Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

Domain: biology
Published: 2025-12-31
Editorial outcome: major_revision

Abstract
Low-protein diets can mitigate renal fibrosis, yet the critical amino acids responsible for this benefit and its underlying mechanism remain unclear. Using exclusively male mice throughout this study, we screen 15 amino acid–restricted diets in a unilateral ureteral obstruction model and identify methionine restriction (MetR) as the most effective intervention. Integrating transcriptomic and cistromic analyses identify Hoxc8 as a central pro-fibrotic transcription factor downstream of TGF-β–Smad3 signaling. Hoxc8 reinforces its own expression and drives fibrotic gene programs through recruitment of the P-TEFb transcriptional elongation complex. HOXC8 is elevated in fibrotic human kidneys, and fibroblast-specific Hoxc8 deletion protects mice from renal fibrosis. MetR suppresses this pro-fibrotic circuit by reducing active histone marks at the Hoxc8 locus, thereby attenuating Hoxc8-dependent transcription. Together, these findings identify the TGF-β–Smad3–Hoxc8/P-TEFb axis as a key driver of renal fibrosis and highlight MetR as a promising therapeutic strategy.

Introduction
Chronic kidney disease (CKD) is an escalating global health concern, affecting an estimated 13.4% (11.7–15.1%) of the world’s population. CKD arises from a wide range of underlying conditions that cause sustained damage to renal function and structure. Common contributors include diabetes, hypertension, and chronic inflammatory conditions such as nephritis. Despite this diverse etiology, renal fibrosis, characterized by the excessive buildup of extracellular matrix (ECM) within the renal interstitium, emerges as the shared pathological pathway that drives CKD progression, regardless of the initial cause. Fibrosis leads to a gradual replacement of functional kidney tissue with scar tissue, severely impairing filtration capacity. This progressive loss of kidney function ultimately results in end-stage renal disease (ESRD), necessitating dialysis or transplantation for survival. Fibrosis is thus a critical target in CKD management, as its inhibition could delay or prevent the onset of ESRD, significantly improving patient outcomes and quality of life.

The activation of ECM-producing myofibroblasts is a crucial event in the pathogenesis of renal fibrosis. These myofibroblasts originate from diverse sources, including kidney pericytes, fibroblasts, circulating cells, and even epithelial or endothelial cells within the kidney. Recent studies have highlighted kidney fibroblasts and pericytes as major contributors to the myofibroblast population, emphasizing their role in fibrotic tissue remodeling. Transforming growth factor-β (TGF-β) is essential for myofibroblast activation. Upon binding to its receptor, TGF-β activates the Smad signaling pathway, inducing the expression of α-smooth muscle actin (α-SMA) and ECM proteins. This cascade drives myofibroblast differentiation, leading to excessive ECM deposition that gradually replaces functional kidney tissue with scar tissue, ultimately impairing kidney function. Currently, anti-fibrotic strategies for treating CKD primarily focus on inhibiting the TGF-β signaling pathway to reduce ECM deposition. However, TGF-β plays a complex role in numerous physiological processes, including immune regulation, cell growth, differentiation, and tissue homeostasis. Directly targeting this pathway may lead to unintended adverse effects. Therefore, there is an urgent need to develop therapeutic strategies specifically tailored to renal fibrosis that minimize systemic side effects.

Nutritional interventions are crucial in the management of CKD, offering dietary strategies that can slow disease progression and reduce the risk of comorbidities and mortality. Among these approaches, regulating protein intake has shown significant impact on CKD outcomes. High-protein diets are linked to increased glomerular pressure and damage, which accelerates CKD progression. In contrast, low-protein diets reduce kidney workload by lowering glomerular pressure, alleviating stress on the kidneys. Studies have demonstrated that low-protein diets can reduce proteinuria by 20–50% in CKD patients, showing a potential benefit in preserving kidney function. However, long-term adherence to low-protein diets raises concerns about protein-energy malnutrition and protein-energy wasting, particularly in patients with advanced CKD who are already at risk of nutritional deficiencies. This challenge has prompted growing interest in targeted amino acid restrictions as a strategy to reduce renal fibrosis and slow CKD progression, while avoiding the adverse effects associated with broad protein restriction.

Methionine is critical during embryonic development in both animals and humans, serving as an essential component for rapid cell division and organ formation. However, its physiological demand declines significantly in adulthood. Methionine restriction (MetR) has been demonstrated to extend lifespan across various animal models and has shown promise in reducing age-related diseases, suppressing tumor growth, and enhancing the efficacy of cancer treatments. It also plays a significant role in gene expression by influencing methylation levels on RNA, DNA, and histones. MetR has shown promise in alleviating renal fibrosis, as evidenced by its protective effects in a subtotal nephrectomy model of CKD. This finding establishes the therapeutic potential of MetR; however, its efficacy compared with other types of amino acid restriction remains unclear, and the precise molecular mechanisms underlying its benefit are still largely unknown.

In this study, a systematic screen of 15 amino acid-restricted diets in unilateral ureteral obstruction (UUO) mice revealed MetR as the superior strategy for alleviating renal fibrosis, a finding confirmed in folic acid nephropathy (FAN) mouse model. We identified Hoxc8 as a key transcription factor responsive to MetR, mediating TGF-β-induced fibrotic gene expression. TGF-β-Smad3 activates Hoxc8, triggering its self-activation and interaction with P-TEFb to enhance fibrotic gene expression. MetR reduces fibrotic gene expression by decreasing histone modifications (H3K4me3 and H3K36me3) required for Hoxc8 activation. Fibroblast-specific Hoxc8 knockout mice showed reduced fibrosis following UUO surgery. Additionally, elevated HOXC8 expression was observed in patients with kidney fibrosis, including those with IgA nephropathy and autosomal dominant polycystic kidney disease (ADPKD).

Results
Systematic screening of amino acid restriction diets reveals MetR as the most effective in reducing renal fibrosis
To assess the impact of amino acid restrictions on renal fibrosis, we administered 15 distinct amino acid restriction diets in a unilateral ureteral obstruction (UUO) mouse model of renal fibrosis. Following UUO surgery, the mice were randomly assigned to 16 groups: one group was maintained on a standard diet, while the remaining groups were placed on diets restricting individual amino acids, including methionine (MetR), arginine (ArgR), leucine (LeuR), lysine (LysR), proline (ProR), histidine (HisR), cysteine (CysR), aspartic acid (AspR), tryptophan (TrpR), glutamine (GlnR), threonine (ThrR), valine (ValR) and isoleucine (IleR). Additionally, some groups were subjected to diets restricting pairs of amino acids that share the same metabolic pathway, specifically serine and glycine (Ser&GlyR), tyrosine and phenylalanine (Tyr&PheR). The specific restriction levels for each amino acid diet are detailed in Supplementary Table S1. These formulations were selected based on established dietary models from prior studies, which have demonstrated their metabolic efficacy and tolerability. After 14 days, kidney samples were collected for fibrosis analysis (Fig. 1A). To ensure that the dietary restrictions did not adversely impact overall health, we closely monitored the general condition and body weight of each mouse. As shown in Fig. S1, all mice, including those on the standard diet, experienced a slight decrease in body weight by day 7 post-surgery, with most regaining their preoperative weight by day 14. However, mice in the ThrR, Ser&GlyR, Tyr&PheR, ValR, and IleR groups continued to lose weight by day 14 and appeared thin and unhealthy, so we excluded these groups from further analysis. Masson staining revealed that LysR, ProR, HisR, CysR, AspR, TrpR, and GlnR had no notable effect on renal fibrosis, while MetR, ArgR, and LeuR, led to varying degrees of fibrosis reduction (Fig. 1B). Among these, MetR showed the most pronounced effect in reducing fibrosis (Fig. 1C), highlighting its potential as an effective intervention.

To further evaluate the impact of MetR diet on renal fibrosis, we assessed the expression of a key fibrotic marker, α-smooth muscle actin (α-SMA), in UUO mice fed either a standard or MetR diet. Immunohistochemical staining revealed a significant reduction in α-SMA expression in UUO mice on the MetR diet (Fig. 1D). Additionally, mRNA levels of Acta2 (encoding α-SMA), as well as two other fibrotic markers, collagen type I alpha 1 (Col1a1) and collagen type III alpha 1 (Col3a1), were notably decreased in UUO mice on the MetR diet (Fig. 1E). These findings demonstrate that MetR effectively suppresses the expression of fibrotic markers, underscoring its potential to slow fibrosis progression.

Both pre- and post-UUO MetR interventions effectively attenuate renal fibrosis
We next investigated the impact of the timing of MetR treatment on its therapeutic efficacy in renal fibrosis. MetR was administered either one week prior to UUO surgery or two days after. In the first approach, MetR was initiated one week before surgery, with renal fibrosis assessed 14 days post-surgery (Fig. 2A). This strategy models early-stage intervention, aimed at reducing the risk of renal fibrosis before significant damage occurs. Fibrosis evaluation, including Masson’s trichrome staining (Fig. 2B), immunohistochemical staining for α-SMA (Fig. 2C), and RT-qPCR analysis of Acta2, Col1a1, and Col3a1 mRNA levels (Fig. 2D), demonstrated that pre-treatment with MetR markedly attenuated UUO-induced fibrosis.

In the second approach, MetR was introduced two days after UUO surgery (Fig. 2E), modeling a clinical scenario where treatment is initiated shortly after the onset of disease or injury. As shown in Fig. 2F–H, post-surgical MetR treatment also substantially reduced Masson’s trichrome staining, immunohistochemical staining for α-SMA, and mRNA levels of Acta2, Col1a1, and Col3a1 in UUO mice. These findings indicate that both pre- and post-UUO MetR interventions effectively attenuate renal fibrosis. Notably, the results were comparable to those observed when MetR was administered on the day of UUO surgery, suggesting that the timing of MetR intervention—whether preventive or therapeutic—does not significantly impact its effectiveness in reducing UUO-induced renal fibrosis.

