Atherosclerosis drives cardiovascular morbidity in diabetes, with endothelial-to-mesenchymal transition (EndMT) as a key contributor. Whereas epigenetic regulators are increasingly implicated in atherosclerotic progression, the specific role of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, in EndMT in diabetes-associated atherosclerosis remains unclear. We show that EZH2-mediated H3K27 (histone H3 at lysine-27) trimethylation is elevated in carotid plaques from patients with diabetes and in the aortic endothelium of diabetic Apoe-/- mice. Pharmacologic EZH2 inhibition with GSK-126 attenuated EndMT and reduced atherosclerotic burden in diabetic mice. In human aortic endothelial cells exposed to high glucose/tumor necrosis factor-α or serum from patients with coronary artery disease, EZH2 blockade via GSK-126 or short hairpin RNA suppressed EndMT and reversed transcriptional programs assessed by RNA sequencing, including COL4A1 and NR2F2. These findings identify EZH2 as a driver of EndMT in diabetes-associated atherosclerosis and highlight EZH2 inhibition as a potential therapeutic strategy to limit vascular pathology.

Mitochondrial alterations are increasingly recognized as central to the pathogenesis of chronic kidney disease (CKD), contributing to impaired energy metabolism, oxidative stress, and maladaptive cellular responses. This review highlights recent advances in our understanding of mitochondrial remodeling in both diabetic and nondiabetic CKD (NDKD), including changes in bioenergetics, dynamics, redox balance, and biogenesis. We discuss state-of-the-art approaches to assess mitochondrial health, ranging from high-resolution respirometry and metabolomic profiling to transcriptomic analysis and advanced imaging techniques such as functional magnetic resonance imaging (MRI) and positron emission tomography (PET) radiotracers with metabolic readouts. Therapeutically, several agents show promise in modulating mitochondrial pathways, including sodium/glucose cotransporter 2 (SGLT2) inhibitors, glucagon-like peptide 1 receptor agonists (GLP-1 RA), and nonsteroidal mineralocorticoid receptor antagonists (nsMRAs), as well as emerging interventions such as glucose-dependent insulinotropic polypeptide and glucagon receptor agonist, nicotinamide adenine dinucleotide (NAD+) boosters, and coenzyme Q10 (CoQ10) derivatives. Multi-omics integration and spatial profiling are enabling precision phenotyping and the development of mitochondrial health scores to guide individualized therapy. Targeting mitochondrial adaptation offers a compelling avenue to improve outcomes in CKD.

Chronic low-grade inflammation underlies many microvascular complications of diabetes, including in diabetic kidney disease (DKD). Lipoxins (LX), an endogenously produced family of lipid mediators, resolve inflammation and protect against renal scarring as occurs in DKD. This study examined the mechanism by which LXs protect against DKD, focussing on the regulation of VCAM-1 and the recruitment of macrophages to the diabetic glomerulus. Lipoxin A4 (LXA4) and two fourth generation mimetics were assessed in diabetic ApoE KO mice, followed by in vitro studies in the main renal cell populations, including podocytes, proximal tubular, mesangial and glomerular endothelial cells. LXs attenuated albuminuria, mesangial expansion, and collagen and fibronectin deposition as both a preventative and delayed intervention in experimental DKD. LXs also attenuated the TNF-α-induced expression of inflammatory markers with VCAM-1 gene expression consistently reduced in all renal cell populations. Further analysis identified the renoprotection was in part mediated by an epigenetic modification of the VCAM-1 gene through H3K4 monomethylation, that did not appear to be dependent on NFkB activation in human glomerular endothelial cells. LXs protect against DKD by modulating glomerular endothelial cell inflammation and via a novel LX-mediated epigenetic mechanism regulating the VCAM-1 promoter in these cells.

