TGF-β ligands comprise three distinct isoforms, namely TGF-β1, TGF-β2 and TGF-β3 (9). These isoforms are ubiquitously distributed across various cellular and tissue contexts, with TGF-β1 being the most prevalent (10). TGF-β ligands initially emerge as precursor proteins, which undergo a cleavage process at the N-terminal region (11). The cleavage process results in the formation of the latency-associated peptide, which stays bound to the mature TGF-β homodimer at the C-terminal (Fig. 1). This complex association with the latent TGF-β-binding proteins ensures that TGF-β remains inactive, commonly termed latent TGF-βs (12,13). To become biologically active, these latent forms require the intervention of certain environmental triggers such as specific enzymes or an acidic milieu (5). In the extracellular environment, associations with the ECM and subsequent cleavage by various proteases, including plasmin and specific matrix metalloproteinases, facilitate the liberation and activation of the TGF-β ligands (11).

Upon engagement with the TGF-β1 ligand, the TGF-β receptor (TGF-βR) II triggers the activation of TGF-βRI through phosphorylation (5). This series of activations subsequently culminates in the activation of Smad2/3 transcription factors through phosphorylation, commencing the standard signaling process (5). Integral to this cascade is Smad4, which associates with Smad2/3 after phosphorylation, directing the Smad2/3/4 complex towards the nucleus (7) (Fig. 1). This migration to the nucleus is a pivotal step for transcribing genes, which includes key genes such as NADPH oxidase 4 (NOX4), connective tissue growth factor (CTGF) and others [receptor interacting protein kinase 3 and proto-oncogene tyrosine-protein kinase Src (Src)] involved in tissue repair and cellular regulation (14–21). Furthermore, Smad7, an inhibitory molecule, becomes active in response to Smad3, and engages in competitive binding with TGF-βRI, thereby hindering the phosphorylation process of Smad2 and Smad3 (22). The transcriptional modulation orchestrated by Smad3-containing complexes is influenced by an array of non-Smad co-activators, including the transcriptional coactivator p300, activator protein 1 and yes-associated protein, as well as co-suppressors such as SKI-like proto-oncogene (SKI) and Ski-like oncoprotein N (SnoN) (23–26). The transcriptional effects of TGF-β1 necessitate the involvement of these accessory factors, driving structural alterations in Smad2/3 to interact effectively with target motifs (8). In addition, TGF-β1 activates an array of pathways independent of Smad signaling, which contribute to its biological activities. These routes include TGF-β-activated kinase 1, phosphatidylinositol 3-kinase/Akt and Rho-like GTPase signaling pathways (27,28).

Activation of myofibroblasts and the ensuing accumulation of ECM are pivotal events in RF (29). The activated myofibroblast serves as the key driver of RF, given its significant capacity to produce the majority of the matrix (29). While myofibroblasts are scarce under normal conditions, their numbers surge in fibrotic kidneys (7,29). Proposed precursors for myofibroblasts include pericytes, cells of epithelial and endothelial origin, circulating cells derived from bone marrow and local fibroblasts (30–35). For epithelial cells, research has clarified the fibrosis-promoting influence of TGF-β1, emphasizing the critical roles of key molecules in the Smad signaling pathway, such as TGF-βRI, TGF-βRII and Smad3, in epithelial-to-mesenchymal transition (EMT) (36–39). Furthermore, a number of miRNAs and long non-coding RNAs (lncRNAs) have been identified that are reliant on Smad3 function in different capacities to regulate EMT (7,40–42). For circulating bone marrow-derived cells, mounting evidence indicates that macrophages originating from bone marrow can directly transition into myofibroblasts (MMT) (43,44). In fibrotic kidneys, the recruited Smad3-deficient macrophages do not differentiate into myofibroblasts (44). Additionally, a series of factors transcriptionally regulated by Smad3, such as Pou4f1, P2Y12 and Src, have been proven to be involved in the MMT process (20,45–48). This suggests that the progression of MMT is closely governed by the TGF-β/Smad3 signaling pathway. For endothelium, in models of RF triggered by either folic acid or unilateral ureteral obstruction, the specific reduction of TGF-βRII levels in endothelial cells led to a mitigation of fibrotic remodeling and an inhibition of endothelial-to-mesenchymal transition (49). For fibroblasts, researchers discovered that half of the myofibroblasts arise from the proliferation of local resident fibroblasts, while an additional 35% originate from bone marrow fibroblasts (50). The TGF-β pathway appears instrumental in this process, as evidenced by the fact that specific deletion of TGF-βRII in α-smooth muscle actin (+) cells leads to a marked decrease in fibroblast numbers (50). Furthermore, initiating the conditional deletion of Smad2 in fibroblasts under the influence of the fibroblast-specific protein 1 promoter, diminishes RF in streptozotocin (STZ)-triggered diabetic nephropathy (DN) (51). In summary, the TGF-β/Smad pathway plays a crucial role in guiding cellular dynamics and transitions vital for RF.

