAT10 in AKI, we performed immunohistochemical and immunofluorescence analyses using tissues from patients with AKI. Immunohistochemical and immunofluorescence double staining results showed that NAT10 protein levels were predominantly localized in the nuclei of the renal tubules of patients with AKI and were significantly higher than those in control tissues (Fig. 1 E–G).

Fig. 1.

ac4C modifications increase in human biopsy, mouse models, and TECs in response to proinflammatory stimuli via a CREB1-dependent mechanism. (A) Uniform manifold approximation and projection (UMAP) of cell type clustering in human kidneys. Cell types include proximal tubule segment 1 (PT S1), proximal tubule segment 2 (PT S2), proximal tubule segment 3 (PT S3), thick ascending limb cell (TAL), descending thin limb cell (DTL), distal convoluted tubule cell (DCT), connecting tubule cell (CNT), principal cell (PC), intercalated cell (IC), podocyte (Pod), endothelial cell (EC), Immune cells (IMM). (B) UMAP plots displaying expression levels of NAT10 in each dataset group. (C) The expression levels of NAT10 in renal tubule cells of health and patients with AKI in single-cell data from Lake et al. (21). (D) The ac4C level in the human kidneys were determined using ac4C dot blot assay (n = 6 biological replicates). (E) Photomicrographs of PAS staining and NAT10 staining in kidney sections of patients with AKI. (Scale bar, 50 μm and 20 μm.) The red arrow indicates protein casts, the yellow triangle indicates tubular dilation, the black arrow indicates preserved proximal tubule with brush border, and the asterisks indicate shedding tubule cells. (F) NAT10 and LTL expression in kidney biopsies from patients with AKI was detected using immunofluorescence staining. LTL was used to stain the proximal tubules. (Scale bars, 50 μm.) (G) Quantifications showing NAT10 expression in the kidney from patients with AKI (n = 6 for normal subjects, n = 12 for patients with AKI, two-tailed unpaired Student’s t test). (H) The ac4C level in the I/R mouse model after 24 h were determined using ac4C dot blot assay. (I) The ac4C level in the IRI mouse model were determined using LC–MS/MS analysis (n = 3 biological replicates of mice, two-tailed unpaired Student’s t test). (J) Representative immunofluorescence staining for NAT10 and LTL in I/R-induced AKI after 24 h. (Scale bars, 50 μm.) (K and L) Western blotting (K) and real-time PCR (L) analyses of NAT10 in I/R (24 h)- and cisplatin (3 d)-induced AKI mouse models (n = 6 biological replicates of mice, two-tailed unpaired Student’s t test). (M) NAT10 was upregulated in response to different stimuli, including H/R (hypoxia 12 h and reoxygenation 6 h) and cisplatin (24 h) in HK2 cells. (N) The ac4C level in H/R- and cisplatin-induced HK2 cells were determined using ac4C dot blot assays. (O) Venn diagram showing the overlap of transcription factors (TFs) of NAT10 predicted by JASPAR, PROMO, and ChIPBase. (P) mRNA expression levels of NAT10 and CREB1 in HK2 cells were determined by real-time PCR analysis (n = 4 biological replicates, one-way ANOVA with Tukey’s multiple comparisons test). (Q) Western blotting of pCREB1 and CREB1 protein expression in H/R-induced HK2 cells. (R) Protein abundance of NAT10 in H/R-treated HK2 cells with CREB1 knockdown. (S) Luciferase activity of full-length or truncated NAT10 promoter constructs co-transfected with control or CREB1-overexpressing plasmids (n = 3 biological replicates, two-tailed unpaired Student’s t test). (T) Luciferase activity of NAT10 promoter constructs containing WT or mutated sites (Mut 1, 2, and 3) when co-transfected with control or CREB1 knockdown in HEK293T cells under H/R stimulation (n = 3 biological replicates, two-tailed unpaired Student’s t test). (U) ChIP assays of the binding of CREB1 to the NAT10 promoter in the presence or absence of H/R stimulation. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Similar results were obtained in ischemia/reperfusion (I/R)-induced AKI mouse model after 24 h. Dot blot assays and liquid chromatography-mass spectrometer/mass spectrometry indicated that ac4C levels significantly increased in I/R-induced AKI at this time point (Fig. 1 H and I). We then performed double immunostaining for NAT10 and various tubular markers by immunofluorescence using the following segment-specific tubular markers: proximal tubule, lotus tetragonolobus lectin (LTL), distal convoluted tubule, calbindin D28k (CD28K), collecting duct, and aquaporin 3 (AQP3). NAT10 mainly colocalized with the proximal and distal tubular segments and collecting ducts in I/R-induced AKI mouse model (Fig. 1J and SI Appendix, Fig. S1D). Protein and mRNA levels of NAT10 were analyzed. NAT10 expression was upregulated in both I/R- and cisplatin-induced AKI compared to controls (Fig. 1 K and L). Similar results were obtained for hypoxia/reoxygenation (H/R) and cisplatin-treated HK2 cells (Fig. 1M and SI Appendix, Fig. S1E). The ac4C dot blot assay indicated that ac4C levels increased in H/R (hypoxia 12 h/reoxygenation 6 h) and cisplatin (24 h)-treated human kidney TEC line (HK2) cells (Fig. 1N).

