Targeting posttranslational modifications of oxidative stress pathways for the treatment of diabetic nephropathy

Abstract

Diabetic nephropathy (DN), a major microvascular complication of diabetes, is the leading cause of chronic kidney disease (CKD). Oxidative stress has emerged as a central driver of DN, making its associated signaling pathways promising therapeutic targets. When reactive oxygen species (ROS) exceed the cell's antioxidant capacity, they disrupt homeostasis and trigger inflammation, apoptosis, necrosis, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. In the kidneys, these events accelerate fibroblast activation and collagen deposition, leading to fibrosis, glomerulosclerosis, and progressive loss of function. Recent studies identify post-translational modifications (PTMs) as key regulators in this process. By altering protein structure, function, and interactions, PTMs modulate many of the pathological mechanisms underlying DN. Targeting oxidative stress pathways regulated by PTMs therefore offers a promising therapeutic approach. In this review, we first introduce the role of PTMs in DN and outline the contribution of oxidative stress to disease progression. We then examine how PTMs regulate oxidative stress pathways, highlight novel modifications with potential relevance to DN, and discuss emerging therapeutic strategies targeting these pathways.

Graphical abstract

Highlights

• •Oxidative stress damages lipids, proteins, and DNA, driving inflammation, ECM accumulation, and fibrosis in DN.
• •PTMs regulate oxidative stress in DN by modulating signaling pathways that control cellular responses to oxidative injury.
• •Modulating PTMs offers a promising strategy to reduce oxidative damage and protect renal function in DN.

1. Introduction

Diabetic nephropathy (DN), also known as diabetic kidney disease (DKD), is one of the most common and severe microvascular complications of diabetes. With the global prevalence of both type 1 and type 2 diabetes rising, DN poses an increasing burden on healthcare systems [1]. By 2045, diabetes is projected to affect 693 million people worldwide, with nearly 30% expected to develop DN [2]. Pathologically, DN is characterized by diffuse thickening of the glomerular basement membrane, mesangial matrix expansion with diffuse and nodular (Kimmelstiel-Wilson) sclerosis, tubulointerstitial fibrosis, and hyaline arteriolosclerosis. These structural changes lead to proteinuria and progressive loss of kidney function, often culminating in end-stage renal disease (ESRD). Clinically, DN is defined by elevated urinary albumin-to-creatinine ratio (UACR > 30 mg/g) and a persistent decline in estimated glomerular filtration rate (eGFR < 60 mL/min/1.73 m²). Without timely intervention, these abnormalities almost inevitably progress to ESRD [3,4]. The onset and progression of DN are shaped by hypertension, chronic inflammation, oxidative stress, and genetic susceptibility, with oxidative stress serving as both a physiological regulator and a major contributor to kidney injury. Despite improvements in glycemic control and overall diabetes care, DN remains highly prevalent and difficult to manage [5]. For the past two decades, treatment has relied primarily on angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) [6]. However, DN arises from a complex interplay of processes, including oxidative stress, inflammation, programmed cell death, impaired autophagy, and endoplasmic reticulum (ER) stress, that complicate the development of targeted therapies [7,8]. Oxidative stress occurs when the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) exceeds the capacity of antioxidant defenses, resulting in cellular damage [9]. Under normal conditions, ROS act as signaling molecules, but when overproduced, they damage lipids, proteins, and DNA, thereby accelerating DN progression. Thus, maintaining redox balance is essential for kidney health. Post-translational modifications (PTMs), chemical changes to proteins after synthesis, are critical regulators of cellular stress responses [10]. Modifications such as acetylation, ubiquitination, and glycosylation influence gene expression, signaling, and cell cycle regulation. Because many PTMs are reversible, they provide cells with flexibility to adapt to changing conditions. Enzymes such as glycosyltransferases and glycosidases add or remove these modifications, fine-tuning protein function in both health and disease [11]. PTMs are closely linked to oxidative stress pathways, modulating the activity and stability of antioxidant proteins. For example, acetylation can enhance the function of enzymes that detoxify ROS, thereby strengthening cellular resilience under stress [12]. This review explores the role of PTMs in regulating oxidative stress pathways in DN and highlights emerging therapeutic strategies that target these modifications to reduce oxidative injury and restore cellular homeostasis.

2. Roles of PTMs in DN

Over 450 PTMs have been identified, including enzymatic processes such as phosphorylation, ubiquitination, acetylation, glycosylation, and palmitoylation, as well as non-enzymatic changes like nitration [13]. Unlike transcriptional or translational regulation, PTMs enable rapid and flexible control of protein function, allowing cells to respond swiftly to physiological signals. Increasing evidence highlights the central role of PTMs in DN. For example, stabilization of sirtuin 1 (SIRT1) reduces both expression and acetylation of hypoxia-inducible factor 1-alpha (HIF-1α), suppressing extracellular matrix (ECM) accumulation, apoptosis, and inflammation, thereby protecting against renal tubular fibrosis in DN [14]. Similarly, deletion of insulin-like growth factor binding protein 5 (IGFBP5) inhibits NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation, preventing endothelial-mesenchymal transition and slowing DN progression [15]. Conversely, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation promotes renal injury and interstitial fibrosis, while tripartite motif-containing 22 (TRIM22) exacerbates DN by enhancing ubiquitination of optic atrophy 1 (OPA1), driving mitochondrial dysfunction and apoptosis in renal tubular epithelial cells (RTECs) [16,17]. Acetylation of forkhead box O3a (FOXO3a) further increases oxidative stress and aggravates renal injury in DN [18]. Phosphorylation of the alpha kinase 1/nuclear factor kappa-B (ALPK1/NF-κB) pathway contributes to tubular injury and interstitial inflammation in DN by activating the caspase-1-Gasdermin D (GSDMD) pyroptosis cascade [19]. PTMs also regulate ferroptosis, a key driver of advanced DN [20]. The E3 ligase pre-messenger RNA (pre-mRNA) processing factor 19 (PRPF19) accelerates ferroptosis in DN by promoting ubiquitination and proteasomal degradation of the vitamin D receptor (VDR), while lysine acetyltransferase 2A (KAT2A) promotes inflammation and ferroptosis through histone H3 lysine 79 (H3K79) succinylation and SAT2 transcription [21,22]. Notably, non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), have emerged as key regulators of PTMs in DN. Silencing miR-150-5p mitigates high glucose (HG)-induced podocytes injury and streptozocin (STZ)-induced DN by enhancing SIRT1-tumor protein p53 (p53) interaction and reducing p53 acetylation [23]. In contrast, lncRNA ARAP1 antisense RNA2 (ARAP1-AS2) promotes HG-induced renal tubular injury and fibrosis by sustaining epidermal growth factor receptor (EGFR)/transforming growth factor-beta (TGF-β)/SMAD family member 3 (Smad3) activation through reduced EGFR ubiquitination [24]. lncRNA SPAG5 antisense RNA1 (SPAG5-AS1) facilitates SPAG5 deubiquitination via interaction with ubiquitin-specific peptidase 14 (USP14), which in turn activates the protein kinase B (AKT/PKB)/mechanistic target of rapamycin (mTOR) pathway, suppresses autophagy, and induces podocyte apoptosis [25]. Conversely, lncRNA SOX2 overlapping transcript (SOX2OT) is downregulated in DN, and its upregulation via forkhead box A2 reduces oxidative stress and apoptosis in RTECs by promoting SIRT1 expression [26]. Collectively, these findings demonstrate that PTMs play a critical role in DN (Fig. 1).

Fig. 1.

The role of post-translational modifications (PTMs) in diabetic nephropathy (DN). Dysregulated PTMs, such as phosphorylation, acetylation, ubiquitination, and glycosylation, can disrupt the proteins involved in apoptosis, mitochondrial function, endoplasmic reticulum (ER) stress, fibrosis, autophagy, and cell cycle regulation. These aberrant modifications may activate pro-apoptotic signaling pathways, induce mitochondrial damage, and exacerbate ER stress by impairing chaperone activity or obstructing the clearance of misfolded proteins. Additionally, PTM dysregulation contributes to pathological tissue remodeling, promoting fibrosis, hindering autophagic clearance, and enforcing inappropriate cell cycle arrest, all of which worsen cellular dysfunction, thereby resulting in DN. Ac: acetylation; La: lactylation; SUMO: small ubiquitin-like modifier; Succ: succinylation; Cr: Crotonylation; Ub: ubiquitination; Palm: palmitoylation; P: phosphorylation; ROS: reactive oxygen species; EMT: epithelial-mesenchymal transition. Created by www.biorender.com.

3. Overview of oxidative stress in DN

Under normal conditions, mitochondria produce small amounts of ROS as byproducts of energy metabolism. At physiological levels, ROS function as signaling molecules that regulate vascular tone, endothelial function, and tissue repair, thereby maintaining vascular integrity. These ROS are typically neutralized by antioxidant defenses, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) [27]. In DN, this antioxidant system is compromised, tipping the balance toward oxidative stress [28]. The kidneys are particularly susceptible due to their high mitochondrial content and reliance on fatty acid β-oxidation and oxidative phosphorylation for energy [29]. HG levels accelerate the tricarboxylic acid (TCA) cycle, generating excess NADH and flavin adenine dinucleotide (FAD) and reduced form of FAD (FADH₂) that overload the mitochondrial electron transport chain. This causes electron leakage and excessive ROS production, resulting in mitochondrial damage, impaired adenosine triphosphate (ATP) synthesis, and a self-perpetuating cycle of oxidative stress [30]. Additional ROS sources include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, advanced glycation end products (AGEs), and ER stress [31]. ER stress, through the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor 2A (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP-homologous protein 10 (CHOP) pathway, can induce podocyte apoptosis and reduce protein synthesis [32]. Loss of protective factors, such as the VDR or DNA damage-inducible 45α (GADD45α), further exacerbates mitochondrial injury, lipid peroxidation, and oxidative stress by impairing the nuclear factor erythroid 2-related factor 2 (Nrf2)/kelch like ECH associated protein 1 (Keap1) antioxidant system and lipid metabolism [33]. Excess ROS activate multiple signaling pathways, including p38 mitogen-activated protein kinase (p38/MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), AKT/PKB, NF-κB, and protein kinase C-α/β (PKC-α/β), which amplify inflammation, fibrosis, and cell death [34]. These processes contribute to proteinuria, lipid accumulation, ECM deposition, and reduced tubular protein reabsorption. In the glomerulus, oxidative stress drives mesangial expansion, podocyte injury, and glomerulosclerosis, whereas in the tubulointerstitium, it promotes fibrosis and apoptosis in RTECs [35]. Targeting oxidative stress has been shown to slow DN progression. For example, nanosized formulations of carvedilol (CVL) delivered via self-nanoemulsifying drug delivery systems (SNEDDS) improve glycemic control and reduce renal fibrosis, inflammation, and oxidative damage in DN [36]. Current antioxidant strategies include phytochemicals such as resveratrol, curcumin, α-lipoic acid, α-tocopherol, vitamin C, and selenium, while mitochondria-targeted antioxidants have recently emerged as a promising approach to mitigate oxidative damage in DN [37]. Collectively, these findings highlight oxidative stress as a central contributor to DN pathogenesis (Fig. 2).