Sterile inflammation, characterized by the inflammatory response devoid of any infectious agents or specific immunogens, serves as a key trigger for the development of RF (52). TGF-β is instrumental in the formation, balance, diversification and tolerance of immune cells (53,54). Diminishment of TGF-β1 can result in the hyperactivation of immunocytes and trigger the occurrence of autoimmune diseases, a phenomenon noted in mice lacking either TGF-β1 or its receptors. In such cases, excessive inflammatory responses with massive lymphocyte and macrophage infiltration were observed in many organs, primarily in the heart and lungs (55,56). Consequently, broadly blocking upstream TGF-β signaling might exacerbate renal inflammation.

Smad3 acts as a central regulator in renal inflammation, uniquely modulating its dynamics by either inhibiting or promoting the functions of macrophages and T cells (8). As a crucial effector molecule, Smad3 is involved in the TGF-β1-driven suppression of macrophage activation, as demonstrated by its capability to inhibit the regulatory actions of the inducible nitric oxide synthase and matrix metalloproteinase-12 promoters within these cells (57). Furthermore, Smad3 has a critical function in preserving the equilibrium between Treg and Th17 immune responses. This is verified by the observation that a lack of Smad3 results in diminished forkhead box P3 induction, while simultaneously enhancing Th17 cell generation by inhibition of retinoid acid receptor-related orphan receptor γt transcriptional activity within both controlled (in vitro) and living (in vivo) contexts (58). Conversely, Smad3 potentially facilitates macrophage recruitment during renal inflammation through its chemotactic effects, as it has been found to engage with macrophage chemotactic protein-1 (MCP-1), in turn amplifying the renal inflammation driven by macrophages (59). Furthermore, LRNA9884, a lncRNA that is transcriptionally regulated by Smad3 has been shown to influence DN in db/db mice by stimulating the synthesis of MCP-1, further amplifying renal inflammation (60). Additionally, in obstructed nephropathy, AT-rich interaction domain 2 intronic transcript, facilitated by TGF-β/Smad3, enhances the inflammation triggered by IL-1β via the NF-κB pathway, without influencing the fibrosis modulated by the TGF-β/Smad3 process (61). Notably, Smad7 serves as a negative regulator of TGF-β/Smad signaling (62). Previous investigations have further demonstrated that Smad7 functions as a key adjuster by stimulating IκBα, a suppressor of NF-κB, consequently mitigating renal inflammation (63,64). The observed phenomenon suggests that a deficiency in Smad3 inhibits renal inflammation, which is driven by NF-κB in the unilateral ureteral obstruction (UUO) model (65). Presumably, the removal of Smad3 inhibits the breakdown of Smad7 by E3 ubiquitin-protein ligases, such as Smad ubiquitin regulatory factor (Smurf)1/Smurf2 (66). In summary, Smad3 has been identified as a key controller in renal inflammation, orchestrating various molecular interactions and pathways to either amplify or mitigate inflammatory responses.