Diabetic kidney disease remains a major clinical challenge despite advances in renoprotective therapies. In this issue of Kidney International, Qi et al. report that selective modulation of the vascular endothelial growth factor (VEGF) system differentially affects diabetic kidney disease progression across multiple advanced diabetic models. Whereas VEGF-A or VEGF receptor 2 inhibition exacerbated albuminuria, targeted VEGF receptor 1 blockade consistently conferred renoprotection, including in established disease. These findings clarify prior conflicting data and identify VEGF receptor 1 inhibition as a therapeutic strategy for diabetic kidney disease.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in adults with type 2 diabetes mellitus (T2DM). Cardiac dysfunction and decreased exercise capacity are common in people with T2DM, even in those without overt heart failure. Empagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, reduces major cardiovascular events and mortality in people with T2DM and established CVD, though the underlying mechanisms are not fully elucidated. This study aimed to assess changes in cardiac function and cardiorespiratory fitness (CRF) at rest and during exercise with empagliflozin treatment in people with T2DM. This double-masked, single-centre, randomised, placebo-controlled analysis combined two studies involving adults (≥ 18 years) with T2DM comparing empagliflozin 25 mg daily vs. placebo, each over three months. VO₂ peak was measured using a ramp protocol peak exercise test on a bicycle ergometer, and echocardiography assessed changes in cardiac structure and function. Of 65 recruited participants, 57 completed the study (70.2

Endothelial to mesenchymal transition (EndMT), the transformation of endothelial cells into a mesenchymal-like state, is regulated by various factors, including transcription factors such as activator protein 1 (AP-1). While recent studies have confirmed the role of EndMT in atherosclerosis, the involvement of AP-1 in EndMT, particularly in the context of human diabetes, remains unclear. This study aimed to elucidate the role of the AP-1 transcription factor complex in EndMT associated with atherosclerosis in diabetes, utilising both an in vivo preclinical model and an ex vivo model using patient-derived serum for translational relevance. Additionally, it sought to profile gene expression changes following AP-1 inhibition in an EndMT model under high glucose conditions. Serum from patients with and without type 2 diabetes mellitus (T2DM) was used to assess EndMT in primary human aortic endothelial cells (HAECs) in the presence and absence of the AP-1 inhibitor T-5224. EndMT was evaluated through immunofluorescent staining of these cells and of aortic sections from a murine model of diabetes-associated atherosclerosis in a preclinical early intervention study. Furthermore, HAECs were used to explore the effects of AP-1 inhibition on the transcriptional signature of EndMT. Patient-derived serum induced EndMT in HAECs, which T-5224 effectively prevented, as confirmed by immunofluorescent staining. Immunofluorescent analysis of the aortic sinus also revealed that T-5224 treatment inhibited EndMT, leading to reduced atherosclerosis in Apoe−/− mice. In parallel, in the HAECs-based in vitro EndMT model, T-5224 mitigated TNF-α and high glucose-induced EndMT. RNA sequencing identified 242 differentially expressed genes (DEGs) associated with EndMT under high glucose conditions, with T-5224 treatment restoring the expression of 77 DEGs. This study identifies AP-1 inhibition with T-5224 as a potential therapeutic approach for EndMT resulting in reduced atherosclerosis in diabetes. The use of human serum underscores the translational relevance of these findings.