To determine the TFs that directly regulate NAT10 expression, we analyzed the ENCODE chromatin immunoprecipitation sequencing (ChIPseq) data in ChIPBase, PROMO with 5 % maximum matrix dissimilarity rate, and JASPAR database. Among the 55, 68, and 280 factors identified by ChIPBase, PROMO, and JASPAR database, respectively, six factors including cAMP responsive element binding protein 1 (CREB1), YY1 TF (YY1), activating TF 2 (ATF2), CCAAT enhancer binding protein alpha (CEBPA), MYC proto-oncogene, bHLH TF (MYC), and Hypoxia-inducible factor 1 alpha (HIF1α) were overlapping between the three databases (Fig. 1O). We compared the expression levels of these factors in H/R-induced HK2 cells. Our data showed that CREB1 were significantly upregulated in response to H/R in a time-dependent manner, preceding an increase in NAT10 expression (Fig. 1P and SI Appendix, Fig. S1F). Importantly, we observed a significant increase in the phosphorylation of CREB1 in both the renal tissues of mice and HK2 cells treated with I/R or H/R (Fig. 1Q and SI Appendix, Fig. S1 G and H). CREB1 mRNA levels also increased in I/R-induced AKI mice (SI Appendix, Fig. S1I). We knocked down the expression of CREB1 in HK2 cells using a specific siRNA (SI Appendix, Fig. S1J). Our data showed that CREB1 knockdown suppressed the protein and mRNA expression of NAT10 in H/R-induced HK2 cells (Fig. 1R and SI Appendix, Fig. S1K). To investigate whether CREB1 binds to the promoter of NAT10, we first performed luciferase reporter assays to examine the regulation of NAT10 by CREB1. Two truncates of NAT10 promoter were constructed with a luciferase reporter; we cloned two fragments of human NAT10 promoter into the pGL3 vector, including −500/+1 and −2,000/+1. The results showed that CREB1 overexpression significantly stimulated the luciferase activity of the NAT10 promoter in the −500 and −2,000 bp regions under H/R stimulation, whereas the −500 to +1 bp region in the NAT10 promoter was not affected by CREB1 (Fig. 1S). Bioinformatics analysis was performed to predict the possible binding sites for TFs in the promoter region of NAT10 (−500 to −2,000 bp) via TFBIND bioinformatics software (https://tfbind.hgc.jp/) and JASPAR (https://jaspar.genereg.net/). We identified three CREB1 potential binding sites (SI Appendix, Fig. S1L). We then constructed mutants and deleted the three binding sites of promoter reporter of NAT10 to generate pGL-NAT10-Mut1, pGL-NAT10-Mut2, and pGL-NAT10-Mut3 vectors. The results shown in Fig. 1T demonstrate that si-CREB1 significantly decreased luciferase levels in pGL-NAT10-WT, pGL-NAT10-Mut2, and pGL-NAT10-Mut3 but not in pGL-NAT10-Mut1 under H/R stimulation. These observations suggested that CREB1 enhanced NAT10 transcription by binding directly to the −1,308/−1,296 promoter region of NAT10 (Fig. 1T). Furthermore, chromatin immunoprecipitation (ChIP)-qPCR assays demonstrated that CREB1 significantly enriched the NAT10 promoter compared to the normal immunoglobulin G (IgG) control in H/R-induced HK2 cells (Fig. 1U and SI Appendix, Fig. S1M).

NAT10 cKO Protects Against I/R-, Cisplatin-, and Cecal Ligation and Puncture (CLP)-Induced Renal Dysfunction, Injury, and Inflammation in Mice.

To explore the role of NAT10 in AKI, we constructed a mouse model of NAT10 cKO, specifically in the renal tubules. We verified the efficacy of the NAT10 knockout in mice using western blotting and real-time PCR (SI Appendix, Fig. S2 A and B). Immunofluorescence staining confirmed the lack of NAT10 signaling in the renal tubules of NAT10Flox/Flox/KspCre (NAT10^FF/KspCre) mice, whereas it was detected in NAT10FF mouse kidneys (SI Appendix, Fig. S2C). We subsequently induced ischemia/reperfusion injury and killed the mice 24 h later (Fig. 2A). The ac4C dot blot revealed that ac4C acetylation further decreased at this time point in the I/R-NAT10 cKO compared with that in I/R-induced AKI mice (SI Appendix, Fig. S2D). As shown in Fig. 2B, NAT10 deficiency decreased serum blood urea nitrogen (BUN) and serum creatinine (CRE) concentrations in ischemia/reperfusion injury after 24 h. Periodic acid–Schiff (PAS) staining revealed that NAT10 cKO attenuated I/R-induced renal damage (Fig. 2 C and D). Furthermore, the abundance of the kidney injury molecule-1 (KIM-1) protein increased in I/R-induced nephropathy and was downregulated in NAT10 cKO mice (Fig. 2E and SI Appendix, Fig. S2E). Furthermore, enzyme-linked immunosorbent assay (ELISA) indicated that the concentrations of KIM-1 and lipocalin 2 (NGAL) in the serum were significantly decreased in I/R-induced AKI mice after 24 h with NAT10 deletion (Fig. 2F). These data show that the cKO of NAT10 protects against I/R-induced renal damage.

Fig. 2.

NAT10 deficiency protects against renal dysfunction, injury, and inflammation in I/R-induced AKI mice. (A) Schematic illustrating experiment groups. (B) Serum BUN and serum creatinine concentrations in control and NAT10-cKO mice exposed to I/R-induced injury after 24 h (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (C) PAS score in NAT10 cKO mice with I/R-induced AKI (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (D) PAS staining and immunohistochemistry staining of F4/80⁺ and Ly6G. (Scale bars, 100 and 50 μm.) The red arrow indicates protein casts, the yellow triangle indicates tubular dilation, the black arrow indicates preserved proximal tubule with brush border, and the asterisks indicate shedding tubule cells. (E) Western blotting of KIM-1, pp65, and p65 after NAT10 deficiency in I/R-induced AKI after 24 h. (F) ELISA analysis of the production of KIM-1 and NGAL in I/R-induced AKI with NAT10 knockout (n = 6 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test). (G) Relative mRNA expressions of CCL2 were determined by real-time PCR (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (H) The proportions of infiltrated Ly6C^hi MDMs and Ly6G⁺ neutrophils in the kidney were detected by flow cytometry. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