MetR attenuates folic acid-induced renal fibrosis
To further evaluate the therapeutic potential of MetR, we employed the folic acid nephropathy (FAN) model, another established murine model of renal fibrosis (Fig. S2A). Compared to mice on a normal diet, those on the MetR diet exhibited significantly reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels (Fig. S2B), indicating improved renal function. Histological analysis revealed a notable reduction in fibrotic areas, as evidenced by Masson staining (Fig. S2C), along with a significant decrease in α-SMA staining, suggesting suppressed myofibroblast activation (Fig. S2D). Additionally, MetR markedly downregulated the mRNA expression of key fibrosis marker genes, including Acta2, Col1a1, and Col3a1 (Fig. S2E). Together, these findings demonstrate the effectiveness of MetR in attenuating renal fibrosis and preserving kidney function in the FAN mouse model.

MetR inhibits fibrotic gene expression during myofibroblast activation
The activation of myofibroblasts is a key event in the progression of renal fibrosis, with the TGF-β signaling pathway playing a critical role in this process. Fibroblasts are one of the primary sources of myofibroblasts, and during renal fibrosis, local fibroblasts are activated by growth factors such as TGF-β, leading to their transformation into myofibroblasts. To determine whether MetR alleviates renal fibrosis by modulating TGF-β-induced myofibroblast activation, we first assessed the influence of MetR on gene expression during the TGF-β-induced transformation of NRK-49F cells, a commonly used renal fibroblast line, into myofibroblasts (Fig. 3A). As shown in Fig. S3A, TGF-β treatment resulted in substantial alterations in gene expression, with 714 genes upregulated and 991 genes downregulated. Gene ontology (GO) analysis revealed that the upregulated genes were predominantly associated with extracellular matrix remodeling, actin dynamics, and TGF-β signaling, whereas the downregulated genes were linked to cell cycle regulation, chromosome segregation, and stress response pathways (Fig. S3B).

Notably, MetR selectively targeted genes upregulated by TGF-β, reversing the expression of 317 genes, which we classified as “MetR-suppressed genes.” In contrast, 397 genes remained unaffected and were categorized as “MetR non-suppressed genes” (Fig. 3B). Interestingly, MetR exerted minimal effects on genes downregulated by TGF-β, indicating that its primary action is on genes implicated in fibrosis progression. GO analysis revealed that MetR-suppressed genes were significantly enriched in fibrotic pathways, including growth factor signaling, extracellular matrix organization, and collagen biosynthesis. In contrast, the MetR non-suppressed genes were associated with non-fibrotic processes such as tube morphogenesis, angiogenesis, and cell migration (Fig. 3C).

At the protein level, MetR treatment suppressed the TGF-β-induced upregulation of key myofibroblast markers, including α-SMA, fibronectin, and Col1a1 (Fig. 3D). Similarly, TGF-β treatment robustly upregulated the mRNA expression of key fibrotic markers, including Acta2, Col1a1, and Col3a1, while the MetR treatment effectively attenuated these increases (Fig. 3E). Collectively, these findings suggest that MetR suppresses TGF-β-induced myofibroblast activation by modulating the expression of fibrosis-related genes.

MetR suppresses fibrotic gene expression by inhibiting Hoxc8
Transcription factors (TFs) are essential regulators of gene expression. To identify the candidate TFs mediating the suppression of fibrotic gene expression by MetR, we applied three criteria: (1) TF motifs enriched in the regulatory regions of TGF-β-induced fibrotic genes, (2) TFs whose expression was upregulated by TGF-β treatment, and (3) TFs whose expression was suppressed by MetR treatment. As shown in Fig. 4A, motif analysis of TGF-β-induced genes revealed significant enrichment of motifs from several TF families, including MADs, TBP, and Homeobox (Hox). We further identified the top 10 TFs from these families, which showed substantial upregulation in response to TGF-β treatment (Fig. 4B). Based on these findings, we selected four TFs—Hoxc8, Hoxc9, Prrx2, and Hoxb9—for further validation, as their expression was notably downregulated by MetR. RT-qPCR analysis confirmed that the mRNA expression of Hoxc8, Prrx2, and Hoxb9 was induced by TGF-β and subsequently suppressed by MetR (Fig. 4C). However, Hoxc9 did not show significant changes in response to either TGF-β or MetR and was thus excluded from further analysis.

To further investigate the functional role of the three candidate TFs—Hoxc8, Prrx2, and Hoxb9—in regulating fibrotic gene expression, we evaluated the effects of their knockdown on the expression of three key fibrotic markers: Acta2, Col1a1, and Col3a1. Among these TFs, knockdown of Hoxc8 exerted the most significant impact, markedly reducing the TGF-β-induced expression of these fibrotic markers (Fig. 4D). The protein expression of Hoxc8, consistent with its mRNA, was induced by TGF-β and suppressed by MetR (Fig. 4E). Crucially, Hoxc8 depletion effectively abolished the TGF-β-driven upregulation of α-SMA, fibronectin, and Col1a1 proteins (Fig. 4F).

To determine whether Hoxc8 is essential for MetR-mediated suppression of fibrotic gene expression, we conducted RNA-seq analysis to evaluate the effects of Hoxc8 knockdown on fibrotic gene expression. As shown in Fig. 4G, knockdown of Hoxc8 reversed the expression of 330 TGF-β-upregulated genes, referred to as “siHoxc8-suppressed genes,” while 384 genes remained unaffected. An integrated analysis of the MetR- and siHoxc8-suppressed genes revealed an overlap of 192 genes, which were predominantly involved in key fibrotic pathways, such as extracellular matrix organization and collagen formation (Fig. 4H). We selected two representative genes from this dataset—Vim and Ccn1—and performed RT-qPCR validation. The results confirmed that their TGF-β-induced upregulation was markedly attenuated upon Hoxc8 knockdown (Fig. S4A). Collectively, these findings suggest that both MetR and Hoxc8 knockdown converge on the same fibrotic gene regulatory networks to suppress TGF-β-induced fibrotic gene expression.

Notably, overexpression of Hoxc8 in NRK-49F cells reversed the MetR-mediated downregulation of fibrotic markers, including Acta2 and Vim (Fig. 4I). Consistently, Hoxc8 overexpression also prevented the reduction in α-SMA, fibronectin, and Col1a1 protein levels induced by MetR (Fig. 4J). Together, these results indicate that MetR suppresses fibrotic gene expression by inhibiting Hoxc8.

MetR suppresses Hoxc8 and fibrotic gene expression by reducing H3K4me3 and H3K36me3 histone modifications
Methionine, as a precursor to S-adenosylmethionine (SAM), serves as a methyl donor for histone methylations that play crucial roles in gene regulation. We then investigated the impact of MetR on histone methylations in NRK-49F cells treated with TGF-β. As shown in Fig. 5A, MetR markedly reduced global levels of H3K4me3 and H3K36me3—modifications strongly associated with gene activation—while having minimal impact on H3K9me3 and H3K27me3, which are linked to gene repression.

To further examine the effects of MetR on the distribution of H3K4me3 and H3K36me3 at TGF-β-induced fibrotic genes, we conducted ChIP-seq analyses in NRK-49F cells treated with TGF-β and MetR. As shown in Fig. 5B, C, TGF-β treatment led to increased enrichment of H3K4me3 and H3K36me3 at TGF-β-upregulated genes, which was substantially reversed by MetR. Figure 5D presents ChIP-seq tracks for three representative fibrotic genes, Acta2, Col1a1, and Col3a1. Notably, MetR also reduced TGF-β-induced enrichment of H3K4me3 and H3K36me3 on Hoxc8 gene loci (Fig. 5E).

Consistent with these in vitro findings, we observed that MetR significantly reduced the levels of H3K4me3 and H3K36me3 in the kidney tissues of UUO mice (Fig. S5A, B). This further supports the role of epigenetic regulation in controlling Hoxc8 expression and fibrotic gene activation in vivo.

Taken together, these findings suggest that MetR downregulates Hoxc8 and fibrotic gene expression by reducing histone methylation modifications that are associated with transcriptional activation. By reversing the TGF-β-induced increase in H3K4me3 and H3K36me3 on Hoxc8 and other fibrotic genes, MetR effectively suppresses the fibrotic transcriptional program, highlighting a potential mechanism for its anti-fibrotic effects.

Smad3 is essential for TGF-β-induced Hoxc8 activation
To investigate how TGF-β induces Hoxc8 activation, we performed a motif analysis of the Hoxc8 gene, which revealed a Smad3 binding site on the Hoxc8 promoter (Fig. S6A), suggesting that Smad3 may regulate Hoxc8 transcription. To test this, ChIP-qPCR analysis of Smad3 was conducted in NRK-49F cells. As shown in Fig. S6B, TGF-β treatment significantly enhanced Smad3 occupancy at Hoxc8 promoter, indicating that Smad3 is recruited to this region in response to TGF-β signaling.

Furthermore, TGF-β treatment markedly upregulated both Hoxc8 and α-SMA expression at the protein and mRNA levels, while Smad3 knockdown effectively suppressed these increases (Fig. S6C, D). Consistently, treatment with SIS3, a Smad3 phosphorylation inhibitor, significantly reduced TGF-β-induced expression of Hoxc8 and α-SMA at both the protein and mRNA levels (Fig. S6E, F). Collectively, these findings indicate that Smad3 is indispensable for TGF-β-induced Hoxc8 activation.