Fig. 2.

The role of oxidative stress in diabetic nephropathy (DN). (A) Oxidative stress disrupts essential cellular processes, including mitochondrial respiration. (B) Oxidative stress disrupts endoplasmic reticulum (ER) stress regulation. (C) Oxidative stress activates several signaling pathways, such as p38 mitogen-activated protein kinase (p38/MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase B (AKT/PKB), nuclear factor kappa-B (NF-κB), protein kinase C-α/β (PKC-α/β), which promote lipid peroxidation, deoxyribonucleic acid and protein damage, and mitochondrial dysfunction. These interconnected mechanisms lead to excessive reactive oxygen species (ROS) production, accelerating both the onset and progression of DN. CoQ: coenzyme Q; NADH/NAD⁺: nicotinamide adenine dinucleotide; FAD/FADH₂: flavin adenine dinucleotide (FAD) and reduced form of FAD; ADP: adenosine diphosphate; Cyt c: cytochrome C; ATP: adenosine triphosphate; PERK: protein kinase R (PKR)-like endoplasmic reticulum kinase; eIF2α: eukaryotic translation initiation factor 2A; IRE11α: endoplasmic reticulum to nucleus signaling 1; ATF4: activating transcription factor 4; CHOP: C/EBP-homologous protein 10; BAX: Bcl-2-associated X protein; P: phosphorylation; CAT: catalase; MDA: malondialdehyde; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; ECM: extracellular matrix; H⁺: hydrogen ion; e⁻: electron. Created by www.biorender.com.

4. PTMs of oxidative stress pathways in DN

PTMs are essential regulators of kidney function, particularly under oxidative stress. They modulate proteins involved in antioxidant defenses, ROS generation, and cellular damage responses, processes that contribute to DN [38]. In DN, chronic hyperglycemia alters PTMs, including glycosylation, S-nitrosylation, and ubiquitination, impairing the function of critical enzymes and signaling proteins. These modifications disrupt redox homeostasis and amplify oxidative stress, creating a self-perpetuating cycle that accelerates DN progression (Table 1) [18,[39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69]].

Table 1.

Post-translational modifications (PTMs) influence diabetic nephropathy (DN) by affecting oxidative stress pathways.

PTMs	Protein	Expression	Biological functions	Refs.
Acetylation	FOXO3a	Increase	In DN, SIRT1 deacetylase activity is markedly reduced, leading to increased acetylation of FOXO3a, which impairs its antioxidant function and promotes oxidative stress-induced injury in RTECs.	[18]
Acetylation	PGC-1α	Decrease	In DN, IPA expression is significantly reduced. Exogenous IPA increases SIRT1 activity, promotes the deacetylation and nuclear translocation of PGC-1α, alleviating oxidative stress, reducing proteinuria, thereby ameliorating renal injury.	[39]
Acetylation	p65,NF-κB, STAT3	Increase	In DN, SIRT1 expression is markedly reduced. Loss of SIRT1 activity enhances phosphorylation and acetylation of p65 and NF-κB/STAT3, promoting apoptosis and inflammation in RTECs, thereby triggering oxidative stress and renal fibrosis.	[40]
Acetylation	MPC2	Increase	In DN, downregulation of SIRT3 promotes MPC2 hyperacetylation at Lys19 and Lys27, triggering podocyte apoptosis, increasing mitochondrial ROS production, reducing mitochondrial membrane potential, decreasing ATP levels, contributing to podocyte injury.	[41]
Acetylation	H3, H4	Increase	In DN, HDAC2 activity is significantly elevated. This increase promotes hyperacetylation of histones H3 and H4, enhancing EMC deposition and driving EMT in RTECs. Elevated ROS levels further activating HDAC2, creating a positive feedback loop that exacerbates renal injury.	[42]
Acetylation	H3	Increase	Inhibiting HDAC4 increases H3K9 acetylation and reduces podocyte apoptosis under HG conditions, highlighting potential therapeutic opportunities for advanced DN.	[43]
Acetylation	Atp5f1c	Increase	Podocyte-specific loss of Rheb1 in diabetic mice causes hyperacetylation of Atp5f1c, reduced ATP production, mitochondrial dysfunction, and accelerated senescence.	[44]
Ubiquitination	Nrf2	Increase	Overexpression of CKIP-1 downregulates Smurf1, reducing K48-linked polyubiquitination and enhancing K63-linked polyubiquitination of Nrf2, while producing the opposite effect on Keap1. These changes stabilize Nrf2, activate the Nrf2/ARE pathway, limit ROS accumulation in GMCs and diabetic kidneys, and suppress fibrotic protein expression.	[45]
	Keap1	Decrease
Ubiquitination	Smurf2	Increase	In SV40 MES13 cells, LPA stimulation increases ROS production and enhances Traf4-mediated ubiquitination of Smurf2. This modification upregulates ChREBP and downstream fibrotic factors, thereby promoting fibrogenic responses.	[46]
Ubiquitination	Smurf1	Increase	In HG-induced GMCs, Cx32 overexpression promotes Smurf1-mediated ubiquitination while reducing ubiquitination of SIRT1 at Lys335. Stabilized SIRT1 then facilitates FOXO3a nuclear translocation and induces MnSOD expression, thereby alleviating oxidative stress and renal fibrosis.	[47]
	SIRT1	Decrease
Ubiquitination	Nox4	Decrease	In DN, reduced Cx32 decreases K48-linked ubiquitination of Nox4, leading to elevated oxidative stress and exacerbated renal fibrosis.	[48]
Ubiquitination	SIRT1	Increase	In DN, elevated Fyn phosphorylates c-Cbl at Tyr731, enhancing its interaction with SIRT1 and promoting K48-linked polyubiquitination of SIRT1 at Lys377 and Lys513, diminishing FOXO3a-mediated antioxidant activity and exacerbating oxidative stress and fibrosis.	[49]
Ubiquitination	PHB2	Increase	TIPE1 drives ubiquitination and proteasomal degradation of PHB2, a key regulator of mitochondrial autophagy. TIPE1 knockout restores mitochondrial function, enhances autophagy, and reduces EMT and interstitial fibrosis.	[50]
Ubiquitination	FPN1	Increase	In DN, activation of the STING pathway promotes ubiquitination-mediated destabilization of FPN1, triggering ferroptosis and oxidative stress. Conversely, STING inhibition restores FPN1 stability and alleviates renal injury.	[51]
Ubiquitination	TAK1	Increase	Deubiquitinating enzyme OTUD5 alleviates DN by deubiquitinating TAK1, thereby reducing podocyte inflammation and injury.	[52]
Ubiquitination	Nrf2	Increase	USP9X deubiquitinates Nrf2, preventing its degradation and promoting the activation of the Nrf2-ARE antioxidative pathway, which prevents ECM accumulation and reverses diabetic renal fibrosis.	[53]
SUMOylation	Podocin	Increase	Restoring podocin SUMOylation through SHP-1 deletion can reverse DN progression.	[54]
SUMOylation	RBMX	Decrease	The level of RBMX is elevated in DN, and its deSUMOylation can promote renal tubulointerstitial fibrosis in DN by regulating exosome cargo sorting and mitochondrial damage.	[55]
SUMOylation	HIF-1α	Increase	SAE1 promotes SUMOylation of HIF-1α, stabilizing its expression. Stabilized HIF-1α binds to the hypoxia-response element on the PFKFB3 promoter, upregulating PFKFB3 transcription. This cascade enhances glycolysis, thereby exacerbating renal fibrosis and injury.	[56]
Glycosylation	O-GlcNAc	Increase	Hyperglycemia-induced O-GlcNAc glycosylation accelerates DN progression by inhibiting AKT/eNOS phosphorylation and HSP72-mediated protective signaling.	[57]
Glycosylation	EZH2	Increase	OGT interacts with and glycosylates EZH2 to enhance its stability, thereby regulating the HES1/PTEN pathway and suppressing fibrosis in DN.	[58]
S-nitrosylation	Myo9A, RhoA actin	Decrease	In diabetic mice, glomerular Myo9A expression and S-nitrosylation are markedly reduced, and under hyperglycemia with elevated NO, decreased S-nitrosylation of Myo9A-interacting proteins RhoA and actin leading to increased RhoA activity and impaired podocyte migration.	[59]
S-nitrosylation	β3-integrin, laminin	Increase	In DN, reduced VEGF-A leads to increased S-nitrosylation of β3-integrin and laminin, which in turn promote glomerulosclerosis and progressive kidney failure.	[60]
Isoprenylation	Rho	Decrease	Simvastatin improves HG induced mesangial cell proliferation by inhibiting Rho protein isoprenylation and blocking the Rho GTPase/p21 pathway.	[61]
Lactylation	LARS1	Increase	In DN patient podocytes, lactylation of LARS1 at Lys970 activates mTORC1, increases the autophagy substrate p62, suppresses autophagy, and accelerates apoptosis, driving DN progression.	[62]
Lactylation	TRIM65	Increase	Lactylation at TRIM65 K206 weakens its dual inhibitory effects on ferroptosis and glycolysis, thereby accelerating the progression of DN.	[63]
Lactylation	ACSF2	Increase	ACSF2, a protein involved in lipid metabolism, is upregulated in DN. Lactylation of ACSF2 at Lys182 increases ROS production and impaired mitochondrial function, thereby contributing to renal injury.	[64]
Crotonylation	H3	Increase	NaCr increases H3K18cr, reduces blood sugar and lipid levels, improves inflammation and fibrosis in DN.	[65]
Succinylation	NEK7	Increase	Reduced SIRT5 expression increases succinylation of NEK7, thereby promoting podocyte pyroptosis and oxidative stress in DN.	[66]
Succinylation	HSD17B10	Decrease	In DN, succinylation of HSD17B10 at Lys99 is reduced. Mutation of K99R induces oxidative stress, disruptes HSD17B10 interactions with CPT1A and MRPP1, triggering proximal tubular injury.	[67]
Palmitoylation	CD36	Increase	Diabetic APT1 deficiency promotes palmitoylation of CD36 at the Cys466 site by reducing APT1 expression, leading to lipid overload and podocyte damage.	[68]
Butyrylation	H3	Decrease	In DN, H3K9bu and p300 levels are reduced, exacerbating inflammation and fibrosis.	[69]