Cellular senescence describes the process wherein cells lose their ability to replicate and permanently exit the cell cycle after repeated duplications (67). These senescent cells resist apoptosis and consistently release a diverse secretome, termed the senescence-associated secretory phenotype, which includes pro-inflammatory and pro-fibrotic mediators (67). In recent studies, cellular senescence in renal tubular epithelial cells has been identified as a primary contributor to the onset of RF, and consequently, delaying this senescence presents an effective intervention to curb RF and offers a crucial strategy for decelerating the progression of CKD (67–69). A previous study indicates that the TGF-β/Smad pathway promotes cellular senescence by reducing histone 4 lysine 20 tri-methylation through miR-29, impacting DNA repair and genome stability (70). Furthermore, a recent study has demonstrated that ubiquitin-specific protease 11 (USP11) promotes cellular senescence and fibrosis regulated by the Smad/P53 complex, by inhibiting the ubiquitination of TGF-βRII (71). In the context of the aging kidney, heightened stimulation of the TGF-β/Smad3 pathway in podocyte-specific TGF-β overexpressing mice leads to cell senescence through processes that involve transference of p16 and initiation of p21 (72).

Autophagy, a preserved lysosomal pathway, facilitates the degradation of cytoplasmic constituents (73). Yet, its role in kidney fibrosis, whether protective or pathological, remains ambiguous (73). Moreover, the exact mechanisms and signaling pathways that govern autophagy responses across various kidney cell types and disease spectra require further elucidation (73). Recent studies highlighted the influence of Smad3 on autophagy and its prospective role as a treatment focus for fibrotic diseases. A study has demonstrated that Smad3 contributes to lysosomal depletion by inhibiting transcription factor EB-mediated lysosome biogenesis, resulting in impaired autophagy during the advancement of DN (74). Moreover, TGFβ, through an epigenetic mechanism that involves the Smad3-mediated decrease of histone acetyltransferase KAT8 (also termed as MYST1), activates autophagy which promotes fibrotic diseases, including dermal and pulmonary fibrosis, suggesting a potential therapeutic target (75).

Preventing the death of renal tubular epithelial cells is crucial in halting the progression of CKD (76). It is widely acknowledged that TGF-β is known to facilitate cell death by the interruption of the cell cycle at its G1 phase, orchestrated through the Smad pathway (77–79). A previous study revealed that Smad3 contributes to acute kidney injury (AKI) by directly interacting with cyclin-dependent kinase inhibitor proteins p21 and p27 (78). This interaction results in the death of renal tubular epithelial cells (TECs) due to G1 phase cell cycle arrest (79). Moreover, a recent study has indicated that Smad3 can also transcriptionally activate the receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein necroptosis pathway, subsequently resulting in the death of TECs (21).

Ferroptosis, a regulated form of cell death induced by oxidative stress and dependent on iron-mediated lipid peroxidation, is intricately linked with numerous renal and fibrotic diseases, including AKI, CKD and diabetic kidney diseases (80,81). However, the precise mechanisms driving RF through ferroptosis are yet to be fully understood. Recently published studies demonstrated that Smad3 induces ferroptosis in TECs, primarily through the modulation of NOX4 gene transcription (18,82,83). It also works in conjunction with activating transcription factor 3 (ATF3) to suppress the gene expression of solute carrier family 7 member 11 (SLC7A11), thereby modulating the ferroptosis process (84). In light of these findings, the present research group has made some interesting discoveries. Preliminary investigations have revealed that active natural compounds exhibit specificity towards key components involved in the ferroptosis process. For example, tectorigenin has been found to specifically target Smad3, regulating NOX4 and thereby inhibiting the ferroptosis pathway, suggesting a potential therapeutic role against RF (18). Formononetin, on the other hand, suppresses the binding of the SMAD3/ATF3 complex, promoting the expression of SLC7A11 and highlighting its potential as a novel modulator for ferroptosis in RF (84). Lastly, kaempferitrin has demonstrated the ability to bind with NOX4, leading to an improvement in tubular ferroptosis within the kidneys, proposing a new approach to the treatment of RF through its impact on ferroptosis (83). These promising steps forward provide valuable insights into how natural compounds can potentially be utilized in the modulation of ferroptosis and treatment of RF.