TGF-β induces Hoxc8 self-activation and recruitment to fibrotic genes
To investigate how Hoxc8 regulates TGF-β-induced fibrotic genes, we conducted Hoxc8 ChIP-seq in NRK-49F cells treated with TGF-β. As shown in Fig. 6A, TGF-β treatment substantially enhanced Hoxc8 recruitment to chromatin regions. Gene Set Enrichment Analysis (GSEA) revealed that genes with increased Hoxc8 binding following TGF-β treatment were enriched among TGF-β-upregulated genes (Fig. 6B), as well as genes suppressed by MetR treatment (Fig. 6C) and Hoxc8 knockdown (Fig. 6D).

Interestingly, Hoxc8 ChIP-seq analysis revealed that Hoxc8 binds to its own gene locus (Fig. 6E), suggesting a potential self-regulatory mechanism. Further investigation of the Hoxc8 locus identified two potential Hoxc8 binding sites within regions enriched for Hoxc8 ChIP-seq signals (Fig. 6F). To test Hoxc8’s self-regulation capability, we constructed luciferase reporters containing either wild-type (WT) or mutant versions of these Hoxc8 binding sequences (Fig. 6F). Overexpression of Hoxc8 significantly activated reporters with WT Hoxc8 sequences, whereas those with mutant sequences were not activated (Fig. 6G), demonstrating that Hoxc8 can directly activate its own expression through these binding sites. To further validate Hoxc8’s self-activation, we overexpressed exogenous human HOXC8 (TY1-HOXC8) in NRK-49F cells. This led to a marked increase in endogenous rat Hoxc8 expression, both at the mRNA level (Fig. 6H) and protein level (Fig. 6I). Collectively, these findings suggest that TGF-β induces Hoxc8 expression, which in turn self-activates to amplify its own expression and drive the activation of fibrotic genes.

Hoxc8 interacts with P-TEFb to facilitate the transcription elongation of fibrotic genes
To further explore the mechanisms by which Hoxc8 regulates fibrotic gene expression, we performed immunoprecipitation (IP) followed by mass spectrometry in FLAG-HOXC8 stably expressing cells. As shown in Fig. S7, we identified cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CycT1), key components of the transcription elongation factor P-TEFb, which were enriched in the Hoxc8-IP samples. P-TEFb is known to play a critical role in transcription elongation and has been reported to modulate TGF-β signaling. To validate this interaction, we performed IP assays in 293T cells, confirming the binding of Hoxc8 to P-TEFb (Fig. 7A, B). Co-immunoprecipitation (Co-IP) in NRK-49F cells treated with TGF-β further demonstrated that endogenous Hoxc8 interacts with both CDK9 and CycT1 (Fig. 7C). Together, these results suggest that Hoxc8 forms a functional complex with P-TEFb, potentially influencing transcriptional regulation in response to TGF-β signaling.

P-TEFb promotes transcriptional elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II) at serine 2, facilitating its release from a paused state and enabling efficient gene transcription. Given its interaction with P-TEFb, we hypothesized that Hoxc8 enhances transcriptional elongation of fibrotic genes. To test this, we performed ChIP-seq for Hoxc8 and phosphorylated Pol II at serine 2 (Pol II Ser2P), a marker of active elongation. ChIP-seq analysis revealed reduced Hoxc8 binding and diminished Pol II Ser2P enrichment on fibrotic genes in Hoxc8 knockdown cells (Fig. 7D, E). To further assess transcriptional pausing, we calculated the pausing index, defined as the ratio of Pol II enrichment at the gene promoter to that at the gene body. An elevated pausing index indicates a delay in Pol II release from its paused state, hindering the transition to productive elongation. As shown in Fig. 7F, Hoxc8 knockdown resulted in a marked increase in the pausing index of siHoxc8-suppressed genes, suggesting that in the absence of Hoxc8, Pol II remains stalled at the promoter, impairing transcription elongation. These findings underscore that Hoxc8 is crucial for maintaining active transcription elongation of fibrotic genes.

Based on these findings, we propose a model (Fig. 7G) in which TGF-β induces Hoxc8 expression through Smad3 activation. Once upregulated, Hoxc8 undergoes self-activation and interacts with P-TEFb to promote transcriptional elongation of fibrotic genes, thereby amplifying the fibrotic response. MetR interferes with this process by downregulating key histone modifications, such as H3K4me3 and H3K36me3, which are essential for Hoxc8 expression. This TGF-β-Smad3-Hoxc8/P-TEFb self-reinforcing axis contributes to the progression of renal fibrosis by sustaining the expression of fibrotic genes.

Fibroblast-specific Hoxc8 knockout alleviates renal fibrosis in UUO mice
To further investigate the role of Hoxc8 in renal fibrosis in vivo, we first confirmed its upregulation in the UUO mouse model. Hoxc8 expression was notably elevated in the kidney of UUO mice compared to sham controls, and treatment with MetR resulted in a marked reduction in Hoxc8 levels (Fig. 8A).

To determine whether inhibiting Hoxc8 could alleviate myofibroblast activation in vivo, we generated fibroblast-specific Hoxc8 knockout (Hoxc8FKO) mice by inducing gene deletion with tamoxifen administration over five consecutive days in 5- to 7-week-old mice (Fig. 8B). Immunofluorescence analysis revealed that in Hoxc8 WT UUO mice, Hoxc8 expression was notably upregulated and co-localized with PDGFR-α-positive cells, indicating that Hoxc8 is upregulated in myofibroblasts during renal fibrosis (Fig. 8C). In contrast, Hoxc8FKO mice exhibited a complete absence of Hoxc8 in PDGFR-α-positive cells, accompanied by a marked reduction in PDGFR-α-positive cells, suggesting that fibroblast-specific Hoxc8 deletion effectively reduces myofibroblast activation (Fig. 8C).

In line with the reduction in myofibroblast activation, immunohistochemical staining for α-SMA showed a marked decrease in Hoxc8FKO mice compared to controls (Fig. 8D). Masson trichrome staining revealed decreased collagen deposition in Hoxc8FKO mice, indicating a reduction in renal fibrosis (Fig. 8E). Moreover, quantitative RT-PCR analysis demonstrated a marked downregulation of fibrotic markers, including Acta2, Col1a1, Col3a1, Vim, and Ccn1, in Hoxc8FKO mice (Figs. 8F and S4B). Collectively, these results suggest that fibroblast-specific deletion of Hoxc8 effectively alleviates renal fibrosis in the UUO model by reducing myofibroblast activation and collagen deposition.

Fibroblast-specific Hoxc8 deletion attenuates renal fibrosis in a 5/6 nephrectomy model
The UUO model is widely used to simulate rapid fibrotic processes induced by acute urinary tract obstruction, while the 5/6 nephrectomy model serves as a classical system for studying chronic progressive fibrosis. To further evaluate the pathological role of Hoxc8 in chronic fibrotic progression, we conducted functional validation in the 5/6 nephrectomy model (Fig. S8A). Results demonstrated that compared with wild-type mice, Hoxc8FKO mice showed significantly attenuated renal dysfunction after surgery, as evidenced by markedly reduced levels of BUN and serum creatinine (Fig. S8B, C). Histological and molecular analyses further revealed that Hoxc8 deletion effectively ameliorated renal fibrosis progression, manifested by improved fibrosis metrics in Masson staining and α-SMA immunohistochemistry, along with significantly downregulated mRNA expression of key fibrotic markers (Acta2, Col1a1, and Col3a1) (Fig. S8D–F). These consistent findings confirm that fibroblast-specific knockout of Hoxc8 can effectively improve renal function and suppress fibrosis progression in the 5/6 nephrectomy model, further establishing Hoxc8 as a key pro-fibrotic regulator across different kidney injury models.

Genetic ablation of tubular Hoxc8 attenuates renal fibrosis progression
Apart from fibroblasts, renal tubular epithelial cells are also considered crucial players in the progression of renal fibrosis. We observed that in fibroblast-specific Hoxc8 knockout mice, the UUO-induced upregulation of Hoxc8 expression was not completely abolished by the genetic deletion in fibroblasts (Fig. 8C). Given the established role of renal tubular epithelial cells in renal fibrosis, we hypothesized that the residual Hoxc8 signal might primarily originate from these cells. To test whether pro-fibrotic signaling can directly regulate Hoxc8 expression in tubular cells, we conducted experiments using the rat renal tubular epithelial cell line NRK-52E. As demonstrated in Fig. S9A, B, TGF-β treatment robustly induced Hoxc8 expression in these cells (Fig. S9A, B). Furthermore, Hoxc8 knockdown effectively counteracted TGF-β-mediated profibrotic responses, as evidenced by reduced protein levels of α-SMA, fibronectin, and Col1a1, and concomitant downregulation of Acta2, Col1a1, and Col3a1 transcripts (Fig. S9A, B). Importantly, MetR treatment mirrored these antifibrotic effects, suppressing both the protein expression and mRNA levels of these fibrotic markers (Fig. S9C, D).

To further elucidate the role of Hoxc8 in tubular cells during fibrosis progression, we generated tubular epithelial cell-specific Hoxc8 knockout mice (Hoxc8fl/fl; PEPCK-Cre, Hoxc8TKO). Immunofluorescence analysis confirmed cell-specific deletion, showing complete absence of Hoxc8 in SGLT2-positive tubular cells of Hoxc8TKO mice following UUO, whereas wild-type littermates exhibited strong Hoxc8 staining (Fig. S10A). This genetic ablation resulted in substantial protection against renal fibrosis, with Hoxc8TKO mice displaying attenuated α-SMA-positive myofibroblast activation (Fig. S10B), reduced collagen deposition (Fig. S10C), and significantly lower expression of fibrotic markers at the transcriptional level (Fig. S10D) compared to wild-type controls. Collectively, these findings demonstrate that Hoxc8 in tubular epithelial cells plays a critical role in promoting renal fibrosis progression.