In addition, immunohistochemistry, western blotting, and real-time PCR showed that the absence of NAT10 reduced F4/80⁺ macrophage and Ly6G⁺ neutrophils infiltration (Fig. 2D), inhibited the p65 nuclear factor κB (p65NF-κB) activation (Fig. 2E and SI Appendix, Fig. S2E), and reduced proinflammatory cytokine production (Fig. 2G and SI Appendix, Fig. S2F). Furthermore, the proportions of infiltrating myeloid cells in the kidneys, specifically monocyte-derived macrophages (MDMs; CD45⁺CD11b⁺Ly6G^-Ly6C^hi) and neutrophils (CD45⁺CD11b⁺Ly6G⁺), were analyzed using flow cytometry (SI Appendix, Fig. S2G). A significant decrease in the population of Ly6C^hi MDMs and Ly6G⁺ neutrophils was observed in NAT10 cKO mice after I/R challenge (Fig. 2H and SI Appendix, Fig. S2H). Next, we examined the function of NAT10 in two other models of AKI, namely those induced by cisplatin and CLP. In the cisplatin model, NAT10^FF/KspCre mice displayed a marked reduction in serum BUN and serum CRE concentrations compared to NAT10^FF mice after cisplatin one injection sustained for 3 d (Fig. 3 A and B). Specific depletion of NAT10 notably improved cisplatin-induced renal dysfunction and tubular damage, as demonstrated by PAS staining and western blotting (Fig. 3 C and D and SI Appendix, Fig. S3A). Similarly, immunohistochemistry, western blotting, and flow cytometry revealed that the absence of NAT10 reduced the population of infiltrating macrophages and Ly6G⁺ neutrophils (Fig. 3 D and E and SI Appendix, Fig. S3B), inhibited p65NF-κB activation (SI Appendix, Fig. S3A). Similarly, in the CLP mouse model, specific depletion of NAT10 notably improved CLP-induced renal dysfunction and tubular damage, as confirmed by serum BUN and serum CRE levels, and PAS staining, reflecting the severity of renal damage harvested 24 h after CLP surgery (Fig. 3 F–H and SI Appendix, Fig. S3C). NAT10 deficiency significantly reduced KIM-1 expression and NF-κB activation (SI Appendix, Fig. S3D) and attenuated the production of inflammatory cytokines (SI Appendix, Fig. S3E). Furthermore, immunohistochemistry and flow cytometry showed that the cKO of NAT10 also reduced the CLP-induced population of Ly6C^hi MDMs and Ly6G⁺ neutrophils (Fig. 3 I and J and SI Appendix, Fig. S3 F and G). These data indicated that NAT10 promotes the recruitment of inflammatory cells and finally facilitates I/R-, cisplatin-, and CLP-induced AKI.

Fig. 3.

NAT10 deletion in renal tubular cells relieved cisplatin- and CLP-induced renal dysfunction, injury, and inflammation. (A) Schematic illustrating experiment groups. (B) Serum BUN and serum creatinine concentrations in control and NAT10-cKO mice exposed to cisplatin-induced injury sustained for 3 d (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (C) PAS score in NAT10-cKO mice with cisplatin-induced AKI (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (D) PAS staining and immunohistochemistry staining of F4/80+ and Ly6G. (Scale bars, 100 and 50 μm.) The red arrow indicates protein casts, the yellow triangle indicates tubular dilation, the black arrow indicates preserved proximal tubule with brush border, and the asterisks indicate shedding tubule cells. (E) The proportions of infiltrated Ly6C^hi MDMs and Ly6G⁺ neutrophils in the kidney were detected by flow cytometry. (F) Schematic illustrating experiment groups. (G) Serum BUN and creatinine concentrations 24 h after in control and NAT10-cKO mice exposed to CLP-induced injury (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (H) PAS score in NAT10-cKO mice with CLP-induced AKI (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (I) Immunohistochemistry staining of F4/80+ and Ly6G. (Scale bars, 50 μm.) (J) The proportions of kidney Ly6C^hi MDMs and Ly6G⁺ neutrophils were detected by flow cytometry. Data represent the mean ± SEM. ***P < 0.001.

Tubular epithelial cell (TEC)-specific overexpression of NAT10 promote renal damage and inflammation.

To further elucidate the role of NAT10 in TECs during kidney injury, we established NAT10-cKI mice (SI Appendix, Fig. S4 A and B). NAT10-cKI mice showed significantly higher NAT10 expression in kidney tissues than in the Cre-negative littermate control (NAT10^FF) mice, as demonstrated by immunofluorescence staining, western blotting, and real-time PCR (SI Appendix, Fig. S4 C–E). Subsequently, NAT10-cKI and NAT10^FF mice were subjected to ischemia reperfusion injury and euthanized 24 h later (Fig. 4A). Obviously, I/R-cKI mice exhibited worse renal function and more severe renal damage (Fig. 4 B–D) and more expression of KIM-1 and p65NF-κB phosphorylation following 24 h ischemia reperfusion injury (Fig. 4E). ELISA analysis indicated that the concentrations of KIM-1 and NGAL in the serum were significantly increased in I/R-induced AKI mice with NAT10 overexpression at the same time point (Fig. 4F and SI Appendix, Fig. S4F). Immunohistochemistry staining and flow cytometry analysis showed that overexpression of NAT10 promoted Ly6C^hi MDMs and Ly6G⁺ neutrophil infiltration (Fig. 4 D, G, and H). Generally, NAT10 overexpression aggravated kidney injury and inflammation after ischemia reperfusion.

Fig. 4.

Kidney tubular cell knock-in of NAT10 aggravates renal injury and inflammation in I/R-induced AKI. (A) Schematic illustrating experiment groups. (B) Serum BUN and creatinine concentrations in NAT10-cKI mice with I/R-induced AKI after 24 h (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (C) Quantification of PAS staining in NAT10-cKI mice with I/R-induced AKI (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (D) PAS staining and immunohistochemistry staining of F4/80+ and Ly6G. (Scale bars, 100 and 50 μm.) The red arrow indicates protein casts, the yellow triangle indicates tubular dilation, the black arrow indicates preserved proximal tubule with brush border, and the asterisks indicate shedding tubule cells. (E) Western blotting of KIM-1, pp65, and p65 in NAT10 overexpression on I/R-induced AKI (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (F) ELISA analysis of the production of KIM-1 and NGAL in I/R-induced AKI with NAT10 overexpression (n = 6 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test). (G and H) Flow cytometry analysis and quantitative data (n = 4 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

NAT10 Promotes the Inflammatory Response and Injury in H/R- and Cisplatin-Treated HK2 Cells.