Phosphorylation is a process occurring after protein translation, characterized by the addition of a phosphate group to certain amino acids in a protein, typically serine, threonine or tyrosine residues (87). Normally, Smad2 and Smad3, which are among the receptor-regulated Smads (R-Smads), undergo activation via ligand-induced phosphorylation at two serine residues within their carboxy-terminal SSXS motif, mediated by TGF-βRI (88,89). Beyond the established TGF-β signaling pathway involving Smad2 and Smad3, the TGF-β/Smad signaling pathway is further influenced by multiple kinases, providing further refinement, expansion or modulation of the signaling output (90). The mitogen-activated protein kinase (MAPK) family, comprising three primary kinases including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), has been shown to be activated in humans with both acute and CKD, as indicated by a number of studies (91–93). In addition, through employing pharmacological and genetic interventions, the blocking of p38 and/or JNK effectively mitigates RF across diverse animal studies (94–96). Notably, each of the three MAPKs are capable of phosphorylating specific sites within the linker regions of both Smad2 and Smad3 (97). These regions are situated in the interdomain space that separates the mad homology domain 1 (MH1) and MH2 domains within a Smad protein, subsequently altering the transcription of Smad3-dependent genes (97). Hence, besides being activated by TGF-β1, Smad3 activation can be triggered by various stress-inducing agents such as angiotensin II (Ang II), advanced end products (AGE) and C-reactive protein (CRP) through MAPK-dependent pathways. Specifically, research reveals that Ang II has the capability to directly stimulate Smad3, thereby promoting the upregulation of both CTGF and collagen I via the AT1-ERK/p38 MAPK interaction route (98). In addition, there is a strong link between AGEs and the expression of CTGF, and EMT. Through the Smad3 pathway that functions independently of TGF-β1, introducing AGEs induces CTGF expression in TECs devoid of TGF-β1, evidenced by the swift activation of Smad2/3, ERK1/2 and p38 (99). Furthermore, CRP, reported as an inflammatory marker and mediator, is known to regulate the fibrotic process, primarily by modulating the CD32b-ERK/p38 MAPK pathway, subsequently activating Smad3 (100).

Protein dephosphorylation, mediated by protein phosphatases, serves as a key control process that influences the operation of a number of proteins within signal transduction routes (87). Chen et al (101) discovered that loss of integrin α1β1, a regulator for collagen synthesis, exacerbated RF in a UUO model via a TGF-β/Smad-dependent manner. In terms of the mechanism, integrin α1β1 promotes the recruitment of the phosphatase, T cell protein tyrosine phosphatase (TCPTP) to TGF-βRII which results in the dephosphorylation of tyrosine residues in the TGF-βRII cytoplasmic tail, subsequently impairing TGF-βR-dependent fibrotic signaling transmission (101). In addition, protein phosphatase magnesium-dependent 1A (PPM1A) facilitates the dephosphorylation of TGF-β-activated Smad2/3 within their carboxy-terminal SSXS motif, subsequently promoting their nuclear export (102). In obstructive and aristolochic acid-induced nephropathy, a decrease in PPM1A levels within the tubulointerstitium has been noted and this diminution plays a role in enhancing Smad3 phosphorylation, leading to subsequent RF (103). In obstructive nephropathy, maxacalcitol, an analog of vitamin D, enhances the function of the PPM1A/vitamin D receptor complex, resulting in the dephosphorylation of Smad3, thereby reducing tubulointerstitial fibrosis (104). Furthermore, PPM1A and PTEN collaboratively work to diminish the phosphorylation of Smad3 and the activation of genes associated with fibrosis (105).

In all organ tissues, intracellular proteins undergo continuous turnover through degradation and synthesis (106). The ubiquitin-proteasome system (UPS) primarily degrades intracellular proteins (106). This multi-enzyme process covalently attaches ubiquitin to a substrate protein, which the proteasome, a core proteolytic complex of the UPS, then recognizes and degrades (106).