FOXO3a: forkhead box O3a; SIRT1: sirtuin 1; RTECs: renal tubular epithelial cells; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; IPA: indole-3-propionic acid; p65: nuclear factor kappa B subunit p65; NF-κB: nuclear factor kappa-B; STAT3: signal transducer and activator of transcription 3; MPC2: mitochondrial pyruvate carrier 2; STAT1: signal transducer and activator of transcription 1; Lys19: lysine 19; Lys27: lysine 27; ROS: reactive oxygen species; ATP: adenosine triphosphate; H3: histone H3; H4: histone H4; HDAC2: histone deacetylase 2; EMT: epithelial-mesenchymal transition; HDAC4: histone deacetylase 4; H3K9: histone H3 lysine 9; HG: high glucose; Atp5f1c: ATP synthase f1 subunit gamma; Rheb1: ras homolog enriched in brain 1; Nrf2: nuclear factor erythroid 2-related factor 2; Keap1: kelch-like ECH-associated protein 1; CKIP-1: casein kinase 2-interacting protein-1; K48: lysine 48; K63: lysine 63; ARE: antioxidant response element; GMCs: glomerular mesangial cells; Smurf2: smad ubiquitination regulatory factor 2; SV40 MES13: simian virus 40-transformed mouse glomerular mesangial cells 13; LPA: lysophosphatidic acid; Traf4: tumor necrosis factor (TNF) receptor associated factor 4; ChREBP: carbohydrate-responsive element-binding protein; Smurf1: smad ubiquitination regulatory factor 1; Cx32: connexin 32; Lys335: lysine 335; MnSOD: manganese superoxide dismutase; Nox4: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4; c-Cbl: casitas b-lineage lymphoma; Tyr731: tyrosine 731; Lys377: lysine 377; Lys513: lysine 513; PHB2: prohibitin 2; TIPE1: TNF-α-induced protein 8-like 1; FPN1: ferroportin 1; STING: stimulator of interferon genes; TAK1: transforming growth factor-β-activated kinase 1; OTUD5: ovarian tumor deubiquitinase 5; USP9X: ubiquitin-specific protease 9X; ECM: extracellular matrix; SHP-1: src homology region 2 domain-containing phosphatase 1; RBMX: RNA binding motif protein X-linked; HIF-1α: hypoxia-inducible factor 1-alpha; SAE1: SUMO-activating enzyme subunit 1; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; O-GlcNAc: O-linked β-N-acetylglucosamine; AKT: protein kinase B; eNOS: endothelial nitric oxide synthase; HSP72: heat shock protein 72; EZH2: enhancer of zeste homolog 2; OGT: O-linked N-acetylglucosamine transferase; HES1: hairy and enhancer of split 1; PTEN: phosphatase and tensin homolog; NO: nitric oxide; Myo9A: MyoIXA; RhoA: ras homolog family member A; VEGF-A: vascular endothelial growth factor-A; Rho: ras homologous; GTPase: guanosine triphosphatase; p21: cyclin-dependent kinase inhibitor 1A; LARS1: leucyl-tRNA synthetase 1; Lys970: lysine 970; mTORC1: mechanistic target of rapamycin complex 1; p62: sequestosome 1; TRIM65: tripartite motif containing 65; K206: lysine 206; ACSF2: acyl-CoA synthetase family member 2; Lys182: lysine 182; NaCr: sodium crotonate; H3K18cr: histone H3 lysine 18 crotonylation; NEK7: never in mitosis A-related expressed kinase 7; SIRT5: sirtuin 5; HSD17B10: 17β-hydroxysteroid dehydrogenase type 10; Lys99: lysine 99; K99R: lys99 to arginine; CPT1A: carnitine palmitoyl transferase 1 A; MRPP1: mitochondrial ribonuclease P protein 1; CD36: cluster of differentiation 36; APT1: acyl-protein thioesterase 1; Cys466: cysteine 466; H3K9bu: histone H3 lysine 9 butyrylation; p300: E1A binding protein p300.

4.1. Acetylation

Protein acetylation is a reversible PTM in which an acetyl group is added to or removed from a protein. Acetyltransferases catalyze acetylation using acetyl-CoA, whereas deacetylases reverse this process. Histone acetyltransferases (HATs), also called KATs, modify both histones and non-histone proteins, including p53, tubulin, and high-mobility group proteins. Acetylation primarily occurs at the N-terminal α-amino group or the ε-amino group of lysine residues. N-terminal acetylation typically targets small, uncharged residues (Ser, Ala, Thr, Gly, Val, Cys), though acidic residues or initiator methionine can also be modified depending on sequence context and enzyme specificity [70]. Lysine deacetylation is mediated by Zn²⁺-dependent histone deacetylases (HDACs) and NAD⁺-dependent SIRTs, which regulate protein function by removing acetyl groups [71]. Proteomic analyses have identified over 6000 acetylated lysine sites in the kidney, many on proteins with multiple modifications. A substantial fraction localizes to the plasma membrane, influencing ion transport. In diabetic rat kidneys, 37 lysine-acetylated proteins are uniquely expressed under hyperglycemia [72]. In DN, acetylation regulates oxidative stress by modulating enzymes, transcription factors, bioactive and structural proteins. HATs such as E1A binding protein p300 (p300)/cAMP response element-binding protein (CBP) and general control non-repressed 5 protein (GCN5) respond to metabolic and oxidative cues by remodeling chromatin and promoting histone modifications. These modifications, such as histone H3 lysine 4 (H3K4) trimethylation and histone H3 lysine 18/27 (H3K18/27) acetylation, contribute to the acceleration of renal fibrosis in DN [73,74]. Elevated transforming growth factor-beta 1 (TGF-β1) in HG-treated glomerular mesangial cells (GMCs) and diabetic mouse glomeruli recruit p300/CBP to target genes, including PAI-1 and cyclin-dependent kinase inhibitor 1A (p21), increasing histone H3 lysine 9 and lysine 14 (H3K9/14) acetylation and enhancing Smad2/3 and specificity protein 1 (SP1) acetylation [75]. Similarly, inhibition of histone deacetylase 1 (HDAC1) enhances NF-κB p65 phosphorylation, leading to increased expression of inflammatory mediators, including monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), accompanied by elevated H3K9 and H3K18 acetylation [76]. Other chromatin regulators also drive DN progression. Myocardin-related transcription factor A (MRTF-A), upregulated in RTECs under HG, recruits WD repeat-containing protein 5 (WDR5), a component of the p300/H3K4 methyltransferase complex, to the collagen promoter, thereby enhancing H3K18 and H3K27 acetylation and promoting fibrosis [74]. Elevated bromodomain-containing protein 4 (BRD4) in RTECs and db/db mice activates the BRD4-p300/H3K27 acetylation (H3K27ac)/polo like kinase 1 (PLK1) pathway, stimulating the NLRP3 inflammasome and promoting pyroptosis, inflammation, and fibrosis [77]. Together, these findings demonstrate that acetylation-dependent chromatin remodeling amplifies inflammatory and fibrotic signaling in DN. Beyond histones, acetylation also modulates transcription factors and signaling proteins that regulate oxidative stress. For example, SIRT1 reduces oxidative damage by deacetylating FOXO transcription factors, promoting antioxidant enzyme expression. In diabetic rats, decreased SIRT1 activity increases FOXO3a acetylation, elevating catalase, SOD, ROS, and malondialdehyde [18]. SIRT1 also maintains mitochondrial homeostasis via peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which activates mitochondrial transcription factor A (mtTFA). In DN mice, disrupted tryptophan metabolism reduces serum indole-3-propionic acid (IPA), destabilizing SIRT1. IPA supplementation prevents SIRT1 degradation, allowing PGC-1α deacetylation and nuclear translocation, which enhances superoxide dismutase 2 (SOD2) and mtTFA expression, increases mitochondrial biogenesis, reduces oxidative stress, and lowers proteinuria [39]. In DN, phosphorylation and acetylation of NF-κB p65 and signal transducer and activator of transcription 3 (STAT3) are elevated, correlating with oxidative stress, altered antioxidant enzyme expression, apoptosis, and upregulation of pro-inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), VCAM-1, cyclooxygenase-2 (COX-2)). These modifications also promote epithelial-mesenchymal transition (EMT), evidenced by increased TGF-β1 and α-smooth muscle actin (α-SMA) and decreased E-cadherin [40]. SIRT3 regulates mitochondrial function by deacetylating mitochondrial pyruvate carrier 2 (MPC2) at K19 and K27, preserving membrane potential and ATP production. Loss of SIRT3 increases ROS, disrupts mitochondria, and induces podocyte apoptosis [41]. ROS can indirectly influence acetylation through pro-fibrotic signaling. ROS-induced TGF-β1 activation elevates histone deacetylase 2 (HDAC2) expression, altering histone acetylation patterns to promote ECM accumulation and EMT [42]. Inhibiting histone deacetylase 4 (HDAC4) increases H3K9 acetylation and reduces podocyte apoptosis under HG conditions, highlighting potential therapeutic opportunities for advanced DN [43]. Acetylation also regulates small GTPases, such as ras homolog enriched in brain 1 (Rheb1), which controls mitochondrial metabolism and ATP synthase acetylation. Podocyte-specific Rheb1 loss in diabetic mice causes hyperacetylation of ATP synthase F1 subunit gamma (Atp5f1c), reduced ATP production, mitochondrial dysfunction, and accelerated senescence [44]. In advanced DN, characterized by proteinuria, glomerular sclerosis, tubulointerstitial fibrosis, and tubular atrophy, therapies aim to slow fibrosis and preserve kidney function. Selective HDAC inhibitors, particularly targeting HDAC2 and HDAC4, combined with TGF-β pathway inhibitors, have shown promise in downregulating pro-fibrotic gene expression. Overall, these findings position protein acetylation as a central mechanism linking oxidative stress, mitochondrial dysfunction, and fibrosis, highlighting acetylation-dependent pathways as promising targets for stage-specific DN therapies (Fig. 3).

Fig. 3.