Next, we evaluated the function of NAT10 in HK2 cells in response to H/R and cisplatin stimulation. Knockdown and overexpression of NAT10 by transfection of siRNA or overexpression plasmid, respectively, in HK2 cells were verified by real-time PCR (SI Appendix, Fig. S5 A and F). The H/R (hypoxia 12 h and reoxygenation 6 h)-induced ac4C modifications were attenuated by NAT10 knockdown (SI Appendix, Fig. S5B). Western blotting revealed that NAT10 knockdown inhibited KIM-1 expression and p65NF-κB activation (SI Appendix, Fig. S5C). Real-time PCR showed that NAT10 knockdown alleviated the H/R-induced production of proinflammatory cytokines (TNF-α and IL-1β), chemokines (CCL2), and KIM-1 mRNA levels at this time point (SI Appendix, Fig. S5D). Furthermore, to clarify the effects of NAT10 knockdown HK2 cells on macrophages migration, we seeded human leukemic cell line (THP-1) into the upper chamber of the Transwell chambers. The results showed that NAT10 knockdown in HK2 cells inhibited the migration of THP-1 cells (SI Appendix, Fig. S5E). In contrast, NAT10 overexpression promoted H/R-induced cell injury and inflammatory responses. Western blotting revealed that overexpression of NAT10 increased the expression of KIM-1 and p65NF-κB phosphorylation compared with H/R-stimulated HK2 cells (SI Appendix, Fig. S5G). Additionally, transwell assays revealed that NAT10 overexpression facilitated the migration of THP-1 cells (SI Appendix, Fig. S5H). We confirmed these findings in a cisplatin-induced cell injury model. Knockdown of NAT10 decreased and overexpression of NAT10 increased cisplatin-induced KIM-1 expression and p65NF-κB activation (SI Appendix, Fig. S6 A and D) and inflammation after cisplatin treatment for 24 h (SI Appendix, Fig. S6B). Consistently, NAT10 knockdown attenuated THP-1 transwell migration capacity (SI Appendix, Fig. S6C).

Identification of NAT10 Downstream Targets with ac4C-RIP-Sequencing and RNA-Sequencing.

To investigate the underlying mechanisms of NAT10 in renal inflammation, we performed ac4C-RIP-seq and RNA-seq analyses in H/R (hypoxia 12 h and reoxygenation 6 h)-exposed HK2 cells with or without NAT10 silencing (Fig. 5A). We found that ac4C peaks were mainly located in the protein-coding region (CDS) of mRNA transcripts in H/R-stimulated HK2 cells with or without NAT10 silencing, as previously reported (Fig. 5B) (10, 23). Consistent with previous research (10), the sequential analysis of ac4C peaks showed that ac4C modifications were highly enriched within a C-rich sequence characterized by three or four obligate cytidines separated by two nonobligate nucleotides (CXXCXXCXXCXX), which is similar to previously reported results (Fig. 5C). Through ac4C-RIP-seq analyses, we found 636 hypoacetylated peaks and 213 hyperacetylated peaks (|log2FC| ≥ 2) on NAT10 knockdown. Meanwhile, RNA-seq revealed that 963 genes were downregulated, and 127 genes were upregulated (|log2FC| ≥ 2) in response to NAT10 knockdown (SI Appendix, Fig. S7 A and B). NAT10 promotes ac4C modification and RNA stability; therefore, we chose hypoacetylated and downregulated genes to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. As Fig. 5D shows, differentially hypoacetylated genes were enriched in inflammation-related signaling pathways, including TNF-α signaling pathways, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, cytokine–cytokine receptor interaction. GO analysis of the differentially hypoacetylated genes indicated that many genes were associated with cytokine production (SI Appendix, Fig. S7C). Additionally, KEGG enrichment analyses of differentially downregulated genes in RNA-seq indicated that many genes were associated with TNF-α signaling pathways (Fig. 5D). Most notably, TNF-α signaling pathways were the top 1 signaling pathways in both differentially hypoacetylated genes and differentially downregulated genes. We therefore investigated potential targets using a combination of acRIP-seq and RNA-seq in TNF-α signaling pathways. By overlapping the differentially hypoacetylated and downregulated genes, we identified 16 candidate genes that showed concomitantly decreased mRNA acetylation and reduced transcriptional levels in NAT10 knockdown cells (Fig. 5E). Therein, some key proinflammatory cytokines and chemokines like TNF-α, CCL2, and CXCL1 may be potential targets of NAT10 (Fig. 5F). Next, we performed real-time PCR assays for those potential genes.

Fig. 5.