Hoxc8 expression is elevated in CKD patients with renal fibrosis
To further explore the clinical relevance of Hoxc8 signaling in renal fibrosis, we examined HOXC8 expression in kidneys from patients with CKD associated with fibrosis. Immunohistochemical analysis of kidney biopsies from patients with immunoglobulin A nephropathy (IgAN) and autosomal dominant polycystic kidney disease (ADPKD), using para-carcinomatous tissues as controls, revealed elevated HOXC8 expression in both IgAN and ADPKD patients (Fig. 8G). Notably, in IgAN patients, HOXC8 expression negatively correlated with eGFR (Fig. 8H), suggesting that higher HOXC8 levels are associated with greater impairment of renal function.

Taken together, these findings demonstrate the critical role of Hoxc8 in renal fibrosis, both in animal models and human diseases. Fibroblast-specific deletion of Hoxc8 significantly reduces renal fibrosis, while its elevated expression in fibrotic kidney conditions highlights its potential as a therapeutic target to mitigate fibrosis and preserve renal function.

Methods
Study approval
This study was designed and conducted in compliance with Chinese regulations and the principles of the Declaration of Helsinki. The protocol involving ADPKD patient samples received approval from the Ethics Committee of Shandong Provincial Hospital (approval number: 2020-013), while the use of IgAN patient samples was authorized by the Ethics Committee of Peking University First Hospital (approval number: 2021KY404). Written informed consent was obtained from all human participants. Additionally, all animal experiments performed in this research were approved by the Ethics Committee of Tianjin Medical University (approval number: TMUaMEC 2022018). Animal euthanasia was performed in accordance with the approved institutional animal care and use protocol and the Guidelines for Euthanasia of Laboratory Animals (GB/T 39760-2021). Mice were placed in a dedicated chamber and euthanized by carbon dioxide (CO₂) inhalation administered by trained personnel. Following loss of consciousness, death was ensured using a secondary physical method. Death was confirmed prior to tissue collection by the absence of spontaneous breathing, cardiac activity, and reflex responses.

Animal models
Eight-week-old male C57BL/6 mice were sourced from Beijing Charles River (Beijing, China). The mice were housed under controlled conditions, including a 12-h light-dark cycle at 21–25°C with 30–70% humidity. All amino acid-restricted diets were purchased from Jiangsu Xietong Pharmaceutical Bio-engineering, China (Supplement Table 1). Hoxc8 flox/flox mice were generated by Cyagen, China, and subsequently bred with Col1a2-CreERT mice and PEPCK-Cre mice to produce fibroblast-specific Hoxc8 knockout mice and tubular cells-specific Hoxc8 knockout mice, respectively. For Hoxc8FKO mice, tamoxifen (75 mg/kg body weight, 20 mg/ml in corn oil) was administered intraperitoneally to the mice for five consecutive days at 5–7 weeks of age, as previously described. Following tamoxifen treatment, the mice were assigned to experimental groups randomly. Unilateral ureteral ligation (UUO) was performed according to established protocols. Subsequently, the mice were placed on different amino acid-restricted diets immediately after UUO surgery. After a 14-day period, the mice were euthanized, and obstructed kidneys were collected for the subsequent experiments described below. The folic acid (FA) mouse model was established through intraperitoneal administration of folic acid (F8758, Sigma) at a dosage of 200 mg/kg body weight. At the end of a 8-weeks period, the mice were sacrificed, and the kidneys were harvested for the subsequent experiments detailed below. For the five-sixths nephrectomy (5/6 Nx) model, a two-step surgical procedure was performed as follows: Initially, the left kidney was exposed, and its upper and lower poles were resected; the surgical site was then covered with a hemostatic gelatin sponge to control bleeding. Seven days later, the right kidney was completely removed through a flank incision. Mice were euthanized at 8 weeks after the second surgery. Following deep anesthesia with isoflurane, mouse kidney tissues were harvested. Prior to harvest, blood was collected via cardiac puncture and the kidneys were perfused with PBS.

Masson staining
Renal specimens were fixed in formalin overnight and embedded in paraffin. Four-micrometer sections were prepared for Masson staining. The relative area of renal tubulointerstitial fibrosis was evaluated at ×400 magnification. To assess renal interstitial fibrosis, ten non-overlapping fields of vision were randomly selected in each section, and the percentage of the tubulointerstitial fibrosis area relative to the total tubulointerstitial area within the same field was determined. The average percentage was calculated to represent the relative area of renal interstitial fibrosis for each section.

Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution). Deparaffinized and rehydrated sections underwent citrate antigen retrieval. Following incubation with 10% bovine serum, sections were exposed to primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ZLI-9018, ZSGB-BIO, 1:20 dilution). To calculate the H score, the staining intensity of the nucleus (0, 1+, 2+, or 3+) is first assessed for each cell within a designated field. Next, the number of cells corresponding to each staining intensity level is quantified. The H score for nuclear HOXC8 is then derived using the formula: (1 × number of cells with staining intensity 1+) + (2 × number of cells with staining intensity 2+) + (3 × number of cells with staining intensity 3+).

Cell Culture and Treatment
NRK-49F cells and NRK-52E cells were procured from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 4.5 g/L glucose and 10% fetal calf serum (Viva cells) at 37°C in a 5% CO2 atmosphere. Prior to treatment, NRK-49F cells were starved for 24 h with DMEM containing 0.2% FBS and then exposed to 5 ng/μL of TGF-β1 (240-B, R&D Systems). For methionine restriction (MetR) treatment, the medium was composed of Eagle’s balanced salt solution 10× (E7510, Sigma-Aldrich), MEM vitamins solution 100× (M6895, Sigma-Aldrich), NaHCO3, dialyzed FBS (04-011-1B, Vivacells), MilliQ water, and amino acids at the following concentrations: L-methionine (Met, 200 μM for Ctrl, 30 μM for MetR), L-leucine (Leu, 380 μM), L-valine (Val, 170 μM), L-arginine (Arg, 1,160 μM), L-phenylalanine (Phe, 90 μM), L-threonine (Thr, 170 μM), L-lysine monohydrochloride (Lys, 220 μM), L-histidine hydrochloride (His, 100 μM), L-isoleucine (Ile, 380 μM), L-tryptophan (Trp, 20 μM), L-serine (Ser, 285 μM), L-cystine (Cys, 270 μM), L-glutamine (Gln, 2000 μM), L-Tyrosine disodium salt (Tyr), and L-glycine (Gly, 130 μM). All amino acids were obtained from Sigma-Aldrich.

siRNA knockdown assay
siRNAs were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) at a final concentration of 20 nM. Cells treated with TGF-β were simultaneously transfected with siRNA. The siRNA sequences used are listed in the Supplementary Table 2.

RNA Isolation and RT-qPCR
Total RNA was extracted from whole kidney tissue using TRIzol (15596018, Invitrogen). Reverse transcription was performed using a cDNA Synthesis Kit (4897030001, Roche) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted in a 96-well reaction plate to detect mRNA expression. The primer sequences are listed in the Supplementary Table 3.

Immunoblotting
Cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche) and PhosSTOPTM (Roche). Following the addition of SDS loading buffer, the samples were boiled for 10 min. Protein samples were separated by SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with primary antibodies. Antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution), α-SMA (ab5694, Abcam, 1:100 dilution), Col1a1 (BOSTER, BA0325), Fibronectin (Sigma, F3648), GAPDH (MAB347, Millipore), H3K4me3 (07473, Millipore), H3K36me3 (ab9050, Abcam), H3K9me3 (ab8898, Abcam), H3K27me3 (ab6002, Abcam,), H3, FLAG-HRP (F7425, Sigma), HA-tag (3724, Cell Signaling Technology), Cyclin T1 (sc-271348, Santa Cruz), and CDK9 (sc-8338X, Santa Cruz), SMAD3 (9523, Cell Signaling Technology), P-SMAD3 (9520, Cell Signaling Technology) were used.

Co-immunoprecipitation (Co-IP)
Cell pellets were resuspended in ice-cold NP-40 lysis buffer (1% NP-40, 150 mM sodium chloride, 50 mM Tris, pH 8.0) supplemented with a protease inhibitor cocktail (Roche) and then centrifuged at 12,000 × g for 20 min at 4°C. For samples containing FLAG-tagged proteins, the supernatants were incubated with anti-FLAG M2 affinity gel overnight at 4°C. In cases of other immunoprecipitations, the supernatants were subjected to overnight incubation with the specified antibodies at 4°C. Dynabeads Protein G were introduced and allowed to bind for 4 h at 4°C. After 4 washes with NP-40 lysis buffer, SDS loading buffer was added, and the samples were boiled for 10 min. The resulting supernatants were subjected to immunoblotting using antibodies against Hoxc8 (15448-1-AP, Proteintech, diluted 1:200), FLAG-HRP (Sigma, F7425), HA-tag (3724, Cell Signaling Technology), Cyclin T1 (sc-271348, Santa Cruz), and CDK9 (sc-8338X, Santa Cruz).