The role of acetylation in diabetic nephropathy (DN) via oxidative stress. Chronic glucose exposure and streptozotocin treatment alter protein acetylation in both in vivo and in vitro models, disrupting redox balance. Acetylation of redox-regulating proteins affects their activity, shifting the balance between reactive oxygen species (ROS) production and antioxidant defenses. Sirtuin 1 (SIRT1) plays a central role in this process by deacetylating key proteins, including peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α), forkhead box O3 (FOXO3a), and nuclear factor kappa-B (NF-κB), thus maintaining redox homeostasis. When SIRT1 activity is reduced, these proteins become hyperacetylated, triggering pro-inflammatory pathways that foster a cycle of inflammation and oxidative damage, accelerating kidney injury in DN. Additionally, loss of SIRT1 impairs PGC-1α deacetylation and nuclear translocation, which reduces the expression of superoxide dismutase 2 (SOD2) and mitochondrial transcription factor A (mtTFA). This mitochondrial dysfunction increases oxidative stress, exacerbates proteinuria, and drives DN progression. High glucose (HG) further amplifies these effects by promoting NF-κB p65 phosphorylation and inhibiting histone deacetylase 1 (HDAC1). These alterations enhance histone H3 acetylation, repress protective gene expression, and intensify oxidative stress. The resulting oxidative environment contributes to albuminuria, epithelial-to-mesenchymal transition (EMT), fibrosis, and pyroptosis, which are key features of DN progression. TGF-β1: transforming growth factor-beta 1; STZ: streptozocin; HDAC: histone deacetylase; Atp5f1c: ATP synthase F1 subunit gamma; BAX: BCL2 associated X protain; Bcl-2: B cell lymphoma 2; MPC2: mitochondrial pyruvate carrier 2; Rheb1: ras-homolog enriched in brain pseudogene 1; SIRT3: sirtuin 3; STAT3: signal transducer and activator of transcription 3; P300: E1A binding protein p300; MRTF-A: mocardin-related transcription factor A; H3: histone 3; pro-IL-18: pro-interleukin-18; pro-IL-1β: pro-interleukin 1 beta; IL-18: interleukin 18; IL-1β: interleukin 1 beta; N-GSDMD: N-terminal of gasdermin-D; IPA: indole-3-propionic acid; TFs: transcription factors; CAT: catalase; MDA: malondialdehyde; SOD: superoxide dismutase; NLRP3: NOD-like receptor family pyrin domain containing 3; WDR5: WD repeat domain 5; CBP: CREB binding lysine acetyltransferase; SP1: specificity protein 1; Smad2/3: SMAD family member 2 and SMAD family member 3; PAI-1: serpin family E member 1; P21: cyclin-dependent kinase inhibitor 1A; BRD4: bromodomain-containing protein 4; PLK1: polo like kinase 1; P: phosphorylation; Ub: ubiquitination; Ac: acetylation; K9: lysine 9; K19: lysine 19; K18: lysine 18; K14: lysine 14; K27: lysine 27; H3: histone H3. Created by www.biorender.com.

4.2. Ubiquitination

Ubiquitination is an essential and evolutionarily conserved PTM that regulates protein degradation, signaling, and cellular homeostasis [78]. It involves the covalent attachment of ubiquitin, a 76-amino acid protein, to target proteins, modulating their stability, localization, and function. Although ubiquitination is best known for marking proteins for proteasomal degradation, it also plays critical roles in DNA repair, oxidative stress responses, and signal transduction. The ubiquitin-proteasome system (UPS) mediates this process through a three-enzyme cascade involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases [79]. E1 enzymes activate ubiquitin in an ATP-dependent reaction and transfer it to E2, which delivers it to an E3 ligase. E3 ligases provide substrate specificity and catalyze ubiquitin attachment to lysine residues on target proteins [80]. They are classified into three main groups: really interesting new gene (RING), homologous to E6-associated protein C-terminus (HECT), and RING-between-RING (RBR). RING ligases act as scaffolds bring E2-ubiquitin complexes close to substrates, HECT ligases form transient ubiquitin-enzyme intermediates, and RBR ligases combine features of both mechanisms [81,82]. In contrast, deubiquitinases (DUBs) remove ubiquitin chains, reversing ubiquitination and fine-tuning protein stability, signaling, and homeostasis. In the early stages of DN, ubiquitination is protective. By removing misfolded or damaged proteins, the UPS prevents toxic accumulation and maintains proteostasis. It also regulates oxidative stress and inflammation by controlling turnover of signaling proteins, including NF-κB inhibitors. As DN progresses, chronic hyperglycemia sustains inflammation, fibrosis, and cell death. Dysregulated ubiquitination alters levels of profibrotic mediators such as TGF-β and connective tissue growth factor (CTGF), thereby promoting fibrosis, apoptosis, podocyte and tubular epithelial cell loss, glomerulosclerosis, and tubular atrophy. Proteasome dysfunction in advanced disease exacerbates injury by allowing ubiquitinated proteins to accumulate, amplifying cellular stress. A central link between ubiquitination and DN involves the transcription factor Nrf2, a master regulator of antioxidant defenses. Under normal conditions, Nrf2 is degraded by the Keap1-cullin 3 (CUL3)-ring-box 1 (RBX1) E3 ligase complex. In DN, this pathway may be disrupted, either reducing Nrf2 degradation as a compensatory response or worsening oxidative stress. For example, overexpression of casein kinase 2-interacting protein-1 (CKIP-1) suppresses smad ubiquitination regulatory factor 1 (Smurf1), decreasing lysine 48 (K48)-linked and enhancing lysine 63 (K63)-linked polyubiquitination of Nrf2, while producing the opposite effect on Keap1. These changes stabilize Nrf2, activate the Nrf2/antioxidant response element (ARE) pathway, limit ROS accumulation in GMCs and diabetic kidneys, and suppress fibrotic protein expression [45]. Other modulators link ubiquitination to oxidative stress and fibrosis in DN. In db/db mice, renal carbohydrate response element-binding protein (ChREBP) is markedly increased. In GMCs, lysophosphatidic acid (LPA) stimulates ROS production and phosphatidylinositol 3-kinase (PI3K)/AKT signaling, while tumor necrosis factor (TNF) receptor associated factor 4 (Traf4)-mediated ubiquitination of Smurf2 elevates ChREBP and profibrotic mediators including fibronectin, TGF-β, and IL-1β [46]. In HG-treated GMCs, Cx32 overexpression promotes Smurf1 ubiquitination but reduces SIRT1 ubiquitination, thereby stabilizing SIRT1. Stabilized SIRT1 facilitates FOXO3a nuclear translocation, activates manganese superoxide dismutase (MnSOD), and attenuates oxidative stress and fibrosis [47]. Conversely, reduced Cx32 expression decreases K48-linked ubiquitination of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4), leading to ROS accumulation and aggravated fibrosis in DN [48]. Aberrant ubiquitination further drives DN pathology. In diabetic mice, upregulated Src-family kinase Fyn phosphorylates casitas b-lineage lymphoma (c-Cbl), enhancing K48-linked ubiquitination of SIRT1 at Lys377 and Lys513, diminishing FOXO3a-mediated antioxidant activity and exacerbating oxidative stress and fibrosis [49]. Similarly, elevated TNF-α-induced protein 8-like 1 (TIPE1) promotes ubiquitination and proteasomal degradation of prohibitin 2 (PHB2), a key regulator of mitochondrial autophagy. TIPE1 knockout restores mitochondrial function, enhances autophagy, and reduces EMT and interstitial fibrosis in DN [50]. In addition, stimulator of interferon genes (STING) activation in DN destabilizes ferroportin 1 (FPN1) via ubiquitination, promoting ferroptosis and oxidative stress, whereas STING inhibition preserves FPN1 stability and protects against renal injury [51]. Deubiquitinating enzyme ovarian tumor deubiquitinase 5 (OTUD5) alleviates DN by deubiquitinating transforming growth factor-β-activated kinase 1 (TAK1), thereby reducing podocyte inflammation and injury [52]. Similarly, ubiquitin-specific protease 9X (USP9X) deubiquitinates Nrf2, preventing its degradation and promoting the activation of the Nrf2-ARE antioxidative pathway, which prevents ECM accumulation and reverses the progression of diabetic renal fibrosis [53]. Collectively, these findings demonstrate that ubiquitination regulates oxidative stress in DN by controlling the stability and degradation of key proteins, ultimately influencing disease progression (Fig. 4).

Fig. 4.

The role of ubiquitination in diabetic nephropathy (DN) via oxidative stress. Ubiquitination contributes to oxidative stress in DN through several signaling pathways, including the activation of lysophosphatidic acid (LPA) via its receptors LPAR1 and LPAR3, the interaction of advanced glycation end products (AGEs) with toll-like receptors (TLRs), glucose transporter (GLUT) activity, and stimulation of transforming growth factor-beta (TGF-β) receptors. Additionally, tumor necrosis factor (TNF) induces reactive oxygen species (ROS) production through TNF receptors (TNFR). These pathways are closely linked to aberrant ubiquitination, which accelerates protein degradation and exacerbates ROS accumulation under oxidative stress. Key substrates, including smad ubiquitylation regulatory factor-2 (Smurf2), sirtuin1 (SIRT1), and nuclear factor erythroid-2-related factor 2 (Nrf2), are affected by ubiquitination, altering their nuclear translocation and influencing downstream transcriptional activity. This disruption in ROS homeostasis promotes the expression of fibrosis-related genes, driving kidney injury and renal fibrosis in DN. Smurf1: smad ubiquitination regulatory factor 1; RAGE: receptor for advanced glycation end products; P: phosphorylation; Ub: ubiquitination; Traf4: tumor necrosis factor (TNF) receptor associated factor 4; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B; ChREBP: carbohydrate response element binding protein; Cx32: connexin 32; NOX4: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4; FOXO3a: forkhead box O3a; Fyn: FYN proto-oncogene, src family tyrosine kinase; c-Cbl: casitas b-lineage lymphoma; Keap1: kelch like ECH associated protein 1; sMaf: small MAF BZIP transcription factor; ARE: antioxidant response element; CKIP-1: PH domain-containing casein kinase 2 interacting protein-1; STING: stimulator of interferon genes; FPN1: ferroportin 1; TIPE1: tumor necrosis factor alpha-induced protein 8-like protein 1; PHB2: prohibitin 2; CUL3: cullin 3; RBX1: ring-box 1; EMT: epithelial-to-mesenchymal transition; USP9X: ubiquitin-specific protease 9X; K48: lysine 48; K377: lysine 377; K513: lysine 513; K63: lysine 63; Y731: tyrosine at position 731. Created by www.biorender.com.