Characterization of ac4C modification and identification of NAT10 downstream targets through ac4C-RIP-seq and RNA-seq in H/R-treated with or without NAT10 knockdown. (A) Heatmap of ac4C-RIP-seq analysis showing differentially acetylated genes in H/R-induced HK2 cells with or without NAT10 knockdown. (B) Density distribution of ac4C-containing peaks across the mRNA transcripts. (C) The ac4C consensus motif in H/R-exposed cells with or without NAT10 knockdown was identified using HOMER. (D) KEGG enrichment analysis identified the top 10 pathways of hypoacetylated (Left) and downregulated genes (Right) following NAT10 knockdown. (E) Overlapping potential downstream targets of NAT10 in hypoacetylated and downregulated genes. (F) Heatmap showed the hypoacetylated genes (Left) and downregulated genes (Right) in the TNF-α pathway. (G) RNA decay assays with actinomycin D treatment were performed to detect the degradation rates of CXCL1 and CCL2 mRNA in NAT10 knockdown cells. (H) Correlation analysis between CCL2 and NAT10 (Left) and CXCL1 and NAT10 (Right) from single-cell data (GSE274819) from Li et al. (22) in I/R 24 h mice. (I) ELISA analysis of CXCL1 and CCL2 production in I/R-induced AKI with NAT10 knockout and NAT10 overexpression (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (J) The levels of CCR2 and CXCR2 in myeloid cells were detected by flow cytometry. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Either NAT10 knockdown in HK2 cell or NAT10 deficiency in mice were both significantly inhibited H/R-induced and I/R-induced TNF-α, CCL2, CXCL1, C-X-C motif chemokine ligand 2 (CXCL2), and C-X-C motif chemokine ligand 5 (CXCL5) mRNA levels, respectively (SI Appendix, Fig. S7D). Subsequently, RNA decay assays were performed to assess the effects of NAT10-regulated those potential genes. As shown in Fig. 5G and SI Appendix, Fig. S7E, NAT10 knockdown significantly decreased the stability of CCL2 and CXCL1 mRNA stability. According to the Lake et al. (21) (GSE183276), which examined patients with AKI, alongside the study by Li et al. (22) (GSE274819) involving I/R 24 h AKI mice, the mRNA levels of CCL2 and CXCL1 positively correlated with NAT10 expression levels in both human and mouse samples (Fig. 5H and SI Appendix, Fig. S7F). These results were confirmed using ELISA and real-time PCR. The concentrations of CCL2 and CXCL1 in the serum of I/R-induced AKI mice were attenuated by NAT10 deficiency and further increased by NAT10 overexpression (Fig. 5I). Similarly, NAT10 deficiency inhibited cisplatin- and CLP-induced production of CCL2 and CXCL1 (SI Appendix, Fig. S8 A–D). Additionally, we detected the expression of the CCL2 receptor C-C motif chemokine receptor 2 (CCR2) and the CXCL1 receptor C-X-C motif chemokine receptor 2 (CXCR2). Flow cytometry analysis indicated that the absence of NAT10 inhibited CCR2 and CXCR2 levels in myeloid cells (Fig. 5J).

CCL2 and CXCL1 Were Downstream Target Genes of NAT10-Mediated ac4C Modification.

From the ac4C-RIP-seq data, we found that the ac4C peaks were distributed in the CDS of CCL2 and CXCL1 mRNA in H/R-stimulated HK2 cells and were diminished upon NAT10 knockdown (Fig. 6 A and B). Therefore, we focused on CCL2 and CXCL1 as the targets of NAT10. Using acRIP-qPCR, we found that CCL2 and CXCL1 mRNA were enriched by the ac4C and NAT10 antibodies (Fig. 6 C and D). Furthermore, luciferase reporter assays were performed to validate the importance of ac4C modifications in the CDS of CCL2 and CXCL1. Both wild-type and mutant CCL2 and CXCL1 reporter genes were constructed. In the mutant forms of CCL2 and CXCL1, cytosine (C) was replaced with thymine (T) in the ac4C peak. The dual-luciferase assay showed that the wild-type CCL2 and CXCL1 reporters decreased upon NAT10 knockdown, whereas NAT10 inhibition had no obvious impact on the expression of the mutant CCL2 and CXCL1 luciferase reporters (Fig. 6 E–H).

Fig. 6.

CCL2 and CXCL1 are potential downstream target genes of NAT10. (A and B) Attenuation of NAT10 diminished ac4C modification of CCL2 (A) and CXCL1 (B) mRNA in H/R-treated HK2 cells. (C and D) ac4C-RIP-qPCR (C) and RIP-qPCR (D) analysis of alterations in the ac4C modifications of CCL2 and CXCL1 genes in H/R-treated HK2 cells with or without NAT10 knockdown (n = 4 biological replicates, two-tailed unpaired Student’s t test). (E) Wild-type and mutant CCL2 were inserted into pmirGLO reporter vectors. (F) Luciferase reporter assay measured the luciferase activities of CCL2-CDS WT or CCL2-CDS Mut in H/R-treated HK2 cells with or without NAT10 knockdown (n = 4 biological replicates, two-tailed unpaired Student’s t test). (G) Wild-type and mutant CXCL1 were inserted into pmirGLO reporter vectors. (H) Luciferase reporter assay measured the luciferase activities of CXCL1-CDS WT or CXCL1-CDS Mut in H/R-treated HK2 cells with or without NAT10 knockdown (n = 4 biological replicates, two-tailed unpaired Student’s t test). (I) Schematic illustrating the mutation site in the NAT10-G641E. (J) The total RNA ac4C level in wild-type NAT10 and mutant NAT10 was determined using anti-ac4C dot blot. (K and L) Western blotting (K) and real-time PCR (L) analysis of NAT expression in wild-type NAT10 and mutant NAT10 (n = 4 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test). (M) ELISA analysis of the production of CCL2 and CXCL1 when NAT10-wt or NAT10-G641E treatment (n = 4 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test). (N) RNA decay assays with actinomycin D treatment were performed to detect the degradation rate of CCL2 and CXCL1 mRNA in NAT10-wt or NAT10-G641E treatment. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

To provide further evidence that the regulation of CCL2 and CXCL1 by NAT10 depends on its acetyltransferase activity. We then introduced a point G641E mutation in the N-acetyltransferase domain of NAT10 with a FLAG tag (NAT10-G641E) or wild-type NAT10 (NAT10-wt) and transfected them into HK2 cells (Fig. 6I). Dot blot, western blotting and real-time PCR analyses indicated that NAT10-G641E did not improve ac4C modification level but increase on NAT10 expression (Fig. 6 J–L). We found that NAT10-wt, but not NAT10-G641E, increased the production and mRNA stability of CCL2 and CXCL1, suggesting that the N-acetyltransferase domain of NAT10 is required for the regulation of CCL2 and CXCL1 (Fig. 6 M and N).