Ubiquitination alters different elements within the TGF-β signaling pathway (87). The Smurfs, identified as the first ubiquitin E3 ligases for Smads, exemplify such ubiquitin E3 ligases (107). Extensive research has focused on these ligases, which specifically act upon R-Smads, Smad7 and certain Smad-associated proteins for proteasomal degradation (108). Smurf1 and Smurf2 are both E3 ubiquitin ligases for Smad7 (87). Additionally, Smurf2 is capable of initiating the polyubiquitylation and degradation of Smad2, as well as the Smad gene silencing cofactors SKI and SnoN within kidneys undergoing fibrosis (107,109). This is evidenced by the fact that the overexpression of Smurf2 has been shown to encourage the activation of the TGF-β-responsive promoter and induce EMT within human kidney cortical epithelial cells (107). Recent research has demonstrated that flavin-containing monooxygenase 2 can enhance the expression of Smurf2 and facilitate its nuclear translocation, resulting in tubular cell fibrogenesis and paracrine secretion (110). In addition, the E3 ligase, Arkadia/RNF111, regulates TGF-β signaling by degrading Smad7 (111,112). This degradation consequently fostered the advancement of fibrosis in a rat model of tubulointerstitial fibrosis (111,112). Overexpression of latent TGF-β1 has been shown to ameliorate DN through the inhibition of Arkadia-induced Smad7 imbalance, which subsequently provoked renal inflammation and tissue fibrosis in type 1 diabetes mice induced by STZ (113). In addition, neural precursor cell expressed developmentally down-regulated 4-2 (NEDD4-2) has been identified as the ubiquitin ligase that facilitates proteasome-mediated degradation of TGF-β-induced phosphorylated Smad2/3 (114,115). Deficiency of NEDD4-2 in mice results in progressive kidney injury, marked by fibrosis, tubular epithelial cell apoptosis and various characteristics of CKD, including dilated/cystic tubules, and elevated expression of kidney injury markers (116). In conclusion, the UPS may either promote or inhibit fibrotic outcomes, depending on the TGF-β signaling elements that undergo degradation.

Deubiquitination, the counterpart to ubiquitination, is a key cellular procedure encompassing the removal of ubiquitin molecules that have been added to proteins (117,118). This dynamic interplay between ubiquitination and deubiquitination ensures the precise regulation of protein function, stability and interactions within the cell (117). Deubiquitinating enzymes, also known as deubiquitinases (DUBs), constitute a vast group of proteases responsible for removing ubiquitin from proteins (117). Moreover, they aid in the creation of independent entities from freshly translated polyubiquitins and repurpose ubiquitins after the breakdown of polyubiquitinated protein substrates (119). While the control of TGF-β signaling through ubiquitination has been widely studied in the past decades, the function of deubiquitination steered by DUBs in the TGF-β signaling pathway, especially in the context of CKD, has only begun to gain attention recently.

PR-619, a comprehensive DUB inhibitor, mitigated fibrosis in mice undergoing UUO and in rat kidney fibroblast cells, namely NRK-49F cells, triggered by TGF-β1 (120). Furthermore, PR-619 demonstrates an inhibitory effect on Smad4 levels, while it does not affect the production of TGF-βR, Smad2 or Smad3 (120). This indicates that DUBs may regulate fibrosis by modulating Smad4 (120). At present, members of the ubiquitin specific proteases family (USPs), which have been reported to regulate Smad4 deubiquitination, include USP9X (121), USP10 (122), USP13 (123), USP17 (124) and USP25 (125). USPs are currently known as the most extensive and predominant family of enzymes associated with deubiquitination (117). In fact, USP9X is documented to suppress fibrosis triggered by the stimulation of AGEs in mesangial cells, as well as EMT in renal tubular cells (126,127). USP10 has been reported to counteract renal impairment caused by sepsis, primarily by reducing apoptosis in TECs and mitigating oxidative stress (128). Furthermore, recent research has confirmed that USP25 is instrumental in advancing hypertensive renal disease (125). Knockout of USP25 in mice has been shown to reduce kidney malfunctions and fibrotic conditions (125). From a mechanistic viewpoint, USP25 is associated with the regulation of TGF-β signaling activation (125). Specifically, USP25 functions by reducing Smad4 K63-linked polyubiquitination (125). For R-Smads and Smad7, although some DUBs have been reported to regulate their ubiquitination processes (129–133), their functions in CKD remain to be further elucidated.