4.3. Small ubiquitin-related modifier (SUMO)ylation

SUMOylation is a PTM in which SUMO proteins, approximately 11 kDa in size and structurally similar to ubiquitin, are covalently attached to target proteins. Under normal conditions, a balance between SUMOylation and deSUMOylation preserves cellular homeostasis. Hyperglycemia disrupts this balance, activating pathological signaling that contribute to DN [83]. Mammalian cells express three main SUMO isoforms: SUMO1, SUMO2, and SUMO3. HG stimulation has been shown to induce SUMO2/3-mediated SUMOylation of Smad4 in mesangial cells, thereby exacerbating DN progression [84]. The SUMO-conjugating enzyme E2 SUMO-conjugating enzyme (UBC9) plays a central role in this pathway. In pancreatic β-cells, UBC9 mediates SUMOylation of Nrf2 at lysine 525 (K525) and lysine 595 (K595). Loss of UBC9 in diabetic mice impairs Nrf2-driven antioxidant defense, leading to ROS accumulation and oxidative stress [85]. Conversely, in a unilateral ureteric obstruction model of renal fibrosis, UBC9 upregulation promotes SUMOylation of the transcription factor nuclear receptor subfamily 5 group A member 2 (NR5A2), enhancing its binding to the calreticulin promoter and driving fibrosis [86]. In podocytes, restoring podocin SUMOylation through deletion of the src homology region 2 domain-containing phosphatase 1 (SHP-1) has been shown to reverse DN progression [54]. Other regulators further link SUMOylation to renal fibrosis and tubular injury. Elevated RNA binding motif protein X-linked (RBMX) levels in DN promote renal tubulointerstitial fibrosis by influencing exosomal cargo sorting, while its deSUMOylation exacerbates this process [55]. Similarly, the SUMO-activating enzyme subunit 1 (SAE1) stabilizes HIF-1α through SUMOylation, which activates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) driven glycolysis and mesenchymal transformations, contributing to fibrotic remodeling in diabetic kidneys [56]. Together, these findings establish SUMOylation as a key regulator of oxidative stress, fibrosis, and tubular injury in DN, underscoring its potential as a therapeutic target.

4.4. Glycosylation

Glycosylation is a key biochemical process that modifies carbohydrates and their conjugates, supporting essential physiological functions. This process involves the covalent attachment of sugars to specific amino acids, most commonly through N- or O-glycosylation. Less common forms include C-, S-, and P-glycosylation. Some proteins are anchored to cell membranes via glycophosphatidylinositol (GPI) linkages, which connect the protein's C-terminus to phosphatidylinositol through a glycan bridge [87]. Under normal conditions, glycosylation regulates various cellular processes, including protein folding, stability, trafficking, cell signaling, immune recognition, and intercellular communication. In DN, hyperglycemia disrupts O-GlcNAcylation, impairing signaling pathways and contributing to increased oxidative stress. Glycosylation also influences the stability and membrane localization of glucose transporters (GLUTs). As the DN progresses, glycosylation patterns shift partly due to the activity of ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), which enhances ER function and promotes mesangial cell proliferation. In the early stages of DN, moderate increases in O-GlcNAcylation may provide protection against metabolic stress [88]. Glycolytic imbalance, characterized by disruptions in the regulation of glycolysis, leads to excessive or insufficient glycolytic activity, altering the flow of metabolites between glycolysis, mitochondrial respiration, and the pentose phosphate pathway. This imbalance contributes to the accumulation of glycolytic intermediates and lactate, exacerbating oxidative stress [89]. In STZ-induced diabetic rats, O-GlcNAc glycosylation is elevated in the renal cortex. However, hyperglycemia reduces the expression of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in proximal tubular cells, impairing AKT/endothelial nitric oxide synthase (eNOS) phosphorylation and suppressing heat shock protein 72 (HSP72) induction [57]. Upregulation of OGT can stabilize enhancer of zeste homolog 2 (EZH2) through glycosylation, limiting excessive cell proliferation and reducing the levels of fibrotic proteins such as fibronectin and collagen IV, which mitigates kidney injury in DN [58]. As DN advances, chronic hyperglycemia activates the hexosamine biosynthesis pathway, increasing the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and driving excessive O-GlcNAcylation [90]. This over-modification disrupts transcription factors like Nrf2, weakening antioxidant defenses and heightening oxidative stress. Additionally, reduced ENTPD5 expression can cause N-glycosylation defects, severe ER stress, and apoptosis in renal tubular cells [88]. Together, these glycosylation disturbances contribute to inflammation, fibrosis, and irreversible kidney damage in late-stage DN. Understanding the interplay between glycosylation, oxidative stress, and renal function is crucial for elucidating the molecular mechanisms of DN and developing targeted therapeutic approaches.

4.5. S-nitrosylation

S-nitrosylation is a reversible PTM in which nitric oxide (NO) or its derivatives attach to cysteine thiol groups (-SH), forming S-nitrosothiols (-SNO). This modification is regulated by enzymes such as S-nitrosylases, which promote it, and denitrosylases, which remove it. Under normal conditions, S-nitrosylation helps protect cells from oxidative stress [91]. eNOS and inducible nitric oxide synthase (iNOS) produce NO to initiate the process, while S-nitrosoglutathione reductase (GSNOR) and thioredoxin (Trx) reverse it to maintain redox balance [92]. However, chronic hyperglycemia and inflammation can disrupt this balance by increasing iNOS activity, leading to uncontrolled S-nitrosylation. This dysregulation impairs antioxidant regulators like SIRT1, induces ER stress, and elevates ROS [93]. Notably, preventing S-nitrosylation of pyruvate kinase M2 (PKM2) restores glycolytic control and enhances antioxidant capacity through the pentose phosphate pathway, reducing oxidative injury [94]. Beyond redox balance, S-nitrosylation also directly impacts protein function and signaling. In endothelial cells, it regulates tissue transglutaminase 2 (TG2), influencing protein crosslinking and amplifying NO bioactivity. These effects extend to glycolysis, redox regulation, and inflammatory signaling, with implications for chronic kidney disease (CKD) and ischemia-reperfusion injury [95]. Additionally, S-nitrosylation of peroxiredoxin 2 (PRDX2), a major ROS scavenger, inhibits its activity at cysteine 172 (Cys172), reducing mitochondrial dysfunction, oxidative stress, and apoptosis in RTECs [96]. In DN, protein S-nitrosylation is significantly elevated in rat kidneys, accompanied by increased ROS, which activates matrix metalloproteinase 9 (MMP-9) and damages podocytes through alterations in type IV collagen [97]. In contrast, diabetic mice show decreased expression and S-nitrosylation of glomerular MyosinIXA (Myo9A). Under hyperglycemic conditions, NO-rich environments reduce S-nitrosylation of ras homolog family member A (RhoA) and actin, leading to enhanced RhoA activity and impaired podocyte migration [59]. Furthermore, S-nitrosylation is crucial for the integrity of the glomerular filtration barrier, which depends on both NO and vascular endothelial growth factor-A (VEGF-A). In DN, reduced VEGF-A disrupts this process, causing abnormal S-nitrosylation of β3-integrin, laminin, and GSNOR, contributing to glomerulosclerosis and kidney failure [60]. Overall, S-nitrosylation plays a pivotal role in maintaining ROS balance in DN.

4.6. Isoprenylation

Isoprenylation is a PTM in which farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) is covalently attached to cysteine residues near the C-terminus of target proteins. This lipid modification anchors proteins to the plasma membrane and other cellular compartments, ensuring proper localization and function. The process is catalyzed by farnesyltransferase (FTase) and geranylgeranyl transferase types I (GGTase-I) and II (GGTase-II), and plays a crucial role in activating signaling proteins, including oncogenic RAS family members [98]. Although isoprenylation is irreversible, it often works in conjunction with reversible modifications like palmitoylation or phosphorylation, which regulate protein interactions, membrane association, and subcellular targeting [99]. Inhibition of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the isoprenoid pathway, has shown promising results in improving renal outcomes in experimental models. Blocking FPPS reduces serum creatinine and urea nitrogen levels, suppresses fibrotic and inflammatory protein expression, enhances catalase activity, and alleviates renal fibrosis [100]. While prenyltransferases have long been studied as potential cancer therapy targets, both these enzymes and their lipid substrates are increasingly implicated in DN. Statins, commonly used for cholesterol-lowering, also affect isoprenoid metabolism and provide renoprotective benefits in DN. For example, lovastatin reduces glomerular TGF-β1 expression and slows the progression of STZ-induced DN by lowering FPP and GGPP levels in mesangial cells under hyperglycemic conditions [101]. In early-stage DN, HG-driven mesangial cell proliferation accelerates glomerular injury and renal decline. Simvastatin disrupts this process by inhibiting Rho protein isoprenylation and blocking the Rho guanosine triphosphatase (GTPase)/p21 pathway. This cholesterol-independent action highlights statins as potential early therapeutic candidates for DN [61]. Additionally, HMG-CoA reductase inhibition decreases monocyte adhesion to endothelial cells induced by TNF-α and angiotensin II, key drivers of vascular inflammation, and reduces ROS production [102]. Despite these protective effects, the exact mechanisms through which isoprenylation influences oxidative stress in DN remain unclear and require further investigation.

5. Novel PTMs of oxidative stress pathways in DN

Advances in mass spectrometry-based proteomics have revealed numerous previously unknown PTMs, significantly enhancing our understanding of protein regulation. Among these, lysine acylations such as lysine lactylation (Kla), lysine crotonylation (Kcr), lysine succinylation (Ksucc), lysine butyrylation (Kbu), along with lipid-based modifications like palmitoylation, have emerged as key regulatory mechanisms. These PTMs are typically categorized into histone and non-histone acylations, both of which influence critical processes, including gene expression, chromatin remodeling, metabolic signaling, and stress responses. Many of these modifications are closely associated with glycolytic flux and metabolic state, emphasizing their role in cellular adaptation to metabolic stress. Together, these modifications serve as important mediators of oxidative stress and are increasingly linked to the development of DN (Fig. 5).

Fig. 5.