Anti-CCL2 and Anti-CXCL1 IgG Treatment Alleviated NAT10-Driven Inflammation Response.

To directly test the potential contributions of CCL2 and CXCL1 to the NAT10-induced inflammatory response, CCL2- and CXCL1-neutralizing antibodies were used. Strikingly, blocking CCL2 and CXCL1 significantly inhibited NAT10-induced p65NF-κB activation and reduced the proinflammatory cytokines (Fig. 7 A and B). To further examine whether CCL2 and CXCL1 were essential for NAT10-induced macrophage migration, THP-1 cells were incubated in the upper chamber of the Transwell chambers, NAT10-overexpressing HK2 cells were incubated in the lower chamber, and then pretreated with CCL2- and CXCL1-neutralizing antibodies in a Transwell assay (Fig. 7C). The results showed that CCL2 and CXCL1 neutralization abolished the increase in macrophage migration by NAT10 (Fig. 7D).

Fig. 7.

CXCL1 and CCL2 neutralizing antibody or CCR2 and CXCR2 inhibitor rescues NAT10 overexpression-induced renal inflammation. (A) Western blotting analysis of pp65 and p65 protein expression. (B) Real-time PCR analysis of CXCL1 and CCL2 mRNA levels (n = 4 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test). (C) Schematic of in vitro co-culture experiments. (D) Representative images of CXCL1 and CCL2 neutralizing antibody treatment on macrophage migration by transwell assay. (E) Schematic illustrating experiment groups and treatment in four groups. (F) Serum BUN and creatinine concentrations 24 h later in I/R-induced AKI mice (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (G) Western blotting analysis of KIM-1 after CCR2 and CXCR2 inhibitor treatment. (H) ELISA analysis of CXCL1 and CCL2 levels (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (I) Quantification of PAS staining in NAT10-cKI mice with CCR2 and CXCR2 inhibitor treatment (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (J) PAS staining and immunohistochemistry staining of F4/80+ and Ly6G. (Scale bars, 100 and 50 μm.) Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Pharmacologic Inhibition of CCR2 and CXCR2 Inhibits Inflammatory Cell Accumulation After Kidney Injury.

We further confirmed whether CCL2 and CXCL1 function as pivotal chemokines mediating NAT10-induced renal damage and the recruitment of inflammatory cells in vivo. NAT10-cKI mice were pretreated with RS102895 (a CCR2 inhibitor) and SB265610 (a CXCR2 inhibitor) before ischemia reperfusion injury (Fig. 7E). Both RS102895 and SB265610 treatment improved NAT10-cKI-promoted I/R-induced AKI mice renal dysfunction and tubular damage and inhibited the upregulation of proinflammatory cytokines, as demonstrated by the levels of serum BUN and serum CRE, PAS staining, and ELISA analyses (Fig. 7 F–J). These results were confirmed by immunohistochemistry. The numbers of F4/80⁺ macrophages and Ly6G⁺ neutrophils significantly decreased in injured NAT10-cKI mouse kidneys treated with RS102895 and SB265610, respectively (Fig. 7J).

AAV9-Mediated Silencing of NAT10 Protects Against Renal Dysfunction and Inflammation Response in I/R-Induced AKI Murine Models.

Two methods were used to explore the therapeutic potential of NAT10 in the AKI mouse model. We silenced NAT10 in mice using an AAV9-packaged NAT10 knockdown plasmid (Fig. 8A). Western blotting revealed that NAT10 expression was reduced in NAT10 shRNA-transfected kidneys (SI Appendix, Fig. S9A). We found that the disruption of NAT10 protected against I/R-induced renal dysfunction and inflammatory response, as demonstrated by the levels of serum BUN and serum CRE (Fig. 8B), PAS staining (Fig. 8C and SI Appendix, Fig. S9B), western blotting (Fig. 8D), and real-time PCR after ischemia reperfusion 24 h (SI Appendix, Fig. S9C). Immunohistochemistry and ELISA showed that NAT10 silencing reduced F4/80⁺ macrophage and Ly6G⁺ neutrophil infiltration and inhibited chemokine CXCL1 and CCL2 production (Fig. 8 C and E).

Fig. 8.

Therapy targeting NAT10 significantly attenuates renal dysfunction, injury, and inflammation. (A) Schematic illustrating the treatment of AAV9-mediated NAT10 knockdown in I/R-induced AKI. (B) Serum BUN and creatinine concentrations 24 h after in mice with I/R-induced nephropathy and knockdown of NAT10 (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (C) PAS staining and immunohistochemistry staining of F4/80⁺ and Ly6G. (Scale bars, 100 and 50 μm.) The red arrow indicates protein casts, the yellow triangle indicates tubular dilation, the black arrow indicates preserved proximal tubule with brush border, and the asterisks indicate shedding tubule cells. (D) Western blot of KIM-1, pp65, and p65 after NAT10 knockdown in I/R-induced AKI. (E) Serum CXCL1 and CCL2 levels detected by ELISA (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (F) Workflow of the hybrid virtual screening strategy. (G) Molecular docking demonstrated that Cpd-155 physically bound to the catalytic domain of NAT10. (H) RMSD of Cpd-155 during MD simulations. (I) The stabilization of NAT10 in vitro with or without Cpd-155 treatment detected by CETSA analysis. (J) Dot blot assay showing the effect of Cpd-155 treatment on ac4C abundance in H/R-treated HK2 cells. (K) Western blot analysis of KIM-1, pp65, and p65 after Cpd-155 treatment in H/R-induced HK2 cells. (L) Schematic illustrating administration of Cpd-155 before and after induction of I/R-induced AKI. ip, intraperitoneally. (M) Serum BUN and creatinine concentrations 24 h after I/R-induced AKI with Cpd-155 treatment (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). (N) PAS staining in I/R-induced AKI with Cpd-155 treatment. (Scale bars, 100 μm.) (O) ELISA analysis of CXCL1 and CCL2 levels (n = 6 biological replicates of mice, two-way ANOVA with Tukey’s multiple comparisons test). Data represent the mean ± SEM. ***P < 0.001.