ChIP-seq
ChIP assays were performed as previously described. For Hoxc8, Pol II and Pol II S2P, NRK-49F cells were crosslinked with 1% formaldehyde for 15 min at room temperature (RT), and the reaction was quenched with 125 mM glycine for 5 min at RT. Subsequently, cells were lysed in lysis buffer (150 mM NaCl, 0.2% sodium dodecyl sulfonate (SDS), 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM EDTA, 50 mM Tris-HCl pH 8.0) and sonicated to shear DNA into fragments ranging from 200 to 500 bp. For histone modifications, NRK-49F cells were lysed in a lysis buffer containing 0.5% NP-40, 0.5% Tween-20, and 0.1% SDS. The chromatin was then digested into mononucleosomes using micrococcal nuclease (N3577, Sigma Aldrich). The resulting chromatin was diluted with an equal volume of dilution buffer (150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0) and incubated overnight at 4°C with specific antibodies: Hoxc8 (15448-1-AP, Proteintech, 2 µg), Pol II (14958S, Cell Signaling Technology, 2 µg), Pol II S2P (13499S, Cell Signaling Technology, 2 µg), histone modifications antibodies 0.5 µg. Following elution and reverse crosslinking, the DNA fragments were utilized for library amplification using a VAHTS Universal Library Prep Kit (ND601, Vazyme) following the manufacturer’s instructions.

ChIP-seq reads were trimmed using Trim Galore and aligned to the rat reference genome (rn6) using Bowtie (v2.3.5.1). Duplicate reads were removed using Picard (v2.26.10), and alignment quality was assessed using SAMtools (v1.12). Peak calling was performed using HOMER to identify enriched regions, followed by peak annotation and differential peak analysis using HOMER’s annotatePeaks.pl and getDifferentialPeaks.pl tools, respectively. Visualization of ChIP-seq data, including heatmaps and profile plots, was conducted using deepTools (v3.5.1).

ChIP–qPCR
After incubating the SMAD3 antibody according to the above ChIP-seq process, elution and reversal of cross-linking, DNA was purified, and qRT–PCR was performed to detect DNA level. The primer sequences are listed in the Supplementary Table 3.

RNA-seq
Total RNA was extracted from cells using TRIzol, and its integrity was evaluated using a Qubit 4 analyzer. RNA-seq libraries were constructed using total mRNA and sequenced on the Illumina platform to generate paired-end reads. Reads were aligned to the rat reference genome (rn6) using Hisat2, and gene quantification was performed with featureCounts (v1.6.0). Differential expression analysis was conducted using the DESeq2 package, and genes with a fold change > 2 and a false discovery rate (FDR) < 0.05 were considered differentially expressed genes (DEGs). In the TGF-β treatment, genes upregulated in response to TGF-β that showed a decrease in expression by more than 1.5-fold after a subsequent treatment were defined as suppressed genes. Functional enrichment analysis of DEGs was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to explore associated biological processes and pathways. All sequencing data have been deposited in the GEO database and are publicly accessible online.

Luciferase assay
4× Hoxc8 WT binding sites or Mutant binding sites sequence were synthesized by GENEWIZ (China). A total of 500 ng DNA (including 100 ng of 4× Hoxc8 WT binding sites or Mutant binding sites luciferase reporter construct and the 100 ng pcDNA5-Hoxc8 plasmids) was transfected into NRK-49F cells using polyethyleneimine (PolyScience). 24 h later after transfection, the luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega).

Mass spectrometry
To identify proteins interacting with HOXC8, HEK293T cells were transfected with a FLAG-tagged HOXC8-expressing plasmid. Biological replicates = 1. The cells were lysed in NP-40 lysis buffer (150 mM sodium chloride, 1% NP-40, 50 mM Tris pH 8.0) supplemented with a protease inhibitor cocktail (Roche) and then centrifuged at 12,000 × g for 20 min at 4°C. The resulting supernatants were incubated overnight at 4°C with anti-FLAG M2 affinity gel. Following four washes with NP-40 lysis buffer, FLAG protein complexes were eluted using FLAG peptide (Sigma). The eluates were resolved on a NuPAGE 4–12% Bis–Tris gel (Invitrogen) and visualized via silver staining. Gel bands of interest were subjected to in-gel digestion. After desalting, each tryptic digest was redissolved in HPLC solvent A (0.1% (v/v) formic acid in water) and injected into a nano-LC system (EASY-nLC 1200, Thermo Fisher Scientific). Each sample was separated by a C18 column (75 μm inner-diameter × 15 cm, 3 μm) with a 60 min HPLC gradient at a flow rate of 300 nL/min. The HPLC gradient was as follows: 5% to 13% solvent B (0.1% formic acid in 80% acetonitrile and 20% H2O (v/v/v)) in 10 min, 13% to 28% solvent B in 28 min, 28% to 40% solvent B in 12 min, 40% to 100% solvent B in 2 min and hold for 8 min at 100% solvent B. The HPLC elute was electrosprayed directly into a Q-Exactive Plus mass spectrometer (Thermo Scientific). Spray voltage was set to 2.4 kV and ion transfer tube temperature was 320°C. MS raw data files were processed with Proteome Discoverer (v. 3.0) against the UniprotKB/Swiss-Prot Human Proteome database, excluding protein isoforms. Trypsin specificity was applied for the peptide sequence search and a maximum of two missed cleavages was allowed. Carbamidomethylation on cysteine was specified as a fixed modification. Methionine oxidation and acetylation on N-terminal were set as variable modification, respectively. The minimal peptide length was set to six. Mass tolerances for precursor ions were set at ±10 ppm and ±0.02 Da for MS/MS.

Human samples
A total of 23 patients diagnosed with ADPKD (13 males, 10 females) were enrolled from Shandong Provincial Hospital, with a median age of 52 (range: 21–72). Separately, a cohort of 40 patients with IgAN (25 males, 15 females) was recruited from Peking University First Hospital; this group had a median age of 37 (range: 18–68).