The role of novel posttranslational modifications in diabetic nephropathy (DN) via oxidative stress. Excess succinylation of 17 β-hydroxysteroid dehydrogenase type 10 (HSD17B10) increases reactive oxygen species (ROS), while reduced succinylation at lysine 99 (K99) destabilizes mitochondrial RNA ribonuclease P (RNase P) and impairs interactions with mitochondrial rnase p protein 1 (MRPP1), causing proximal tubular injury; reduced sirtuin 5 (SIRT5) further enhances never in mitosis A-related expressed kinase 7 (NEK7) succinylation, leading to podocyte pyroptosis. Hyperglycemia-driven lactate accumulation modifies acyl-CoA synthetase family member 2 (ACSF2), increasing ROS, reducing adenosine triphosphate, and activating autophagy, while lactylation of leucyl-tRNA synthetase 1 lysine 970 (LARS1K970) triggers apoptosis. Lactate also regulates tripartite motif-containing 65 (TRIM65), a protective factor against ferroptosis and glycolysis, but p300-dependent lactylation of TRIM65 at lysine 206 impairs its protective functions. Beyond succinylation and lactylation, other post-translational modifications (PTMs) also contribute: sodium crotonate (NaCr) restores histone H3 lysine18 (H3K18) crotonylation and is protective; reduced histone butyrylation and p300 activity promote inflammation and fibrosis; and downregulated APT1-dependent depalmitoylation of cluster of differentiation 36 (CD36) at cysteine 466 (Cys466) increases fatty acid uptake in podocytes. K970: lysine 970; H3K9: H3 lysine 9; mTOR: mechanistic target of rapamycin; K182: lysine 182; PDK4: pyruvate dehydrogenase kinase 4; IREB2: iron responsive element binding protein 2; NLRP3: NOD-like receptor family pyrin domain containing 3; APT: acyl-protein thioesterase; La: lactylation; Succ: succinylation; Cr: crotonylation; Ub: ubiquitination; bu: butyrylation; Pal: palmitoylation; K206: lysine 206; K466: lysine 466. Created by www.biorender.com.

5.1. Lactylation

Lactate, once considered merely as a byproduct of glycolysis, is now recognized as a key metabolic regulator [103]. Beyond serving as an energy substrate, lactate maintains cellular redox balance and homeostasis. It crosses membranes via monocarboxylate transporters (MCTs) and lactate dehydrogenase (LDH), entering mitochondria to facilitate ATP production and preserve oxidative equilibrium [104,105]. A central mechanism by which lactate regulates cellular processes is through protein lactylation, the covalent attachment of lactate to lysine residues, which modulates metabolism and immune signaling. Disrupted lactate metabolism impairs mitochondrial function, increases oxidative stress, and contributes to disease progression. The discovery of histone lactylation in 2019 revealed that lactate can directly influence gene expression by remodeling chromatin [106]. In the kidney, glycolytic reprogramming of proximal tubular cells through PFKFB3 enhances histone H4 lysine 12 lactylation (H4K12la). This modification accumulates at NF-κB target promoters (Ikbkb, Rela, Relb), driving the expression of inflammatory mediators such as IL-1β, TNF-α, and c-c motif chemokine ligand 2 (CCL2), which promote fibrosis-associated proteins like fibronectin and α-SMA, exacerbating renal injury [107]. Similarly, heat shock protein A12A (HSPA12A) enhances glycolysis-derived lactate via HIF-1, inducing lactylation of c-Myc. Lactylated c-Myc exhibits increased nuclear localization and activates proliferation genes such as Cyclin B1, Rcc1, and Cdk4, while inhibiting this modification reduces RTEC proliferation [108]. Lactylation is also implicated in DN. Hyperglycemia-driven lactate accumulation intensifies oxidative stress by increasing ROS production, damaging mitochondrial DNA and membranes, and amplifying apoptotic signaling [109]. Under hyperglycemic conditions, elevated lactate in podocytes promotes protein lactylation, contributing to cellular injury. In DN patient podocytes, lactylation of leucyl-tRNA synthetase 1 (LARS1) at lysine 970 activates mechanistic target of rapamycin complex 1 (mTORC1), increases the autophagy substrate sequestosome 1 (p62), suppresses autophagy, and accelerates apoptosis, driving DN progression [62]. Lactate also regulates the tripartite motif-containing 65 (TRIM65) gene, with TRIM65 deficiency exacerbating diabetic kidney injury. Overexpression of TRIM65 in RTECs, however, is protective. TRIM65 suppresses ferroptosis by promoting the degradation of iron-responsive element-binding protein 2 (IREB2) via ubiquitination and inhibits glycolysis through the degradation of pyruvate dehydrogenase kinase 4 (PDK4). Notably, lactate induces TRIM65 lactylation at lysine 206 (K206) in a p300-dependent manner, impairing its protective functions [63]. Lactylomics analyses in db/db mice identified 165 upregulated and 17 downregulated proteins, with 356 lysine lactylation sites increased. Many of the lactylated proteins (115 proteins, 269 sites) localize to mitochondria, highlighting lactate's role in mitochondrial function. For instance, acyl-CoA synthetase family member 2 (ACSF2), involved in lipid metabolism, is upregulated in DN, and its lactylation at lysine 182 (K182) increases ROS production and impairs mitochondrial function, underscoring lactate's pathogenic role [64]. In summary, lactylation of both histone and non-histone proteins plays a crucial role in regulating renal function, particularly mitochondrial processes. This modification can trigger excessive ROS production, mitochondrial dysfunction, and inflammatory responses, contributing to renal fibrosis. However, studies on lactylation in DN and its interaction with oxidative stress remain limited, and the underlying molecular mechanisms are not fully understood. Further research is needed to clarify its biological and pathological significance.

5.2. Crotonylation

Crotonylation is a recently identified PTM regulated by a set of enzymes classified as “writers”, “erasers”, and “readers”. Initially observed on histones, crotonylation has since been detected on numerous non-histone proteins, where it influences metabolism, cell cycle progression, and structural organization [110]. Writers are primarily HATs, some of which also function as histone crotonyltransferases (HCTs). These enzymes are categorized into three families, p300/CBP, GCN5-related N-acetyltransferase (GNAT), and MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST), based on structural and sequence similarities. However, the full complement of enzymes responsible for crotonylation remains to be fully defined. Erasers, including HDACs and SIRTs like HDAC1, HDAC3, and SIRT2, remove crotonyl groups from histone and non-histone proteins, thereby regulating crotonylation levels and gene expression. Readers are proteins that recognize crotonylated lysine residues and translate this modification into functional outcomes. Three major recognition domains have been identified: YAF9, ENL, AF9, TAF14, and SAS5 (YEATS), double PHD finger (DPF), and bromodomains, with YEATS being the primary crotonylation-binding motif. Proteins containing YEATS domains, such as AF9, TAF14, and YEATS2, play a central role in this recognition. Crotonylation is also influenced by substrate availability. Crotonyl-CoA, the donor molecule for this modification, is synthesized and degraded by enzymes including acyl-CoA synthetase short chain family member 2 (ACSS2), chromodomain Y-like protein (CDYL), enoyl-coenzyme A hydratase, short chain 1 (ECHS1), glutaryl-coenzyme A dehydrogenase (GCDH), acyl-coenzyme A dehydrogenase short chain (ACADS), acyl-coenzyme A oxidase 1 (ACOX1), and acyl-coenzyme A oxidase 3 (ACOX3). These enzymes collectively regulate Kcr dynamics, influencing chromatin structure, gene expression, and cellular function [111,112]. In renal fibrosis, histone crotonylation is notably increased in RTECs. ACSS2, which produces crotonyl-CoA, can enhance H3K9 crotonylation (H3K9cr) in kidney tissue. Integrated ChIP-seq and RNA-seq analyses have shown that H3K9cr promotes the expression of IL-1β, a pro-inflammatory cytokine that drives fibrosis. Suppressing ACSS2 reduces H3K9cr-mediated IL-1β expression, attenuates macrophage activation, and decreases levels of the senescence marker p53, along with senescence-associated secretory phenotype (SASP) factors like interleukin 6 (IL-6), matrix metallopeptidase 9 (MMP9), and IL-1β, ultimately alleviating renal fibrosis [113]. Further studies in db/db mice and HG-treated RTECs demonstrate that supplementation with sodium crotonate (NaCr) increases histone crotonylation, particularly H3K18 crotonylation (H3K18cr), in DN. NaCr treatment enhances H3K18cr, lowers blood glucose and lipid levels, and reduces pro-inflammatory cytokines (IL-1β, IL-6) and fibrotic markers (TGF-β1, α-SMA). Inhibition of ACSS2 or the HAT p300 reverses these effects, highlighting their essential roles in mediating the protective functions of histone crotonylation [65]. Targeting crotonylation-related enzymes is emerging as a promising therapeutic strategy for DN. However, further studies are required to clarify the specific contributions of crotonylation and to identify effective therapeutic targets within this pathway.

5.3. Succinylation

Succinylation, the addition of a succinyl group (a four-carbon dicarboxylic acid) to a lysine residue, is a PTM that regulates various cellular processes, including metabolism, signaling pathways, and gene expression [114]. First identified in Escherichia coli in 2011, lysine succinylation requires succinyl-CoA as a cofactor and predominantly occurs in mitochondria. This modification can occur through both enzymatic and non-enzymatic mechanisms, with most events happening spontaneously in response to elevated intracellular succinyl-CoA levels. Certain enzymes, such as carnitine palmitoyltransferase 1A (CPT1A) and HAT1, also catalyze lysine succinylation [115]. In renal tissues, protein hyper-succinylation is driven by succinyl-CoA accumulation and the downregulation of desuccinylase SIRT5. The loss of SIRT5 leads to excessive succinylation of parkinsonism-associated deglycase 7 (Park7) at lysine 93 in proximal tubular cells. This shift in fatty acid oxidation towards the peroxisome, while suppressing mitochondrial fatty acid oxidation, reduces oxidative stress and ROS, ultimately mitigating acute kidney injury [116]. Thus, modulating succinylation, either by enhancing SIRT5 activity or correcting metabolic imbalances, presents a potential therapeutic strategy for DN. In HG-treated podocytes, reduced SIRT5 expression increases succinylation of never in mitosis A-related expressed kinase 7 (NEK7), which promotes podocyte pyroptosis and oxidative stress in DN [66]. Similarly, in db/db mice and HG-stimulated RTECs, proteomic analysis of succinylation revealed decreased succinylation at lysine 99 (K99) of 17β-hydroxysteroid dehydrogenase type 10 (HSD17B10). K99 succinylation is crucial for stabilizing mitochondrial RNA ribonuclease P (RNase P). Mutating K99 to arginine (K99R) induces oxidative stress, disrupts HSD17B10 interactions with CPT1A and mitochondrial ribonuclease P protein 1 (MRPP1), and triggers proximal tubular injury [67]. Despite these findings, the precise regulatory mechanisms and broader biological roles of protein succinylation remain incompletely understood. Further research is needed to elucidate its contributions to DN pathogenesis and to identify potential therapeutic targets associated with oxidative stress.