Abstract The kidney is a highly energetic organ, requiring substantial ATP production through mitochondrial oxidative phosphorylation to support tubular reabsorption. Metabolic reprogramming and impaired mitochondrial function are implicated in diabetic kidney disease, yet direct assessment of mitochondrial respiratory flux in the human kidney has been constrained by limited access to freshly obtained tissue. Consequently, much of the evidence supporting altered renal mitochondrial function in diabetes derives from animal models that do not fully recapitulate the human condition. We established a workflow for real-time bioenergetic profiling of fresh kidney cortex obtained during nephrectomy from living individuals with diabetes and preserved kidney function. Mitochondrial respiration, electron transport system activity and tubular mitochondrial morphology were compared with age- and sex-matched, histopathologically normal non-diabetic controls. High-resolution respirometry revealed increased mitochondrial respiratory flux in permeabilised diabetic kidney cortex. In contrast, mitochondria isolated from the same tissue exhibited reduced respiratory capacity and impaired complex I activity. Quantitative analysis of tubular cells demonstrated increased mitochondrial volume density together with greater mitochondrial fragmentation in diabetes. These findings reveal that the human kidney undergoes substantial metabolic adaptation early in diabetes, before measurable loss of kidney function. Increased tissue-level respiratory flux despite intrinsic mitochondrial impairment suggests that expansion and remodelling of the mitochondrial network may initially compensate for reduced organelle efficiency and sustain the kidney’s high energetic demands. This compensatory state may, however, increase metabolic stress and vulnerability to subsequent kidney injury. To our knowledge, this study provides the first direct tissue-level functional evidence that mitochondrial metabolism is reprogrammed in the human kidney in diabetes before measurable kidney dysfunction develops. It defines an early bioenergetic signature characterised by tissue hypermetabolism despite impaired mitochondria-specific respiratory capacity, challenging the concept that diabetes produces a uniform decline in renal mitochondrial function. Failure to sustain this adaptive state may represent a critical transition towards diabetic kidney disease. GRAPHICAL ABSTRACT One Sentence Summary Diabetes drives early metabolic reprogramming of the human kidney before measurable kidney dysfunction

Cell Division Autoantigen 1 (CDA1) has been shown to play a role in enhancing transforming growth factor beta (TGFβ) signaling, leading to fibrosis in diabetic kidney disease (DKD) using mouse strains with global CDA1 gene deletion. In these models, diabetes has been induced, leading to DKD in the absence of CDA1. It is still unknown whether inhibition of CDA1 activity after onset of diabetes in the presence of CDA1 can attenuate renal fibrosis in vivo. Thus, we examined the effect of inducing genetic deletion of CDA1 in adulthood in mice using a tamoxifen-activated estrogen receptor fused cyclization recombinase (ERCre)-Locus of cross-over in P1 (LoxP) system. Male mice at 6-8 weeks of age were rendered diabetic with streptozotocin (STZ) or injected with buffer alone to serve as non-diabetic controls. Five weeks later, genetic deletion of CDA1 was induced by tamoxifen administration in CDA1Flox/ERCre mice, with mice injected with vehicle to serve as CDA1 wildtype controls. Kidney tissues were analyzed 5 weeks after deletion of CDA1. Tamoxifen administration reduced CDA1 gene expression by ~80% in CDA1Flox/ERCre mice. Renal levels of phosphorylated Smad3 and expression of profibrotic genes as well as accumulation of extracellular matrix proteins (ECMs) such as collagens III and IV were increased in diabetic mice, and induced deletion of CDA1 led to attenuation of these parameters. Therefore, targeting CDA1 after onset of diabetes in mice where CDA1 was initially expressed is able to attenuate diabetes-associated renal injury, providing the impetus to target this pathway in order to reduce diabetic kidney disease.

Glucagon-like peptide-1 (GLP-1) was initially considered to be a hormone with a predominant role in regulating glucose metabolism by inducing insulin secretion, reducing glucagon secretion, and ameliorating insulin resistance, with the last effect being largely dependent on the induction of weight loss. In more recent years, the role of this peptide beyond metabolism has progressively been explored, including its impact on kidney physiology and kidney clinical outcomes in people with obesity with or without diabetes. Indeed, despite only modest expression of the GLP-1 receptor in the kidney, the renoprotective actions of GLP-1 and its receptor agonists have become an area of intensive investigation. This Review appraises the current status of GLP-1 peptide and its receptor agonists and focuses on the preclinical as well as recent seminal clinical findings defining the kidney benefits conferred by GLP-1 receptor agonist treatment in people living with type 2 diabetes and obesity.