Treatment with the NAT10 Inhibitor Cpd-155 Exerts Reno-protective Effects In Vitro and In Vivo.

NAT10 may act as an important regulator of acute renal inflammation, therefore, we next performed a structure-based virtual screening of 180,000 compounds from the SPECS library to identify potential NAT10 inhibitors to improve clinical usability. From SPECS, we requested the top 40 candidate compounds that exhibited the highest docking scores to the catalytic pocket of NAT10 (Fig. 8F). Preliminary observations of their renoprotective effects on cisplatin-induced cell activity were made using the cell counting kit 8 (CCK8) assay. These results indicated that Cpd-155 had the strongest renoprotective effect (SI Appendix, Fig. S10). Therefore, Cpd-155 was selected for further validation. Molecular docking analysis based on the crystal structure of NAT10 indicated that Cpd-155 interacted with distinct NAT10 residues, including Thr419, His537, Gln636, and lle629 (Fig. 8G).

Molecular dynamics simulation and cellular thermal shift assay (CETSA) supported the potential interaction between Cpd-155 and NAT10 (Fig. 8 H and I). Moreover, H/R-induced ac4C modifications were attenuated by Cpd-155 at concentrations of 20, 40, and 80 μM in a dose-dependent manner (Fig. 8J). Compared with remodelin, a positive NAT10 inhibitor, Cpd-155 showed a greater renoprotective effect and anti-inflammatory activity. The administration of Cpd-155 in H/R-induced HK2 cells resulted in a significant reduction in KIM-1 expression, NF-κB activation, and proinflammatory cytokine production (Fig. 8K and SI Appendix, Fig. S11A), whereas no significant change was observed in NF-κB activation when Cpd-155 was added to NAT10-G641E cells (SI Appendix, Fig. S11B). We evaluated the renoprotective effects of Cpd-155 on I/R-induced AKI. Considering the safety and applicability of small molecule NAT10 inhibitors. We performed histopathological analysis of the major organs. Hematoxylin and eosin (H&E) staining indicated that Cpd-155 treatment did not result in any significant changes in the heart, liver, spleen, lungs, or kidneys when compared with those of the control group (SI Appendix, Fig. S11C), indicating that Cpd-155 treatment had no obvious toxicity. Mice were pretreated with Cpd-155 (12.5, 25, and 50 mg/kg) or remodelin (50 mg/kg) 12 h before ischemia reperfusion injury (Fig. 8L). Consistently, Cpd-155 showed a greater renoprotective effect at a lower dose than remodeling in vivo. Serum BUN and serum CRE levels reduced following Cpd-155 administration (Fig. 8M). PAS staining demonstrated that Cpd-155 treatment attenuated I/R-induced renal injury (Fig. 8N and SI Appendix, Fig. S11D). Cpd-155 treatment also dose-dependently inhibited KIM-1 expression, NF-κB activation, and chemokine production (CCL2 and CXCL1) (Fig. 8O and SI Appendix, Fig. S11E).

Discussion

Abnormal ac4C modifications are closely related to major human diseases, including autoimmune diseases, neurodevelopmental disorders, infertility, and various cancers. However, few studies have focused on the role of ac4C modifications in kidney diseases, such as AKI. In the present study, we extensively investigated NAT10-medicated ac4C modifications in renal inflammation. In this study, we analyzed two different single-cell databases. By analysis of Lake et al. (21) (GSE183276), which examined patients with AKI, alongside the study by Li et al. (22) (GSE274819) involving I/R 24 h AKI mice, we found NAT10 increased in renal tubular cells in both human and mouse samples. Our data also confirmed that NAT10 was highly induced in renal tubules in I/R- and cisplatin-induced AKI models, as well as in human biopsies and cultured TECs, in a CREB1-dependent manner. NAT10 deficiency protects the kidney from I/R-, cisplatin-, and CLP-induced renal damage and inflammation. According to the combined analysis of acRIP- and RNA-seq, NAT10 effectively aggravated renal inflammation and injury by promoting CXCL1 and CCL2 levels through enhanced CXCL1 and CCL2 mRNA ac4C acetylation and improved mRNA stability. The upregulation of CXCL1 and CCL2 could recruit more infiltrating macrophages and neutrophils, and consequently promote the inflammatory response after AKI. Furthermore, CXCL1- and CCL2-neutralizing antibodies or their receptor inhibitor alleviated NAT10-driven inflammation response. We identified a NAT10 inhibitor, Cpd-155, and found that treatment with Cpd-155 or AAV9-mediated NAT10 silencing protected against renal injury and inflammation. These results collectively demonstrate the NAT10/CXCL1/CCL2 axis. Collectively, these findings suggest that the NAT10/CXCL1/CCL2 axis is a promising therapeutic target for renal inflammation (SI Appendix, Fig. S12).

We have demonstrated that abnormally upregulated NAT10 plays a key role in various diseases, however, the factors that drive NAT10 upregulation remain unclear. A previous study showed that lysine 2-hydroxyisobutyrylation of NAT10 enhances its interaction with the deubiquitinase USP39, thereby increasing NAT10 protein stability (24). Currently, the TFs which can regulate NAT10 expression remain unknown. Our data suggest a mechanism responsible for the upregulation of NAT10 in AKI. We found that CREB1 regulates the transcription of NAT10 to increase its expression by possibly binding to the −1,308 to −1,296 bp NAT10 promoter region.