5.4. Palmitoylation and butyrylation

Protein palmitoylation is a reversible PTM in which palmitate is covalently attached to cysteine residues. This modification is regulated by two main classes of enzymes: palmitoyl S-acyltransferases (PATs) and acyl protein thioesterases (APTs). PATs, also known as Zinc finger DHHC-type containing (ZDHHC) enzymes, catalyze the attachment of palmitate via a conserved DHHC (Asp-His-His-Cys) motif. In mammals, 23 ZDHHC enzymes have been identified, each with distinct substrate specificities and subcellular distributions. Depalmitoylation is mediated by APTs, including acyl protein thioesterase 1 (APT1) and acyl protein thioesterase 2 (APT2), along with other thioesterases like palmitoyl-protein thioesterase 1 (PPT1) and alpha/beta hydrolase domain-containing protein 17 (ABHD17) family members. The dynamic regulation of these enzymes ensures S-acylation homeostasis, which is crucial for protein localization, stability, and signaling [117]. In fibrotic kidneys from both patients and animal models, global S-palmitoylation and Zinc finger DHHC-type palmitoyltransferase 9 (DHHC9) expression are markedly reduced. Under normal conditions, DHHC9 palmitoylates β-catenin, promoting its ubiquitination and proteasomal degradation. However, during fibrosis, upregulation of APT1 leads to depalmitoylation of β-catenin, stabilizing the protein, facilitating its nuclear translocation, and activating profibrotic gene programs. This shift in palmitoylation dynamics exacerbates renal fibrosis. Genetic deletion of APT1 in renal tubules significantly reduces fibrosis, suggesting that DHHC9 and APT1 could be potential therapeutic targets for preventing renal fibrosis [118]. In DN, APT1-dependent depalmitoylation of cluster of differentiation 36 (CD36) at Cys466 is downregulated, promoting enhanced fatty acid uptake in podocytes [68]. Additionally, ZDHHC18 is upregulated in renal fibrosis and palmitoylates harvey rat sarcoma viral oncogene homolog (HRAS), promoting its plasma membrane translocation and activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, further contributing to fibrotic progression [119]. Understanding the specific patterns of palmitoylation in DN may reveal new oxidative stress pathways and therapeutic targets. Butyrylation, a relatively novel PTM, involves the addition of a four-carbon butyryl group to lysine residues. This modification is catalyzed by lysine acyltransferases like p300/CBP, which use butyryl-CoA as the donor. Meanwhile, SIRTs, particularly SIRT2 and SIRT3, act as the primary erasers, with other HDACs playing a secondary role in this process. Though less studied than acetylation or phosphorylation, butyrylation regulates important cellular processes, including gene expression, protein function, and responses to cellular stress [[120], [121], [122]]. Histone butyrylation, in particular, has emerged as a key modification involved in pathophysiological processes. In DN, studies in mouse kidneys and GMCs have shown reduced levels of pan-Kbu, histone H3K9 butyrylation (H3K9bu), and p300, accompanied by increased expression of inflammatory cytokines (IL-6, MCP-1) and fibrotic proteins (TGF-β, fibronectin). Inhibition of p300 further suppressed H3K9 butyrylation, exacerbating inflammation and fibrosis [69]. These findings underscore the importance of butyrylation in DN pathogenesis, highlighting its potential as a promising therapeutic target for preventing or managing renal fibrosis and inflammation.

6. Targeting PTMs of oxidative stress pathways against DN

Current treatments for DN primarily focus on controlling blood glucose and blood pressure to slow kidney damage. First-line pharmacologic therapies, including angiotensin-converting enzyme (ACE) inhibitors and ARBs, effectively reduce proteinuria and lower intraglomerular pressure [123]. Recently, sodium-glucose cotransporter-2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists have emerged as adjunct therapies, providing kidney protection beyond glycemic control [124]. Additional interventions, such as lipid management, lifestyle modification, and diuretics, address fluid overload and hypertension [125]. However, these treatments have limitations. While renin-angiotensin-aldosterone system (RAAS) and SGLT2 inhibitors can slow disease progression, they do not prevent it and may cause side effects like hyperkalemia or volume depletion in advanced kidney disease [126]. Intensive glycemic control carries the risk of hypoglycemia, while GLP-1 receptor agonists are limited by gastrointestinal side effects and high cost [127]. Overall, current therapies rarely reverse established damage, highlighting the need for treatments targeting the underlying molecular mechanisms of DN. Emerging research suggests that modulating PTMs, particularly those involved in oxidative stress, could offer a new therapeutic approach.

Several small molecules have shown promise in protecting against DN by modulating acetylation and other PTMs. Resveratrol, a plant-derived antioxidant, can activate SIRT1 to alleviate renal fibrosis in DN by reducing acetylation of yin yang 1 (YY1) and suppressing EMT in RTECs. Conversely, the SIRT1 inhibitor EX527 exacerbates renal fibrosis by increasing YY1 acetylation [128]. Valproic acid (VPA), a non-selective HDAC inhibitor, modifies histone acetylation at stress-response genes, enhancing H4 acetylation at the glucose-regulated protein 78 (GRP78) promoter while reducing it at CHOP, thereby mitigating ER stress, reducing renal cell apoptosis, and alleviating renal injury in DN [129]. Losartan, an angiotensin II type 1 receptor blocker, decreases H3K9/14 acetylation at the MCP-1 promoter in mesangial cells, improving glomerular hypertrophy, ECM accumulation, and fibrosis in diabetic mice [130]. Natural compounds like curcumin also show benefits by promoting histone H3 acetylation, reducing oxidative stress, downregulating HSP72 and p38, thus slowing STZ-induced DN progression [131,132]. Similarly, its analogue C66 prevents glomerular enlargement and fibrosis in STZ-induced DN by reducing H3K9/14 acetylation at fibronectin and CTGF promoters [133]. Additionally, the MCP-1/CCL2 antagonist mNOX-E36 reduces acetylation at H3K9 and histone H3 lysine 23 (H3K23), promoting glomerular cell proliferation, decreasing sclerosis and proteinuria, and preserving kidney function [134]. N-acetylcysteine (NAC), a glutathione precursor, restores mitochondrial redox balance by inhibiting SOD2 acetylation and activating the SIRT3-SOD2-glutathione peroxidase 4 (GPX4) pathway, thereby reducing oxidative stress and renal injury in DN [135]. Salidroside, a phenolic glycoside from Rhodiola species, can activate the adenosine monophosphate-activated protein kinase (AMPK)/SIRT1 axis, suppress acetylation of forkhead box o 1 (FOXO1) and p53 to reduce ROS production and apoptosis in mesangial cells, thereby alleviating renal damage in DN [136]. Targeting acetyltransferases also shows therapeutic potential. The p300/CBP inhibitor C646 reduces H3K27 acetylation, downregulates NADPH oxidase subtypes, pro-inflammatory mediators, and pro-fibrotic molecules, and lowers ROS generation in diabetic kidneys [137]. Several small molecules also offer protection through ubiquitination pathways. Ginkgolide B, a terpene from Ginkgo biloba, has been shown to improve DN by inhibiting GPX4 ubiquitination, reducing ferroptosis and oxidative stress [138]. Aspirin, a 20S proteasome inhibitor, increases histone H2A lysine 119 (H2AK119) ubiquitination by downregulating swim and MPN domain 1 (MYSM1), which in turn reduces Set7 expression and alleviates renal fibrosis in DN [139]. Schisandra A, derived from Schisandra chinensis, stabilizes adiponectin receptor 1 (AdipoR1) by decreasing its ubiquitination, activating the AdipoR1/AMPK pathway, and mitigating ferroptosis and NLRP3-mediated pyroptosis in DN [140]. Penehyclidine hydrochloride (PHC), an anticholinergic drug, promotes non-degradable ubiquitination of apoptosis inducing factor mitochondria associated 1 (AIFM1), activates Nrf2-dependent parkin rbr e3 ubiquitin protein ligase (PARK2) transcription, induces mitochondrial autophagy, and reduces ROS levels and RTEC apoptosis, thereby inhibiting renal tubular injury in DN [141]. Beyond acetylation and ubiquitination, other histone modifications also influence DN progression. Elevated lactic acid in diabetic mice increases histone H3 lysine 14 (H3K14) lactylation, promoting EMT and fibrosis. Inhibition of kruppel-like factor 5 (KLF5) by ML264 reduces H3K14 lactylation and suppresses EMT and fibrosis in DN [142]. Similarly, GLIS family zinc finger 1 (Glis1) prevents RTEC senescence and renal fibrosis by downregulating H3K14 lactylation in DN. Notably, lactylation enhancers such as rotenone and nala reduce Glis1's protective effects, whereas sodium dichloroacetate enhances them [143]. Shenkang injection (SKI), commonly used for CKD in China, restores lysine crotonylation in redox enzyme-associated proteins, raising crotonylation at NADH:ubiquinone oxidoreductase subunit s4 (Ndufs4) and cytochrome c oxidase subunit 5a (Cox5a) while reducing it at glutathione peroxidase 3 (GPX3), thereby enhancing antioxidant defenses and protecting renal function in DN [144]. Palmitic acid (PA) induces palmitoylation of peroxisomal biogenesis factor 11 beta (PEX11B), disrupting peroxisome homeostasis, whereas fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, restores peroxisome abundance and function in DN mice [145]. The acyl-coenzyme A oxidase 1 (ACOX-1) inhibitor 10,12-tricosadiynoic acid (TDYA) also prevents lipid accumulation and ROS buildup by blocking peroxisomal β-oxidation of dicarboxylic acids, improving DN outcomes [146]. Collectively, these studies highlight those small molecules modulating PTMs, particularly within oxidative stress pathways, offering a promising therapeutic strategy for DN (Table 2) [[128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146]].

Table 2.

Regulatory small molecules of post-translational modifications (PTMs) in diabetic nephropathy (DN): pharmacological mechanisms of action.