Pathological signaling via the receptor for advanced glycation end-products (RAGE) is critical in diabetic kidney disease (DKD) development, while RAGE deletion is reno-protective. Non-coding RNAs (ncRNAs), including microRNAs (miRs), also play key roles in DKD, including renal fibrosis. However, the involvement of ncRNAs in RAGE signaling remains unclear. This study investigated the regulation of ncRNAs by RAGE and assessed renal expression of ncRNAs, miRs, fibrotic/inflammatory markers in diabetic RAGE knockout (KO) and wild-type (WT) mice, as well as in mesangial cells (MCs) obtained from these mice. Diabetes induction in both RAGE-/- and WT mice exhibited elevated renal expression of miR-214 and its host ncRNA, Dnm3os. miR-214 and Dnm3os levels were remarkably higher in RAGE-/- MCs compared to WT MCs. Overexpression of miR-214 in WT MCs reduced fibrotic/inflammatory gene expression, while its inhibition increased these markers. Human DKD tissue demonstrated higher Dnm3os expression compared to controls. Notably, miR-214 targeted the RAGE signaling mediator diaphanous1 (DIAPH1), while Dnm3os had an opposing effect, enhancing fibrosis and inflammation. miR-214 administration in a DKD mouse model significantly reduced renal fibrosis. These findings propose a novel mechanism where miR-214 and Dnm3os act as negative and positive regulators of fibrosis via the RAGE-DIAPH1 axis.

Type 1 diabetes is associated with a progressive decline in kidney function. With improved survival, more individuals with type 1 diabetes are reaching an age where irreversible loss of kidney function can impact health and clinical outcomes. Although current standard of care has markedly reduced the incidence of fast-progressing kidney disease characterized by heavy albuminuria, and improved patient survival, these same interventions have not slowed kidney decline in most patients. An urgent need remains to develop a different foundational approach to type 1 diabetes with early kidney protection considered as important as glucose control. Toward this goal, a number of new renoprotective strategies have emerged over the last decade, including glucagon-like peptide 1 receptor agonists, aldosterone antagonists, and sodium-glucose cotransporter 2 inhibitors. For each therapy unequivocal activity has been demonstrated in patients with type 2 diabetes and kidney disease, which has prompted some clinicians to use agents off-label in patients with type 1 diabetes, so pressing is their need. However, before such important benefits can be translated to patients with type 1 diabetes, the risk of serious adverse effects must be carefully considered and the balance of efficacy and safety verified in clinical trials. In this review we consider the potential role of adjunctive therapy in type 1 diabetes in improving kidney outcomes.

There is a large body of evidence implicating mitochondrial reactive oxygen species (ROS) overproduction and oxidative stress in the development of diabetic kidney disease and the deficiency of mitochondrial antioxidant systems in the kidney, such as manganese superoxide dismutase (MnSOD/SOD2) have been identified. The proximal tubules of the kidney are densely packed with mitochondria thereby providing energy via oxidative phosphorylation in order to drive active transport for proximal tubular reabsorption of solutes from the glomerular filtrate. We hypothesized that maintenance of MnSOD function in the proximal tubules would be critical to maintain kidney health in diabetes. Here, we induced targeted deletion of SOD2 in the proximal tubules of the kidney in Ins2Akita diabetic mice (SODptKO mice) and show that 20 weeks of SOD2 deletion leads to no major impairment of kidney function and structure, despite these mice displaying enhanced albuminuria and kidney lipid peroxidation (8-isoprostanes). Plasma cystatin C, which is a surrogate marker of glomerular filtration was not altered in SODptKO diabetic mice and histological assessment of the kidney cortex revealed no change in kidney fibrosis. Thus, our findings suggest that deletion of SOD2 in the proximal tubular compartment of the kidney induces a more subtle phenotype than expected, shedding light on the involvement of SOD2 and the proximal tubular compartment in the pathogenesis of diabetic kidney disease.

