Tie2 activator 4E2 ameliorates diabetic nephropathy and synergizes with dapagliflozin in a mouse model

Abstract

Diabetic nephropathy (DN), a primary cause of end-stage renal disease, stems from hyperglycemia-induced vascular dysfunction and aberrant angiogenesis. Sodium-glucose cotransporter 2 inhibitors, such as dapagliflozin, improve glycemic control and provide renal protection yet fall short of fully halting DN progression. This study explores 4E2, a Tie2 receptor activator that mimics angiopoietin-1 to stabilize the vascular endothelium, as a novel DN therapy—both independently and in combination with dapagliflozin. In a streptozotocin (STZ)-induced DN mouse model (DBA/2J strain), male mice were treated with weekly intravenous 4E2, daily oral dapagliflozin, or a combination of both for 4 weeks following STZ administration. Dapagliflozin primarily reduced fasting blood glucose with modest renoprotective effects, whereas 4E2 significantly lowered kidney weight, blood urea nitrogen, and urinary albumin while elevating serum albumin, indicating greater renal protection. Histological analysis showed that 4E2 more effectively attenuated glomerular hypertrophy and lesions compared to dapagliflozin. Immunohistochemistry revealed that 4E2 markedly increased VE-cadherin and CD31 expression while decreasing PDGFR-β, reflecting enhanced endothelial stability and reduced vascular remodeling through Tie2-mediated mechanisms. Combination therapy synergistically enhanced these outcomes, achieving superior reductions in glucose levels, glomerular damage, and vascular pathology compared to either treatment alone. In contrast to anti-VEGF therapies, which can worsen proteinuria, 4E2-mediated Tie2 activation normalizes vascular stability without disrupting physiological angiogenesis, providing a safer therapeutic option. These findings establish 4E2 as a promising treatment for DN, especially when combined with dapagliflozin, by leveraging Tie2-driven stabilization and synergistic benefits to meet this critical unmet need.

INTRODUCTION

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD) worldwide, accounting for 30%–40% of ESRD cases and affecting 20%–40% of individuals with diabetes [1]. Despite advancements in medical technology, its prevalence continues to rise [2]. DN is strongly associated with increased cardiovascular morbidity and mortality, highlighting the urgent need for early diagnosis and intervention [3].

The progression of DN is primarily driven by chronic hyperglycemia, which initiates a cascade of structural and functional changes in the kidney. Initially, glomerular hypertrophy and hyperfiltration occur, followed by thickening of the glomerular basement membrane (GBM) and mesangial expansion [4]. Endothelial dysfunction—an early hallmark of DN—progressively worsens, leading to podocyte loss and increased vascular permeability [5]. In advanced stages, these pathological changes culminate in glomerulosclerosis and tubulointerstitial fibrosis [6]. Given the central role of endothelial injury in DN progression, targeting vascular dysfunction presents a promising therapeutic strategy.

Currently, there is no definitive cure for DN, making a multifaceted management approach essential. This includes glycemic and blood pressure control, lifestyle modifications, and cardiovascular risk reduction [7]. Despite intensive treatment with agents such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, renin inhibitors, and lipid-lowering drugs, disease progression often persists, with treatment efficacy varying widely in clinical practice [8]. As renal function declines, patients eventually require dialysis or kidney transplantation; however, these interventions significantly reduce quality of life and offer only limited long-term benefits [9]. Therefore, there is an urgent need for therapies that can effectively restore renal filtration capacity compromised by chronic hyperglycemia.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have emerged as a promising therapeutic strategy for DN, with multiple clinical trials demonstrating their ability to reduce renal injury—particularly glomerular damage—while also improving cardiovascular outcomes [10]. However, due to the multifactorial nature of DN, effective management often necessitates a combination of therapeutic agents [11]. To address this challenge, novel pharmacological approaches targeting inflammation and oxidative stress are under investigation. Additionally, growing evidence suggests that abnormal angiogenesis plays a crucial role in DN progression, making it an increasingly important therapeutic target [12].

Abnormal angiogenesis is a key pathophysiological mechanism in DN, contributing to disease progression. Studies in DN patients have confirmed the presence of this dysregulated process [13]. In diabetic rodent models, aberrant angiogenesis in glomerular and vascular tissues is closely linked to damage in mesangial cells, podocytes, and microvascular structures [14]. This vascular imbalance arises from disrupted regulation between pro-angiogenic and anti-angiogenic factors, leading to impaired vascular homeostasis [15]. Consistently, glomeruli from both diabetic patients and rodent models show increased expression of pro-angiogenic factors alongside decreased levels of anti-angiogenic molecules [16].