Small molecules	Animals/cells	Modification	Targets	Pharmacological mechanisms of action	Refs.
Resveratrol or SRT1720 or EX527	db/db mice and HK-2 cell	Acetylation	SIRT1	Activation of SIRT1 with resveratrol or SRT1720 restores its expression, reversing HG-induced EMT and reducing YY1 acetylation. Conversely, pharmacological inhibition of SIRT1 with EX527 under diabetic conditions slightly increases YY1 acetylation in RTECs, thereby exacerbating renal fibrosis.	[128]
Valproic acid	Diabetic rats	Acetylation	HDAC	HDAC inhibitor alproic acid, enhances H4 acetylation at the GRP78 promoter while reduces it at CHOP, thereby mitigating ER stress, decreasing renal cell apoptosis, and alleviating renal injury in DN.	[129]
Losartan	db/db mice	Acetylation	H3	Losartan reduces H3K9/14 acetylation at the promoters of RAGE, PAI-1, and MCP-1, thereby suppressing inflammation and ECM gene expression.	[130]
Curcumin	Diabetic rats	Acetylation	H3	Curcumin maintains histone H3 phosphorylation, promotes acetylation, reduces oxidative stress, downregulates HSP-27 and p38, and slows STZ-induced DN progression.	[131,132]
C66	Diabetic mice	Acetylation	H3	C66 prevents glomerular enlargement and fibrosis in STZ-induced DN by reducing H3K9/14 acetylation at fibronectin and CTGF promoters.	[133]
mNOX-E36	db/db mice	Acetylation	MCP-1/CCL2	MCP-1/CCL2 antagonist mNOX-E36 reduces acetylation at H3K9 and H3K23, promoting glomerular cell proliferation, decreasing sclerosis and proteinuria, and preserving kidney function.	[134]
NAC	Beagle	Acetylation	Mn-SOD2	NAC, a glutathione precursor, restores mitochondrial redox balance by inhibiting SOD2 acetylation and activating the SIRT3-SOD2-GPX4 pathway, thereby reducing oxidative stress and renal injury in DN.	[135]
Salidroside	Diabetic rats	Acetylation	FOXO1	Salidroside activate the AMPK/SIRT1 axis, suppress acetylation of FOXO1 and p53 to reduce ROS production and apoptosis in mesangial cells, thereby alleviating renal damage in DN.	[136]
C646	Diabetic mice	Acetylation	p300/CBP	The p300/CBP inhibitor C646 reduces H3K27 acetylation levels, downregulates NADPH oxidase subtypes, pro-inflammatory mediators, and pro-fibrotic molecules, and lowers ROS generation in diabetic kidneys.	[137]
Ginkgolide B	Diabetic mice and MPC5 cells	Ubiquitination	GPX4	Ginkgolide B improves DN by inhibiting GPX4 ubiquitination, reducing ferroptosis and oxidative stress.	[138]
Aspirin	Diabetic rats	Ubiquitylation	H2A	Aspirin increases H2AK119 ubiquitination by downregulating swim and MYSM1, which reduces Set7 expression and alleviates renal fibrosis in DN.	[139]
Schisandrin A	Diabetic mice and HRGECs	Ubiquitylation	AdipoR1	Schisandra A stabilizes AdipoR1 by decreasing its ubiquitination, activating the AdipoR1/AMPK pathway, mitigating ferroptosis and NLRP3-mediated pyroptosis in DN.	[140]
PHC	Diabetic mice and HK-2 cell	Ubiquitylation	PARK2	PHC promotes non-degradable ubiquitination of AIFM1, activates Nrf2-dependent PARK2 transcription, induces mitochondrial autophagy, and reduces ROS levels and RTEC apoptosis, thereby inhibiting renal tubular injury in DN.	[141]
ML264	db/db mice and HK-2 cell	Lactylation	KLF5	ML264, reduces H3K14 lactylation by inhibiting KLF5, thereby delaying EMT and alleviating renal fibrosis in DN.	[142]
Rotenone, Nala, sodium dichloroacetate	Diabetic mice and mRTEC	Lactylation	Glis1	Glis1 prevents RTECs senescence and renal fibrosis by downregulating H3K14 lactylation in DN. Lactylation enhancers such as rotenone and nala reduce Glis1's protective effects, whereas sodium dichloroacetate enhances the effects.	[143]
SKI	Diabetic mice	Crotonylation	REDOX enzyme–protein	SKI reverses the abnormal expression of REDOX enzyme-associated protein lysine crotonylation in DN, raising crotonylation at Ndufs4 and Cox5a while reducing it at GPX3, enhancing antioxidant capacity.	[144]
Fenofibrate	Diabetic mice and RSC96 Schwann cells	Palmitoylation	PPARα	PEX11B palmitoylation linked peroxisomal dysfunction to Schwann cell failure in DN. Fenofibrate, a PPARα agonist, can mitigate this injury.	[145]
TDYA	Diabetic mice	Succinylation	ACOX-1	ACOX-1-specific inhibitor TDYA reduces peroxisomal succinyl-CoA and succinic acid levels, leading to decreased ROS production, thereby ameliorating DN.	[146]

EX527: 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; HK-2: human kidney 2 cell; SIRT1: sirtuin 1; HG: high glucose; EMT: epithelial-mesenchymal transition; YY1: yin yang 1; RTECs: tubular epithelial cells; HDAC: histone deacetylase; H4: histone H4; GRP78: glucose-regulated protein 78; CHOP: C/EBP homologous protein; ER: endoplasmic reticulum; H3: histone H3; H3K9/14: histone H3 lysine 9 and lysine 14; RAGE: receptor for advanced glycation end products; PAI-1: plasminogen activator inhibitor-1; MCP-1: monocyte chemoattractant protein-1; ECM: extracellular matrix; HSP-27: heat shock protein 27; p38: p38 mitogen-activated protein kinase; STZ: streptozocin; CTGF: connective tissue growth factor; CCL2: c-c motif chemokine ligand 2; H3K23: histone H3 lysine 23; NAC: n-acetylcysteine; Mn-SOD2: manganese superoxide dismutase 2; SOD2: superoxide dismutase 2; SIRT3: sirtuin 3; FOXO1: forkhead box o 1; p53: tumor protein p53; AMPK: adenosine monophosphate-activated protein kinase; ROS: reactive oxygen species; p300/CBP: E1A binding protein p300/cAMP response element-binding protein; NADPH: nicotinamide adenine dinucleotide phosphate; H3K27: histone H3 lysine 27; nicotinamide adenine dinucleotide phosphate; MPC5: mouse podocyte cell line; GPX4: glutathione peroxidase 4; H2A: histone H2A; H2AK119: histone H2A lysine 119; MYSM1: Myb-like, SWIRM, and MPN domains 1; HRGECs: human renal glomerular endothelial cells; AdipoR1: adiponectin receptor 1; NLRP3: NOD-like receptor family, pyrin domain containing 3; PHC: penehy clidine hydrochloride; PARK2: parkin rbr e3 ubiquitin protein ligase; AIFM1: apoptosis inducing factor mitochondria associated 1; Nrf2: nuclear factor erythroid 2-related factor 2; KLF5: kruppel-like factor 5; H3K14: histone H3 lysine 14; mRTEC: mouse renal tubular epithelial cells; Glis1: glis family zinc finger 1; SKI: shenkang injection; REDOX: reduction-oxidation enzyme-protein; Ndufs4: NADH:ubiquinone oxidoreductase subunit s4; Cox5a: cytochrome c oxidase subunit 5a; GPX3: glutathione peroxidase 3; RSC96 Schwann cells: rat schwann cell line 96; PPARα: peroxisome proliferator-activated receptor alpha; PEX11B: peroxisomal biogenesis factor 11 beta; TDYA: 10,12-tricosadiynoic acid; ACOX-1: acyl-coenzyme A oxidase 1; succinyl-CoA: succinyl coenzyme A.

Accurate evaluation of animal models and in vitro systems is essential for interpreting PTM studies in DN and understanding their relevance to human disease. Commonly used models, such as STZ-induced diabetic rodents, db/db mice, and combined STZ-high-fat diet models, replicate key features of DN, including hyperglycemia, structural kidney injury, and functional decline. While these models have been invaluable for investigating the role of PTMs in disease progression, they each have notable limitations [[147], [148], [149]]. For example, the STZ model primarily mimics type 1 diabetes and fails to capture the gradual onset and chronic course of type 2 diabetes or human DN. In contrast, db/db mice, which model obesity-driven type 2 diabete, differ from humans in metabolic and immune characteristics. In vitro systems, often involving cultured renal cells exposed to HG or AGEs, allow for precise molecular analysis of PTM regulation [150]. However, they lack the complexity of the kidney's structure and physiology, including multicellular interactions and systemic influences. Experimental design also presents challenges, such as the need to balance glucose concentrations and exposure durations to better reflect human physiology. Moreover, identical stimuli can induce a variety of PTMs, such as phosphorylation, acetylation, ubiquitination, SUMOylation, lactylation, and succinylation, leading to overlapping modifications that complicate mechanistic interpretation. Despite these challenges, animal and in vitro models have significantly advanced our understanding of PTMs in DN. Careful interpretation of these results is essential when translating them to clinical settings. Integrating findings across various models and validating them in human tissues or patient-derived samples can enhance their translational relevance. Given that PTMs form a dynamic, interconnected, and often reversible network in vivo, future research should refine experimental systems to more accurately replicate the human DN pathophysiology. Such advancements will be crucial for identifying PTM-related biomarkers and therapeutic targets with genuine clinical potential.

7. Concluding remarks and future perspectives

This review highlights the diverse molecular mechanisms driving DN, with a particular emphasis on how PTMs regulate oxidative stress. Targeting PTM-related enzymes or developing small molecules that modulate their activity offers a novel therapeutic strategy to restore redox balance and limit oxidative injury in the kidney. Emerging evidence on less-characterized PTMs, such as lactylation, underscores their potential to reveal new regulatory pathways and therapeutic opportunities. In summary, PTM-based interventions represent a promising and adaptable approach for managing oxidative stress in DN. Continued progress in this field could lead to precise, mechanism-driven therapies that improve patient outcomes. Advancing treatment will require deeper exploration of PTM signaling networks and careful translation of these insights into clinically relevant applications.

CRediT authorship contribution statement

Li Zhou: Writing – original draft, Investigation, Conceptualization. Xin-Lei Shen: Writing – original draft, Investigation, Conceptualization. Qing-Ru Zhu: Validation, Supervision, Resources. Wen-Kai Yu: Validation, Supervision, Resources. Hang-Chao Li: Writing – review & editing, Funding acquisition, Conceptualization. Gang Cao: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Yi-Ni Bao: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.