High-density lipoprotein (HDL) exhibits multiple metabolic protective functions, such as facilitating cellular cholesterol efflux, antioxidant, anti-inflammatory, anti-apoptotic and anti-thrombotic properties, showing antidiabetic and renoprotective potential. Diabetic kidney disease (DKD) is considered to be associated with high-density lipoprotein cholesterol (HDL-C). The hyperglycemic environment, non-enzymatic glycosylation, carbamylation, oxidative stress and systemic inflammation can cause changes in the quantity and quality of HDL, resulting in reduced HDL levels and abnormal function. Dysfunctional HDL can also have a negative impact on pancreatic β cells and kidney cells, leading to the progression of DKD. Based on these findings, new HDL-related DKD risk predictors have gradually been proposed. Interventions aiming to improve HDL levels and function, such as infusion of recombinant HDL (rHDL) or lipid-poor apolipoprotein A-I (apoA-I), can significantly improve glycemic control and also show renal protective effects. However, recent studies have revealed a U-shaped relationship between HDL-C levels and DKD, and the loss of protective properties of high levels of HDL may be related to changes in composition and the deposition of dysfunctional particles that exacerbate damage. Further research is needed to fully elucidate the complex role of HDL in DKD. Given the important role of HDL in metabolic health, developing HDL-based therapies that augment HDL function, rather than simply increasing its level, is a critical step in managing the development and progression of DKD.

Rationale & Objective: Evidence has demonstrated that albuminuria is a key diagnostic and prognostic marker of diabetic chronic kidney disease, but the impact of its day-to-day variability has not been adequately considered. This study quantified fi ed within-individual variability of albuminuria in people with type 2 diabetes to inform clinical albuminuria monitoring. Study Design: Descriptive cross-sectional analysis. Setting & Participants: People with type 2 diabetes (n = 826, 67.1 [IQR, 60.3-72.4] years, 64.9% male) participating in the Progression of Diabetic Complications (PREDICT) cohort study. Exposure: Four spot urine collections for measurement of urinary albumin-creatinine ratio (UACR) within 4 weeks. Outcome: Variability of UACR. Analytical Approach: We characterized within- individual variability (coefficient i cient of variation [CV], 95% limits of random variation, intraclass correlation coefficient), i cient), developed a calculator displaying probabilities that any observed difference between a pair of UACR values truly exceeded a 30% difference, and estimated the ranges of diagnostic uncertainty to inform a need for additional UACR collections to exclude or confirm fi rm albuminuria. Multiple linear regression examined factors influencing fl uencing UACR variability. Results: We observed high within-individual variability (CV 48.8%; 95% limits of random variation showed a repeated UACR to be as high/low as 3.78/0.26 times the fi rst). If a single-collection UACR increased from 2 to 5 mg/mmol, the probability that UACR actually increased by at least 30% was only 50%, rising to 97% when 2 collections were obtained at each time point. The ranges of diagnostic uncertainty were 2.0-4.0 mg/mmol after an initial UACR test, narrowing to 2.4-3.2 and 2.72.9 mg/mmol for the mean of 2 and 3 collections, respectively. Some factors correlated with higher (female sex; moderately increased albuminuria) or lower (reduced estimated glomerular fi ltration rate and sodium- glucose cotransporter 2 inhibitor/angiotensinconverting enzyme inhibitor/angiotensin receptor blocker treatment) within-individual UACR variability. Limitations: Reliance on the mean of 4 UACR collections as the reference standard for albuminuria. Conclusions: UACR demonstrates a high degree of within-individual variability among individuals with type 2 diabetes. Multiple urine collections for UACR may improve capacity to monitor changes over time in clinical and research settings but may not be necessary for the diagnosis of albuminuria.