Statistics
Statistical significance was determined using the two-tailed unpaired t-test to compare two groups of independent samples with GraphPad Prism Software. P values less than 0.05 were considered statistically significant, and results were presented as means ± SEM for at least three independent experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Liu et al. presents a well-constructed and mechanistically insightful study that explores methionine restriction (MetR) as a dietary intervention to mitigate renal fibrosis. The authors provide compelling evidence for the involvement of the TGF-β–Smad3–Hoxc8/P-TEFb axis in the regulation of fibrotic gene expression and demonstrate how MetR mediates epigenetic reprogramming to suppress this pathway. The data are convincing, and the study offers valuable translational implications. However, several points require further clarification or expansion to strengthen the manuscript’s impact and scientific rigor.:\n1. The authors applied varying degrees of dietary restriction for different amino acids in the UUO model to assess their respective effects on renal fibrosis. However, the rationale behind this multi-level restriction design remains unclear. Was the selection of restriction levels based on prior dietary or metabolic studies? Were preliminary dose-finding or tolerability assessments performed for each amino acid? A brief explanation of the design strategy would help readers better understand the robustness and physiological relevance of the dietary intervention model.\n2. In Figure 4, the changes in Hoxc8 expression are assessed solely at the mRNA level by RT-qPCR. Given the central role of Hoxc8 in the proposed signaling axis, validation at the protein level—e.g., by Western blotting or immunofluorescence staining—would significantly strengthen the conclusions and support the translational relevance of Hoxc8 as a potential therapeutic target.\n3. In Figure 4, the authors present RNA-seq data identifying a subset of genes whose TGF-β-induced expression is suppressed by Hoxc8 knockdown. However, the study does not include independent validation of representative target genes using RT-qPCR. Moreover, the relevance of these transcriptional changes has not been assessed in vivo in mouse models. Incorporating such validation would enhance the robustness of the transcriptomic findings and further substantiate the role of Hoxc8 in regulating fibrosis-associated gene networks.\n4. The authors demonstrate that MetR leads to a significant reduction in activating histone marks (H3K4me3 and H3K36me3), while repressive marks (H3K27me3 and H3K9me3) remain largely unaffected. This observation raises interesting mechanistic questions:Why are only activating marks sensitive to methionine availability? Is this due to differential regulation of histone methyltransferases or demethylases in response to SAM/SAH levels? A discussion of potential mechanisms or relevant literature would enhance the depth and mechanistic insight of the manuscript.\n5. Figures 8C show elevated Hoxc8 expression in renal tubular compartments during fibrosis. However, direct functional evidence linking Hoxc8 activity in tubular epithelial cells to fibrotic outcomes is lacking. The authors are encouraged to incorporate or reference data using tubule-specific Hoxc8 knockout models, which would provide definitive proof of causality and enhance the mechanistic framework of the study.\n6. In Figure 8G, the authors include HOXC8 expression data from both IgAN and ADPKD patient kidney samples. While IgAN is a widely recognized fibrotic kidney disease, ADPKD is not commonly considered a fibrosis-predominant pathology. The rationale for including ADPKD in the context of renal fibrosis should be clarified. Furthermore, the authors should provide histological or molecular evidence of fibrosis in these samples. Moreover, the relevance of these transcriptional changes has not been assessed in vivo using mouse models. Incorporating such validation would enhance the robustness of the transcriptomic findings and further substantiate the role of Hoxc8 in regulating fibrosis-associated gene networks.\n4. The authors demonstrate that MetR leads to a significant reduction in activating histone marks (H3K4me3 and H3K36me3), while repressive marks (H3K27me3 and H3K9me3) remain largely unaffected. This observation raises intriguing mechanistic questions: Why are only activating marks sensitive to methionine availability? Is this phenomenon attributable to differential regulation of histone methyltransferases or demethylases in response to SAM/SAH levels? A discussion on potential mechanisms or relevant literature would greatly enhance the depth and mechanistic insight of the manuscript.", "reviewer_2": "Type of manuscript: original article\nTitle: \" Methionine Restriction Alleviates Kidney Fibrosis through Epigenetic Repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis\"\nAuthors: Yunjing Liu, Zhiheng Liu, Lijun Liu, Jiayuan Chen, Bo Qing, Chen Tang, Rongjie Ji, Xinyi Cao, Junya Jia, Yupeng Chen, Lirong Zhang\nIn this original article entitled \"Methionine Restriction Alleviates Kidney Fibrosis through Epigenetic Repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis\" Liu and colleagues delivered interesting manuscript dealing about the TGF-β-Smad3-Hoxc8/P-TEFb axis downstream MR as a crucial regulator of renal fibrosis. The authors identified TGF-β-Smad3-Hoxc8/P-TEFb signalling pathway as a potential novel therapeutic target for CKD. This study is certainly interesting for the readership of Nature communications.\nThis is a highly interesting and well-written manuscript, but for publication a few points should be re-evaluated and would improve the manuscript.\nMAJOR POINTS.\n1. Previous study of other authors described the contribution of MR in renal fibrosis in response to damage (FASEB J. 2018;32(2):693-702). This study described that MR diet delays the progression of kidney disease regulating fibrosis in an experimental model of CKD (subtotal nephrectomy). This approach showed the capacity of MR to restored renal function. Therefore, the authors´ manuscript is not novel associated to the premise of the improvement of renal damage. However, there is no studies about the signalling pathways implicated in this process and this manuscript suggest a key signalling pathway associated to renal fibrosis modulation by MR. It is necessary that the authors include FASEB J. 2018;32(2):693-702 in the references of the manuscript and include as previous studies in the introduction/discursion.\n2. The authors used a profibrotic renal damage model (UUO model and FAN model), but these models do not resemble the real situation of patients with CKD. Other models, such as subtotal nephrectomy, are more representative of the pathophysiological situation of CKD patients. One important determination to recognize renal damage improvement is the determination of renal function in serum and urine levels. This determination is not possible in the UUO model due to the compensation of renal function carried out by the contralateral kidney. However, the authors used the FAN model to determine renal function; this model is not a model of pure CKD; is a model of AKI to CKD transition that presents with fibrosis, but the aetiology of the damage is not the same as pure CKD model. FAN model is a model of acute kidney injury in which cell death and regeneration are key process implicated in the damage in contrast to the situation in CKD. The authors need to include a subtotal nephrectomy (5/6 nephrectomy) model in Hoxc8fl/fl;Col1a2-cre/ERT mice to demonstrated that the Hoxc8 knockout mice have the same effect as MR in the control of renal function described by FASEB J. 2018;32(2):693-702 . In this model the authors have to determinate creatinine/BUN serum levels and urea/ creatinine ratio in urine and measure profibrotic markers by RT PCR and by western blot such as alpha-SMA, fibronectin or collagen I to demonstrate its beneficial effect in fibrosis.\n3. The fibrotic response is a consequence of a chronic inflammatory response chronified over the time. Is it possible to observe an improvement of the fibrotic response due to a modulation of previous inflammation in the kidney? Can you check the presence of inflammatory infiltration (T lymphocytes and Macrophages) in the renal tissue or expression of proinflammatory factors (MCP-1; IL6 or RANTES) in your UUO model with MR diet and in Hoxc8fl/fl;Col1a2-cre/ERT mice? Is possible that HOXc8 blockade can modulate this inflammation in the kidney? Include this point in the discussion of the results.\n4. In renal damage, not only fibroblasts contribute to fibrosis; tubular cell damage also has a pronounced deleterious effect and is the main cells that are damaged in UUO and FAN model. Tubular cells in response to damage begin to induce profibrotic factors and secrete matrix proteins such as fibronectin and collagen I into the extracellular milieu inducing a profibrotic environment that modify the phenotype of tubular and fibroblast phenotype. This environment contributes to the damage. What happens to tubular cells in response to damage? Do hoxc8 modulation in these cells contribute to fibrosis inducing the secretion of EMC protein to its extracellular medium? Determine protein levels of fibronectin or collagen I into the supernatant of tubular epithelial cells stimulated with TGF-beta and treated with MR and SiRNA against Hoxc8.\n5. In the UUO+ MR diet model, the authors determined the mRNA expression of col1a1 and col3a1 and protein levels by masson trichromic. In mRNA levels the authors observed increased levels of col1 than col3. Is it coincident with protein levels? ¿What is the most expressed collagen in the renal tissue of in UUO model in MR feed mice? You can use picrosirius red to differentiate both collagens. When exposed to polarized light, the birefringence of type I collagen appeared reddish-orange, while that of type III collagen was bluish-green.\n6. In vitro studies: the authors need to indicate the number of independent experiments in each case in figure footnotes; At least 3 independent experiments are needed.\n7. In vitro studies of fibrosis, the authors only determined the protein levels of alpha-SMA by Western blot; what happens with the levels of other EMC proteins such as fibronectin or collagen I?\n8. The discussion of the results is short and not sufficiently deep for the high amounts of results shown by the authors. The authors need to need to expand it by referring to all the premises and conclusions reached in the different sections of the results section by comparing them with previous bibliography."}, "author_response": "Response: We thank the reviewer for the positive appraisal of our work.\n1.The authors applied varying degrees of dietary restriction for different amino acids in the UUO model to assess their respective effects on renal fibrosis. However, the rationale behind this multi-level restriction design remains unclear. Was the selection of restriction levels based on prior dietary or metabolic studies? Were preliminary dose-finding or tolerability assessments performed for each amino acid? A brief explanation of the design strategy would help readers better understand the robustness and physiological relevance of the dietary intervention model.\nResponse: We thank the reviewer for raising this important point. The restriction levels for each amino acid in this study were based on established dietary models from previous literature (references 34-45), with specific doses detailed in Supplementary Table S1. These restriction regimens have been demonstrated in prior studies to possess good metabolic efficacy and animal tolerability. We have added this explanatory information in the revised manuscript (Page 6, Line 124-126).\n2.In Figure 4, the changes in Hoxc8 expression are assessed solely at the mRNA level by RT-qPCR. Given the central role of Hoxc8 in the proposed signaling axis, validation at the protein level—e.g., by Western blotting or immunofluorescence staining—would significantly strengthen the conclusions and support the translational relevance of Hoxc8 as a potential therapeutic target.\nResponse: We thank the reviewer for this suggestion. In response, we have now performed Western blot analysis to evaluate Hoxc8 expression at the protein level. These new data have been incorporated as Figure 4E in the revised manuscript and confirm that Hoxc8 protein expression changes align with the observed mRNA patterns (Page 10, Line 220-221).\nFigure R1. TGF-β-induced upregulation of Hoxc8 is inhibited by methionine restriction.