The most significant finding of the current study is that NAT10 may be a key upstream switch in renal inflammation because it controls the majority of infiltrating inflammatory cells in AKI, such as neutrophils and macrophages. Kidney injury and inflammation are tightly linked (25, 26). An excessive inflammatory response may induce kidney injury through the direct detrimental effects of circulating cytokines and chemokines. Severe or sustained AKI usually causes incomplete repair, leading to renal tubular degeneration, chronic inflammation, renal fibrosis, and ultimately CKD or ESRD (4, 27). A fine balance between the inflammatory and anti-inflammatory factors facilitates tissue repair and restores homeostasis. Thus, targeting the inflammatory response has been suggested as a therapeutic strategy to treat not only AKI, but also CKD (28). Accumulating evidence has shown that kidney TECs regulate both innate and adaptive immune responses. Activated TECs generate and secrete proinflammatory factor, including TNF-α, IL-1β, CXCL1, CXCL2, CXCL5, C-X3-C motif chemokine ligand 1 (CX3CL1), and CCL2 to interact directly with neutrophils, macrophage, that promote inflammatory response in AKI (29, 30). NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated pentraxin 3 (PTX3) mRNA in rheumatoid arthritis, resulting in synovial infiltration by multiple types of immune cells (31). Additionally, NAT10 activates the NF-κB signaling pathway to promote malignant progression of lung cancer (32). These studies confirm the potential role of NAT10 in regulating inflammation; however, the underlying mechanisms and whether it may serve as an anti-inflammatory target are largely unknown; we showed the importance of TEC-specific NAT10 in renal inflammation by using NAT10 kidney-specific cKO and cKI mice with different AKI models. The absence of NAT10 was reduced, and the overexpression of NAT10 promoted the infiltration of macrophages and neutrophils.

To investigate the principal mechanism of NAT10 in renal inflammation, ac4C-RIP-seq and RNA-seq were performed in H/R-stimulated HK2 cells with NAT10 knockdown and in control cells. We identified some key proinflammatory cytokines and chemokines, such as TNF-α, CCL2, CXCL1, and CXCL2, may be potential targets of NAT10, and further demonstrated that NAT10-mediated ac4C acetylation stabilized CCL2 and CXCL1 mRNA are most significant. In the acRIP-seq data, the ac4C modification was distributed in the CDS of CXCL1 and CCL2 mRNA in H/R-stimulated HK2 cells, which was diminished upon NAT10 knockdown. We confirmed the regulation of NAT10 on CXCL1 and CCL2 by applying acRIP-qPCR and luciferase reporter assays. More importantly, only NAT10 promoted CXCL1 and CCL2 transcriptional activity, while the NAT10 N-acetyltransferase domain mutant had no effect. CXCL1 is a CXC chemokine family member, also known as growth-regulated oncogene-alpha (Gro-α) and keratinocyte-derived chemokine, is expressed by immune and epithelial cells, such as renal TECs (33, 34). CXCL1 levels are elevated in the serum, urine, and kidney tissues of renal ischemia and UUO mouse models (34, 35), suggesting that it could be an early biomarker of AKI and kidney fibrosis. It modulates tumorigenesis and metastasis in several types of human cancers (36), and inflammation, especially in neutrophil granulocytes (37), by binding and activating its receptor, CXCR2. CCL2, also known as macrophage chemoattractant protein-1 (MCP-1), is secreted by many cell types, including endothelial cells, activated monocytes, fibroblasts, and renal TECs (38–40). CCL2 and its receptor CCR2 are chemokines widely studied in kidney diseases (41). Clinical evidence has shown that CCL2 is a potential biomarker for AKI, CKD, and diabetes (42–44). To determine whether CXCL1 and CCL2 are key players in kidney inflammation driven by NAT10 and whether blocking CXCL1 and CCL2 could inhibit renal inflammation, CCL2- and CXCL1-neutralizing antibodies were used. In our data, the blocking of CCL2 and CXCL1 significantly inhibited the NAT10-induced p65NF-κB activation, and reduced the proinflammatory cytokines production, and abolished the increased macrophages migration by NAT10.

Considering the significant role of NAT10 in AKI, we tested NAT10-targeted therapy for inflammatory kidney disease using our found NAT10 inhibitor and AAV9-mediated in vivo silencing. A NAT10 structure-based virtual screening strategy was used to identify NAT10 novel inhibitors. Cpd-155 is a promising and novel inhibitor of NAT10. Remodelin, a classical inhibitor of NAT10, has therapeutic effects in various diseases, including acute myeloid leukemia (45), autoimmune disease (31), and cancers (46, 47), suggesting that NAT10-targeting therapy may be a potential therapeutic target in diseases. We found that Cpd-155 had better effects on the restoration of renal dysfunction and inflammation than on remodelin both in vivo and in vitro. Overall, these findings indicate that NAT10 is a promising target for AKI therapy and that Cpd-155 deserves further study to improve its clinical usability.

Collectively, our study showed that NAT10 expression is highly induced in TECs in response to various AKI stimuli in a CREB1-dependent manner and that NAT10 may serve as a key switch for renal inflammation by promoting the ac4C modification of CXCL1 and CCL2. The NAT10/CXCL1/CCL2 axis may serve as a novel potential therapeutic target for future interventions in inflammatory kidney diseases.

Materials and Methods

All the animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals at Anhui Medical University and all animal procedures were approved by the Animal Experimentation Ethics Committee of Anhui Medical University, Anhui, China. The methods in this study included animals models, generated the TEC-specific knock-in and knockout mice, measurement of serum urea nitrogen and serum creatinine concentrations, western blotting, cell lines and culture conditions, dot blot assay, ELISA, real-time PCR analysis, renal histology and immunohistochemistry, immunofluorescent staining, RNA decay assays, RNA immunoprecipitation, overexpression and knockdown of genes, luciferase reporter assay, flow cytometry analysis, ac4C-RIP-Seq, transwell assay. The detailed description of the material and method utilized in this study is provided in SI Appendix, Materials and Methods.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

ac4C-RIP-seq data and RNA-seq data have been deposited into the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database under GSE286352 (48). All other data are included in the manuscript and/or SI Appendix.