Additionally, several DUBs, such as 26S proteasome-associated PAD1 homolog 1 (134) and ubiquitin C-terminal hydrolase 37 (131), have been reported to regulate the deubiquitination process of TGF-βRI, thereby promoting TGF signaling. However, only USP11 has been documented to modulate the fibrotic process in CKD (135,136). Ni et al (71) conducted an intervention using the USP11 inhibitor mitoxantrone on mice experiencing both UUO and folic acid-triggered RF. They discovered that this intervention could inhibit TGF-βRII expression and associated fibrotic and aging phenotypes. The use of USP11 conditional knockout mice further confirmed this phenomenon (135). It is noteworthy that these DUBs often have multiple substrates. For instance, USP11 can regulate the deubiquitination of both TGF-βRII and epidermal growth factor receptor (EGFR) (135,136).

SUMOylation, a process where SUMO covalently attaches to specific protein targets, plays a pivotal role in modulating signal transduction through changes in subcellular localization, influencing enzymatic activity and directing the ubiquitin-mediated breakdown of its target substrates (137). This modification process is driven by a series of enzymes requiring ATP, encompassing the E1 activator, the E2 conjugator known as Ubc9 and various E3 ligases (138). Notably, the function of SUMOylation in the TGF-β signaling pathway is attracting more and more interest (139).

To date, researchers have identified five SUMO proteins, labeled SUMO1-5 (140). Given the considerable sequence resemblance between SUMO2 and SUMO3, they are commonly categorized as SUMO2/3. Among these, SUMO1 and SUMO2/3 are expressed ubiquitously, while the distribution of SUMO4 is confined to specific organs, such as the spleen and kidney (137). The ability for SUMOylation to be reversed is maintained by SUMO-specific proteases (SENPs), which can detach SUMO proteins from their targets (108). Currently, seven distinct SENPs, ranging from SENP1-3 to SENP5-8, have been identified (141,142). Apart from reversing SUMOylation, these enzymes also mature pro-SUMO into a conjugatable form (142).

Within the scope of TGF-β signaling, the SUMOylation process of TGF-βR1 amplifies its capability to interact with Smad3, which promotes the phosphorylation of Smad3 (139). SENP2 counteracts this alteration, and an upsurge in SENP2 expression curtails the EMT instigated by TGF-β (143). The role of Smad4 SUMOylation in TGF-β transcription regulation remains contentious. Some researchers posit a detrimental effect of Smad4 SUMOylation on TGF-β signaling, highlighting that the K113R/K159R mutation curtails the polyubiquitination of Smad4 (144). In the context of renal mesangial cells under high glucose conditions, the SUMO2/3-driven SUMOylation of Smad4 triggers the TGF-β/Smad pathway, subsequently elevating fibronectin levels (145). These contrasting perspectives might arise from distinct cellular contexts (146). Conclusively, adjusting the (de-)SUMOylation of the TGF-β/Smad pathway presents a hopeful approach for CKD treatment.

Protein acetylation is recognized as a significant and reversible post-translational modification, underscoring its various cellular and physiological activities (147). Reversible acetylation is orchestrated by two primary enzyme classes: Acetyltransferases (KATs) and deacetylases (KDACs) (147). KATs enable the addition of acetyl groups onto lysine residues and encompass the general control non-derepressible 5, p300 and MYST families, along with other unclassified KATs (147). Although KATs primarily acetylate histones, enzymes such as p300 also influence the TGF-β/Smad pathway, and are also recognized for enhancing TGF-β activity through the acetylation of Smad2 or Smad3 (148,149). Furthermore, the inhibition of p300 with a novel FATp300 inhibitor, L002, mitigates RF caused by hypertension and opposes fibrogenic responses in fibroblasts (150).