\n3.In Figure 4, the authors present RNA-seq data identifying a subset of genes whose TGF-β-induced expression is suppressed by Hoxc8 knockdown. However, the study does not include independent validation of representative target genes using RT-qPCR. Moreover, the relevance of these transcriptional changes has not been assessed in vivo in mouse models. Incorporating such validation would enhance the robustness of the transcriptomic findings and further substantiate the role of Hoxc8 in regulating fibrosis-associated gene networks.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have performed independent validation of our RNA-seq findings by quantifying the expression of two representative target genes (Vim and Ccn1) using RT-qPCR. This validation was conducted both in NRK-49F cells with Hoxc8 knockdown and in kidney tissues from Hoxc8FKO mice, with results consistently confirming their significant downregulation. These new data have been included as Supplementary Figure 4A (in vitro validation) (Page 10-11, Line 230-233) and Supplementary Figure 4B (in vivo validation) (Page 16, Line 352), thereby strengthening the reliability of our transcriptomic findings and supporting the role of Hoxc8 in regulating fibrosis-associated gene networks.\nFigure R2. Validation of representative siHoxc8 target genes. (A) Quantitative RT-PCR analysis of mRNA levels for Vim and Ccn1 in NRK-49F cells. Data are expressed as mean ± SEM. (B) Quantitative RT-PCR analysis of mRNA levels for Vim and Ccn1 in Hoxc8FKO mice. *P < 0.05, **P < 0.01, ****P < 0.0001.\n4.The authors demonstrate that MetR leads to a significant reduction in activating histone marks (H3K4me3 and H3K36me3), while repressive marks (H3K27me3 and H3K9me3) remain largely unaffected. This observation raises interesting mechanistic questions:Why are only activating marks sensitive to methionine availability? Is this due to differential regulation of histone methyltransferases or demethylases in response to SAM/SAH levels? A discussion of potential mechanisms or relevant literature would enhance the depth and mechanistic insight of the manuscript.\nResponse：We thank the reviewer for this insightful question regarding the selective sensitivity of histone marks. The differential response can be explained by the distinct metabolic requirements and turnover dynamics of these modifications. Unlike the relatively stable repressive marks (e.g., H3K9me3 and H3K27me3), activating marks such as H3K4me3 and H3K36me3 undergo rapid turnover and require continuous SAM-dependent maintenance by their respective histone methyltransferases (HMTs). Under methionine restriction, the resulting SAM depletion preferentially affects these dynamic marks by limiting the substrate availability for their maintenance. This mechanism is supported by emerging evidence: 1) SAM deficiency activates the H3K36me3 demethylase Rph1 (PMID: 30772176), creating an imbalance toward demethylation; and 2) the lifespan-extending effect of metformin involves SAM-limited perturbation of the balance between H3K4 methyltransferases and demethylases (PMID: 35146893). We have incorporated discussion of these points in the revised manuscript to provide deeper mechanistic insight (Page 19-20, Line 434-449).\n5.Figures 8C show elevated Hoxc8 expression in renal tubular compartments during fibrosis. However, direct functional evidence linking Hoxc8 activity in tubular epithelial cells to fibrotic outcomes is lacking. The authors are encouraged to incorporate or reference data using tubule-specific Hoxc8 knockout models, which would provide definitive proof of causality and enhance the mechanistic framework of the study.\nResponse: We thank the reviewer for this insightful suggestion. To directly evaluate the functional contribution of Hoxc8 in tubular epithelial cells to renal fibrosis, we generated proximal tubule-specific Hoxc8 knockout mice (Hoxc8fl/fl; PEPCK-Cre, designated Hoxc8TKO) and subjected them to UUO surgery. As demonstrated in the following Figure R3, tubule-specific deletion of Hoxc8 significantly attenuated UUO-induced renal fibrosis, paralleling the protective effects observed in fibroblast-specific knockout models. These new findings provide direct genetic evidence establishing Hoxc8's pro-fibrotic function within tubular epithelial cells, substantially strengthening the mechanistic framework of our study. These new data have been incorporated into the main text (Page 17-18, Line 386-395, new Supplementary Figure 10).\nFigure R3. Proximal tubule cell-specific Hoxc8 knockout alleviates kidney fibrosis. (A) Immunofluorescence staining of Hoxc8 and SGLT2 in kidney sections from sham or UUO-operated Hoxc8WT and Hoxc8TKO mice. Scale bar = 25 μm. (B) IHC analysis of α-SMA expression. Quantification of α-SMA-positive staining is shown as the percentage of stained area(n=6). Scale bar = 100 μm. (C) Masson staining of kidney sections from sham or UUO-operated Hoxc8WT and Hoxc8TKO mice to assess fibrosis. Quantification of fibrosis is shown as the percentage of the fibrotic area(n=6). Scale bar = 100 μm. (D) Quantitative RT-PCR analysis of mRNA expression for Acta2, Col3a1, and Col1a1 in kidney sections from sham or UUO-operated Hoxc8WT and Hoxc8TKO mice(n=6). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\n6.In Figure 8G, the authors include HOXC8 expression data from both IgAN and ADPKD patient kidney samples. While IgAN is a widely recognized fibrotic kidney disease, ADPKD is not commonly considered a fibrosis-predominant pathology. The rationale for including ADPKD in the context of renal fibrosis should be clarified. Furthermore, the authors should provide histological or molecular evidence of fibrosis in these samples.\nResponse: We thank the reviewer for raising this important point regarding our inclusion of ADPKD samples. While ADPKD is primarily characterized by cyst formation, accumulating evidence indicates that interstitial fibrosis serves as a critical determinant of disease progression and renal function decline in these patients. To directly address this concern, we performed Masson's trichrome staining in ADPKD samples, which clearly demonstrates substantial collagen deposition in the interstitial compartments. These histological findings confirm the presence of significant fibrotic pathology in ADPKD patients.\nFigure R4. Renal Fibrosis in Patients with ADPKD.\nReviewer #2\nIn this original article entitled \"Methionine Restriction Alleviates Kidney Fibrosis through Epigenetic Repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis\" Liu and colleagues delivered interesting manuscript dealing about the TGF-β-Smad3-Hoxc8/P-TEFb axis downstream MR as a crucial regulator of renal fibrosis. The authors identified TGF-β-Smad3-Hoxc8/P-TEFb signalling pathway as a potential novel therapeutic target for CKD. This study is certainly interesting for the readership of Nature communications.\nThis is a highly interesting and well-written manuscript, but for publication a few points should be re-evaluated and would improve the manuscript.\nResponse: We thank the reviewer for this positive appraisal of our work. We have carefully revised the manuscript in response to all the comments and suggestions provided by the reviewers to improve the manuscript.\nMAJOR POINTS.\n1.Previous study of other authors described the contribution of MR in renal fibrosis in response to damage (FASEB J. 2018;32(2):693-702). This study described that MR diet delays the progression of kidney disease regulating fibrosis in an experimental model of CKD (subtotal nephrectomy). This approach showed the capacity of MR to restored renal function. Therefore, the authors´ manuscript is not novel associated to the premise of the improvement of renal damage. However, there is no studies about the signalling pathways implicated in this process and this manuscript suggest a key signalling pathway associated to renal fibrosis modulation by MR. It is necessary that the authors include FASEB J. 2018;32(2):693-702 in the references of the manuscript and include as previous studies in the introduction/discursion.\nResponse: We thank the reviewer for this important suggestion. We fully agree with your perspective that the cited study represents a foundational contribution by first establishing the beneficial role of MetR in mitigating renal fibrosis within a specific CKD model. In response to your suggestion, we have made the following revisions to our manuscript:\nWe have now cited this pioneering work in the Introduction section to properly acknowledge its significance (Page 5, Line 96-100).\nIn the Discussion section, we have incorporated a detailed analysis of this reference, emphasizing that our current study not only confirms the efficacy of MetR but also systematically extends these findings by demonstrating its beneficial effects on renal function and fibrosis in two additional kidney disease models (UUO and FAN) that were not examined in the previous research. Most importantly, our work provides novel insights into the previously unidentified signaling pathways underlying these protective effects (Page 18-19, Line 412-419).\nWe believe these revisions better contextualize our work as both a validation and a substantial extension of prior findings, specifically addressing the crucial question of \"how MetR exerts its effects\" at the mechanistic level.\n2. The authors used a profibrotic renal damage model (UUO model and FAN model), but these models do not resemble the real situation of patients with CKD. Other models, such as subtotal nephrectomy, are more representative of the pathophysiological situation of CKD patients. One important determination to recognize renal damage improvement is the determination of renal function in serum and urine levels. This determination is not possible in the UUO model due to the compensation of renal function carried out by the contralateral kidney. However, the authors used the FAN model to determine renal function; this model is not a model of pure CKD; is a model of AKI to CKD transition that presents with fibrosis, but the aetiology of the damage is not the same as pure CKD model. FAN model is a model of acute kidney injury in which cell death and regeneration are key process implicated in the damage in contrast to the situation in CKD. The authors need to include a subtotal nephrectomy (5/6 nephrectomy) model in Hoxc8fl/fl;Col1a2-cre/ERT mice to demonstrated that the Hoxc8 knockout mice have the same effect as MR in the control of renal function described by FASEB J. 2018;32(2):693-702 . In this model the authors have to determinate creatinine/BUN serum levels and urea/ creatinine ratio in urine and measure profibrotic markers by RT PCR and by western blot such as alpha-SMA, fibronectin or collagen I to demonstrate its beneficial effect in fibrosis.\nResponse: We thank the reviewer for the insightful comments and valuable suggestions regarding the animal models. In our study, the primary objective for selecting the UUO and FAN models was to enable rapid and efficient preliminary screening and mechanistic exploration. The UUO model is widely used to simulate rapid fibrotic processes, providing a robust and highly reproducible fibrotic signal, making it particularly suitable for the systematic screening of the most effective anti-fibrotic regimen from 15 dietary interventions. Meanwhile, the FAN model allowed us to preliminarily observe the effects of MetR on renal function within a system characterized by transition from acute injury to chronic fibrosis.\nWe agree with the reviewer’s comments that the 5/6 nephrectomy model serves as a classical system for studying chronic progressive fibrosis. Therefore, we performed the 5/6 nephrectomy experiment in Hoxc8FKO (Hoxc8fl/fl;Col1a2-cre/ERT) mice to rigorously evaluate the functional role of Hoxc8 in this more clinically relevant CKD model. We assessed key parameters, including renal function, extracellular matrix deposition, and the expression of fibrosis-related genes. As shown in the new Supplementary Figure S8, the loss of Hoxc8 conferred significant protection against subtotal nephrectomy-induced renal fibrosis. This finding not only strongly recapitulates the beneficial effects of MetR described in the cited study (FASEB J. 2018;32(2):693-702) but also genetically demonstrates that Hoxc8 is a key mediator underlying this process. We have now incorporated these data as a new Supplementary Figure S8 in the Results section of our revised manuscript (Page 16-17, Line 357-370).