Background Hyperglycemia influences the development of glomerular endothelial cell damage, and nowhere is this more evident than in the progression of diabetic kidney disease (DKD). While the Set7 lysine methyltransferase is a known hyperglycemic sensor, its role in endothelial cell function in the context of DKD remains poorly understood. Methods Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and short hairpin RNA inhibition inhibition of Set7. Results Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of glomerular endothelial cell populations by transcriptional regulation of the insulin growth factor binding protein 5 (IGFBP5). Chromatin immunoprecipitation assays confirmed that the expression of the IGFBP5 gene was associated with mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). This generalizability was investigated in human kidney and circulating hyperglycemic cells exposed to TGF beta 1. We showed that the highly selective Set7 inhibitor (R)-PFI-2 hydrochloride attenuated indices associated with renal cell damage and mesenchymal transition, specifically (1) reactive oxygen species production, (2) IGFBP5 gene regulation, and (3) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely corresponded in human glomerular endothelial cells with (R)-PFI-2 hydrochloride inhibition or Set7 short hairpin RNA silencing. Conclusions Set7 regulates the phenotypic endothelial-mesenchymal transition switch and suggests that targeting the lysine methyltransferase could protect glomerular cell injury in DKD

Despite advances in treatment, atherosclerotic cardiovascular disease remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus, newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single-cell sequencing approach was used to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin-induced diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells (HAECs) and human THP-1-derived macrophages. We then performed a preclinical study using an activator protein-1 (AP-1) inhibitor in a diabetic Apoe-/- mouse model. Single-cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from individuals with diabetes compared with those without diabetes. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, the inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes. ARTICLE HIGHLIGHTS:

Abstract Background Hyperglycemia-induced oxidative stress is a well-established pathological mediator of vascular complications in diabetes. We assessed plasma oxidant and antioxidant levels in response to acute and chronic hyperglycemia in relation to vascular stiffness and varying degrees of kidney disease in type 1 diabetes individuals. Methods The acute hyperglycemia study included 22 type 1 diabetic individuals with normal albumin excretion rate (AER) and 13 non-diabetic controls. These individuals received an acute glucose challenge during a 120-minute hyperglycemic clamp. The chronic hyperglycemia study included 118 type 1 diabetic individuals with chronically low (n = 60) or high (n = 58) HbA1c concentrations and varying degrees of diabetic kidney disease (DKD) classified as normal, moderate, or severe albuminuria (AER). Levels of malondialdehyde (MDA), reactive oxygen metabolites (ROMs), total antioxidant capacity (TAC), biological antioxidant potential (BAP) and superoxide dismutase (SOD) were measured from plasma or serum samples in the FinnDiane study. Results Levels of MDA (p < 0.01) and ROMs (p < 0.01) were elevated in type 1 diabetes individuals compared to non-diabetic controls at baseline. Acute hyperglycemia further increased MDA levels (p < 0.05) and sustained the elevation of ROMs in type 1 diabetes individuals. Acute hyperglycemic challenge impaired TAC in both non-diabetic (p < 0.05) and type 1 diabetes (p < 0.01) individuals compared to baseline whereas BAP was increased (p < 0.05) with no difference observed in non-diabetic controls. There was a positive association between high circulating MDA and AIx (r2 = 0.611, p = 0.05), and between delta ROMs and delta AIx (r2 = 0.955, p = 0.014) in combined analysis of individuals with type 1 diabetes and non-diabetic controls. Type 1 diabetes individuals with varying status of DKD, showed elevated levels of ROMs in those with high HbA1c compared to their counterpart with low HbA1c (p < 0.05). Individuals with severe albuminuria showed elevated ROM levels (p < 0.01) and depressed antioxidant capacity (p < 0.01) compared to those with normal AER of comparable HbA1c concentrations. Conclusions Biomarkers of oxidative stress are associated with vascular stiffness and DKD following acute and chronic hyperglycemic exposure and may provide added value to HbA1c in understanding disease pathology, predicting risk and assessing the status of secondary complications of type 1 diabetes.