Unlike KATs, KDACs are divided into two main categories: Classical histone deacetylases, which are Zn²⁺-dependent, and sirtuin deacetylases (SIRTs), which rely on NAD⁺ (147). In recent studies, seven mammalian counterparts of the yeast Sir2, specifically labeled as SIRT1 to SIRT7, have been discerned (151). Within the kidney, SIRT1 stands out as the predominant SIRTs under investigation. Primarily localized in the nucleus, it orchestrates the acetylation patterns of nucleosome histones and influences the dynamics of multiple transcriptional regulators (151). As a result, SIRT1 plays a pivotal role in cellular defense by mitigating processes such as apoptosis, inflammation and fibrosis (151). SIRT1 deficiency promotes Smad3 acetylation, subsequently activating TGF-β signaling and exacerbating the progression of CKD (152). Resveratrol intervention facilitates the interaction between SIRT1 and Smad3, thereby attenuating Smad3 acetylation (153). Moreover, elevating SIRT1 levels in tubular cells impedes the transition from AKI to tubulointerstitial fibrosis (151). This also curtails the subsequent accumulation of matrix metalloproteinase-7 in the kidney through the deacetylation of Smad4 (154). Hence, SIRT1 emerges as a promising candidate for therapy in treating CKD (152). Unlike SIRT1, SIRT2 predominantly resides in the cytoplasm and plays a role in hindering fibrosis within renal tubules (155). The specific removal of SIRT2 from TECs aggravates RF, while its deliberate overexpression in these cells reduces RF (155). In terms of mechanism, SIRT2 forms a direct association with Smad2 and Smad3, leading to their deacetylation; this interaction subsequently mitigates the fibrotic effects triggered by TGF-β (155). This highlights the therapeutic potential of SIRT2 in addressing fibrosis. In the context of CKD, there are limited reports on the regulatory roles of other SIRT family members, specifically SIRT3-7, concerning the TGF-β/Smad pathway. To sum up, the complex equilibrium and interaction between these enzymes underscore their potential as therapeutic targets in CKD and fibrotic conditions.

O-GlcNAcylation, a fluctuating and reversible type of PTM, entails the addition of a β-D-N-acetylglucosamine (GlcNAc) molecule onto serine or threonine residues within proteins (156). This modification, predominantly occurring within the cytoplasm and nucleus, is distinct from the conventional N- and O-glycosylations that take place in the endoplasmic reticulum and Golgi apparatus (156). The enzymes O-GlcNAc transferase and O-GlcNAcase are key in controlling O-GlcNAcylation, which is vital for numerous cellular functions such as signal transduction and transcription, as well as maintaining protein stability (156,157). Due to its significance, changes in O-GlcNAcylation are linked to various diseases including diabetes, neurodegenerative conditions and cancer (158–160).

A previous study has indicated that an increase in O-GlcNAcylation levels in kidney tissues following UUO, and that glucosamine-driven O-GlcNAcylation can promote fibrosis in the renal parenchymal cell, HK2 TECs (161). Notably, a previous study observed that Smad4 has been identified as a newly discovered protein undergoing O-GlcNAc modification in human lung cancer cells. In this context, O-GlcNAc hinders the linkage between Smad4 and GSK-3β, interactions that are crucial for the proteasomal breakdown of Smad4 (162). Nevertheless, the precise function of O-GlcNAcylation in fibrosis, along with its interplay with the TGF-β1/Smad3 signaling pathway remains to be further elucidated.

Myristoylation, a post-translational modification process, involves covalently linking myristate (a 14-carbon fatty acid) to the N-terminal glycine residue of the protein via an amide bond (163). This modification is critical in numerous protein signaling systems as it imparts various effects such as modulating protein stability, facilitating protein-protein interactions and enhancing subcellular localization to organelles or the plasma membrane (163).

There is relatively scant research focusing on the role of myristoylation in the progression of CKD. Notably, a previous study noted that myristoylated TGF-βRI and TGF-βRII can induce transcriptional activation of Smad2, suggesting a potential role for myristoylation in the activation of the TGF-β pathway (164). In addition, myristoylation may exert an indirect influence on the regulation of the TGF-β signaling pathway. For instance, the myristoylation of PPM1A could enhance the phosphatase activity of PPM1A, as promoted by the cellular senescence-inhibited gene, thereby inhibiting TGF-β signaling further (165). This suggests that the modulation of myristoylation states could represent a novel strategy for managing TGF-β-driven processes in CKD.