\nFigure R5. Hoxc8FKO alleviates renal fibrosis in the 5/6 nephrectomy model. (A) Schematic view of experimental design in the Hoxc8 conditional knockout mice(n=5). (B) Blood urea nitrogen (BUN) levels in 5/6nephrectomy mice from the indicated groups (n=5). Data are presented as mean ± SEM. (C) Serum creatinine (Scr) levels in 5/6 nephrectomy mice from the indicated groups (n=5). (D) Masson staining showing collagen deposition in mouse kidneys from the indicated groups (n=5). Scale bar = 100 μm. Quantitative analysis of collagen deposition from Masson staining images is shown on the right. (E) IHC staining showing renal α-SMA expression. Scale bar = 100 μm. Quantitative data for each group are shown on the right (n=5). (F) Quantitative RT-PCR analysis of mRNA expression for fibrotic marker genes (Acta2, Col1a1, and Col3a1) (n=5). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\n3. The fibrotic response is a consequence of a chronic inflammatory response chronified over the time. Is it possible to observe an improvement of the fibrotic response due to a modulation of previous inflammation in the kidney? Can you check the presence of inflammatory infiltration (T lymphocytes and Macrophages) in the renal tissue or expression of proinflammatory factors (MCP-1; IL6 or RANTES) in your UUO model with MR diet and in Hoxc8fl/fl;Col1a2-cre/ERT mice? Is possible that HOXc8 blockade can modulate this inflammation in the kidney? Include this point in the discussion of the results.\nResponse: We sincerely thank the reviewer for the insightful comments regarding the relationship between renal fibrosis and chronic inflammation. We fully agree that fibrosis results from the progression of chronic inflammation and believe that investigating the regulatory roles of MetR and Hoxc8 in inflammation will significantly enrich the mechanistic depth of our study.\nIn response to the reviewer’s suggestions, we conducted the following additional experiments. We employed two approaches to analyze renal tissues in UUO models from both MetR-fed and Hoxc8FKO mice. As shown in the following Figure R6, immunohistochemical analysis revealed substantial macrophage infiltration and modest T lymphocyte infiltration in the kidneys following UUO surgery. Both MetR and Hoxc8 deletion significantly attenuated this immune cell recruitment, reducing the infiltration of macrophages and T lymphocytes. Furthermore, these interventions downregulated pivotal pro-inflammatory mediators, including Ccl2 (MCP-1), Ccl5 (RANTES), and Il6.\nCollectively, these data demonstrate that both MetR and Hoxc8 deletion effectively suppress the renal inflammatory response. These findings strongly suggest that Hoxc8 blockade can indeed modulate the inflammatory microenvironment in the kidney. Thus, by inhibiting Hoxc8, the MetR diet likely disrupts the progression from chronic inflammation to fibrosis by early suppression of inflammatory cell infiltration and activation, providing an upstream mechanistic explanation for its renal protective effects. In accordance with the recommendation, we have thoroughly discussed these new findings in the Discussion section of the revised manuscript (Page 21, Line 465-477, new Supplementary Figure S11).\nFigure R6. MetR and Hoxc8FKO alleviates renal inflammation in the UUO mouse model. (A) IHC staining showing macrophages infiltration in mouse kidneys from the indicated groups (n=6). Scale bar = 100 μm. Quantitative analysis of F4/80 from IHC images is shown on the right. (B) IHC staining showing T lymphocytes infiltration in mouse kidneys from the indicated groups (n=6). Scale bar = 100 μm. Quantitative analysis of CD3 from IHC images is shown on the right. (C) Quantitative RT-PCR analysis of mRNA expression for pro-inflammatory factors (Ccl2, Ccl5, and Il6) (n=6). (D) IHC staining showing macrophages infiltration in mouse kidneys from the indicated groups (n=6). Scale bar = 100 μm. Quantitative analysis of F4/80 from IHC images is shown on the right. (E) IHC staining showing T lymphocytes infiltration in mouse kidneys from the indicated groups (n=6). Scale bar = 100 μm. Quantitative analysis of CD3 from IHC images is shown on the right. (F) Quantitative RT-PCR analysis of mRNA expression for pro-inflammatory factors (Ccl2, Ccl5, and Il6) (n=6). *P < 0.05, **P < 0.01, ***P < 0.001.\n4. In renal damage, not only fibroblasts contribute to fibrosis; tubular cell damage also has a pronounced deleterious effect and is the main cells that are damaged in UUO and FAN model. Tubular cells in response to damage begin to induce profibrotic factors and secrete matrix proteins such as fibronectin and collagen I into the extracellular milieu inducing a profibrotic environment that modify the phenotype of tubular and fibroblast phenotype. This environment contributes to the damage. What happens to tubular cells in response to damage? Do hoxc8 modulation in these cells contribute to fibrosis inducing the secretion of EMC protein to its extracellular medium? Determine protein levels of fibronectin or collagen I into the supernatant of tubular epithelial cells stimulated with TGF-beta and treated with MR and SiRNA against Hoxc8.\nResponse: We thank the reviewer for raising this important question regarding the role of tubular epithelial cells in renal fibrosis and the specific function of Hoxc8 in this process. In direct response to the reviewer's suggestion, we examined whether Hoxc8 modulates fibrotic protein secretion by measuring fibronectin and collagen I levels in TGF-β-stimulated NRK-52E cells. As shown in the following Figure R7, both Hoxc8 knockdown and MetR treatment significantly reduced the production of these key ECM proteins. These in vitro data have been included in new Supplementary Figure 9 (Page 17, Line 372-385).\nTo further validate these findings in vivo, we generated tubular cell-specific Hoxc8 knockout mice (Hoxc8TKO). The genetic ablation of Hoxc8 in tubular cells provided substantial protection against renal fibrosis, with Hoxc8TKO mice showing attenuated α-SMA-positive myofibroblast activation, reduced collagen deposition, and significantly lower expression of fibrotic markers at the transcriptional level compared to wild-type controls. These in vivo data have been included in new Supplementary Figure 10 (Page 17-18, Line 386-395).\nCollectively, these results from both in vitro and in vivo experiments demonstrate that Hoxc8 in tubular epithelial cells plays an important role in promoting renal fibrosis through regulation of ECM protein secretion and fibrotic responses.\nFigure R7. Methionine restriction and siHoxc8 inhibits fibrotic gene expression in NRK-52E. (A) Western blot analysis of α-SMA, Col1a1,Fibronectin and Hoxc8 expression in NRK-52E cells treated with TGF-β and MetR. (B) Quantitative RT-PCR analysis of mRNA levels for fibrosis marker genes (Acta2, Col1a1, and Col3a1). (C) Western blot analysis of α-SMA, Col1a1, Fibronectin and Hoxc8 expression in NRK-52E cells treated with siHoxc8. (D) Quantitative RT-PCR analysis of mRNA levels for fibrosis marker genes (Acta2, Col1a1, and Col3a1). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\n5. In the UUO+ MR diet model, the authors determined the mRNA expression of col1a1 and col3a1 and protein levels by masson trichromic. In mRNA levels the authors observed increased levels of col1 than col3. Is it coincident with protein levels? ¿What is the most expressed collagen in the renal tissue of in UUO model in MR feed mice? You can use picrosirius red to differentiate both collagens. When exposed to polarized light, the birefringence of type I collagen appeared reddish-orange, while that of type III collagen was bluish-green.\nResponse: We thank the reviewer for this excellent suggestion regarding collagen composition analysis. Following the reviewer's methodology recommendation, we performed Sirius Red staining on kidney tissues from UUO mice under MetR and examined the sections under polarized light. As the reviewer indicated, collagen I exhibits reddish-orange birefringence while collagen III appears bluish-green. Our analysis revealed that collagen I was indeed more abundant than collagen III in the fibrotic areas, which aligns with our mRNA expression data showing higher Col1a1 than Col3a1 levels. These new findings confirm that collagen I is the predominant collagen type in the UUO model under methionine restriction and further support the modulatory effect of MR on collagen composition during renal fibrosis progression.\nFigure R8. Sirius Red Staining of kidneys in UUO mice.\n6. In vitro studies: the authors need to indicate the number of independent experiments in each case in figure footnotes; At least 3 independent experiments are needed.\nResponse: We thank the reviewer for this important reminder. We have now clearly indicated in the respective figure legends that all in vitro experiments were performed with at least three independent replicates.\n7. In vitro studies of fibrosis, the authors only determined the protein levels of alpha-SMA by Western blot; what happens with the levels of other EMC proteins such as fibronectin or collagen I?\nResponse: We thank the reviewer for raising this important point. In response, we have now performed additional Western blot analyses to examine the protein levels of key ECM components, including fibronectin and collagen I. These new data have been incorporated as new Figure 3D, Figure 4F, and Figure 4J, providing a more comprehensive assessment of ECM protein expression changes in our in vitro fibrosis models.\nFigure R9. Detection of ECM protein in the indicated groups. (A) Western blot analysis of α-SMA, Fibronectin and Col1a1 expression in NRK-49F cells treated with TGF-β and MetR. (B) Western blot analysis of Hoxc8, α-SMA, Col1a1 and Fibronectin expression in NRK-49F cells treated with TGF-β and siHoxc8. (C) Western blot analysis of α-SMA, Col1a1 and Fibronectin and Hoxc8 expression in NRK-49F cells with or without Hoxc8 overexpression and MetR treatment.\n8. The discussion of the results is short and not sufficiently deep for the high amounts of results shown by the authors. The authors need to need to expand it by referring to all the premises and conclusions reached in the different sections of the results section by comparing them with previous bibliography.\nResponse: We sincerely thank the reviewer for this insightful suggestion. We agree that a more in-depth discussion would strengthen the impact of our findings. As recommended, we have expanded the Discussion section to provide a more comprehensive analysis of our results.\nThe key additions include:\n1. Mechanistic Insight into the Epigenetic Specificity of MetR: We have added a dedicated paragraph discussing the intriguing observation that MetR selectively reduces activating histone marks (H3K4me3, H3K36me3) while sparing repressive ones (H3K27me3, H3K9me3). This discussion incorporates potential mechanisms, citing relevant literature (e.g., PMID: 30772176, 35146893), to explain that this specificity likely stems from the distinct metabolic requirements and turnover dynamics of these modifications, particularly the continuous need for SAM by the methyltransferases responsible for maintaining the dynamic activating marks. (Page 19-20, Line 434-449)\n2. Linking Fibrosis and Inflammation: We have explicitly connected our findings on fibrosis with the results on inflammation. The expanded discussion now positions Hoxc8 as a key modulator at the interface of inflammation and fibrosis. We discuss how the anti-fibrotic effects of both MetR and Hoxc8 deletion are associated with the suppression of upstream inflammatory responses (such as reduced macrophage/T lymphocyte infiltration and downregulation of Ccl2, Ccl5, and Il6), and have cited previous literature on the role of chronic inflammation as a driver of fibrosis and on the known anti-inflammatory effects of MetR. (Page 21, Line 465-477)\n3. Cross-Model and Cross-Cellular Consistency: We have expanded the discussion to highlight the significance of our consistent findings across different experimental models (UUO and 5/6 nephrectomy) and different cellular compartments (fibroblasts and tubular epithelia cells). This reinforces the central and multifaceted role of Hoxc8 in renal pathology. (Page 20-21, Line 459-481)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "My concerns have been addressed.", "reviewer_2": "The authors have satisfactorily addressed all my comments and concerns in their response and have made the necessary revisions to the manuscript. I am therefore confident that the paper meets the journal’s standards, and I recommend it for publication in its current form."}, "author_response": ""}]

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