Although vascular endothelial growth factor (VEGF)-targeting therapies have shown potential, they often lead to proteinuria and hypertension, which may further exacerbate renal damage [17]. Moreover, they fail to address the endothelial dysfunction and vascular instability that drive DN progression [18]. These limitations underscore the need for alternative approaches, such as Tie2 activation, which stabilizes endothelial junctions and reduces vascular permeability [19]. By targeting the angiopoietin-2 (Ang2)/Tie2 imbalance, this strategy offers a promising solution to overcoming the shortcomings of current DN treatments [20].

Unlike direct VEGF inhibition, Tie2 activation supports vascular homeostasis by stabilizing endothelial junctions and reducing aberrant permeability in DN [21]. The Ang2/Tie2 imbalance in DN contributes to glomerular endothelial dysfunction and capillary rarefaction, emphasizing the need for alternative therapeutic strategies [22]. 4E2, a Tie2 activator, mimics Ang1 activity and activates the Ang1-Tie2 signaling pathway to promote vascular normalization, potentially addressing the limitations of anti-VEGF therapies [23]. By enhancing vascular endothelial cadherin (VE-cadherin) localization, promoting VEGFR dephosphorylation, and strengthening tight junction integrity, 4E2 is expected to restore endothelial stability and support perivascular cell attachment [24]. As a result, it may provide more sustained and pronounced improvements in glomerular vascular architecture than conventional symptom-focused treatments.

This study assesses the therapeutic potential of 4E2 in DN by leveraging its ability to stabilize vascular structures. We propose an innovative endothelial-targeted approach to address the vascular pathogenesis of DN, overcoming the limitations of current treatments. Using a well-established streptozotocin (STZ)-induced DN mouse model, we investigate whether 4E2 mitigates glomerular endothelial dysfunction and related structural damage. We also examine its potential synergy with dapagliflozin, an SGLT2 inhibitor with known renoprotective benefits. By elucidating the role of Tie2 activation in glomerular vascular normalization, this study provides critical insights into a novel strategy for enhancing renal outcomes in DN patients.

METHODS

Test article

4E2 (mIgG2a, Batch T24001, VBX-00-01144-02), a Tie2 activator, was obtained from PhamAbcine and prepared at a concentration of 6 mg/ml in a vehicle solution. The vehicle comprised of 20 mM sodium acetate, 50 mg/ml sucrose, 10 mM sodium chloride, 0.02% (w/v) polysorbate 80 with the pH adjusted to5.0.

Animals and husbandry

Male DBA/2NCrljOri mice, aged approximately 5 weeks and weighing 16–20 g, were obtained from Orient Bio. The mice were housed in groups of up to five per polycarbonate/polypropylene cages with steel mesh tops and solid bottoms. They had ad libitum access to food and water, except during fasting required for specific procedures. Housing conditions were maintained at a temperature of 19°C–23°C, 40%–70% humidity, and a 12-h light/dark cycle. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Asan Medical Center, Republic of Korea (Approval No. 2021-02-291).

STZ-diabetic nephritis modeling

Following an acclimation period, mice were fasted for 6 h prior to STZ (Sigma-Aldrich) administration on Day 1. STZ was freshly dissolved in 50 mM sodium citrate buffer (pH 4.5) to 6 mg/ml and administered intraperitoneally at 50 mg/kg daily for 5 consecutive days. To reduce the risk of hypoglycemia, 10% sucrose water was provided during this period. On Day 6, the sucrose water was replaced with plain reverse osmosis drinking water. To sustain hyperglycemia and induce DN, the STZ dosing regimen was repeated for an additional 5 days, during which sucrose water was withheld to prevent rapid glucose fluctuations [25]. A visual summary of the study timeline is presented in Fig. 1A.

Fig. 1. Experimental timeline and body weight changes in diabetic nephropathy (DN) study.

(A) Overall study timeline for streptozotocin (STZ)-induced DN induction and test article treatment. (B) Mean body weight changes in the normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups. Body weight changes during the study period are presented as mean ± SEM (n = 10; ^#p ≤ 0.05 vs. normal control).

Combination study of 4E2 with dapagliflozin

At Week 8, mice were randomized into five groups (n = 10 per group): normal control (non-diabetic), vehicle, 4E2, dapagliflozin, and 4E2 + dapagliflozin. 4E2 was administered intravenously at weekly intervals, while dapagliflozin was administered daily by oral gavage, both over a 4-week period. All animals were euthanized at Week 12 for subsequent analysis.

In-life examinations, observations, and measurements

Mortality, abnormalities, and signs of pain or distress were monitored daily throughout the induction and treatment periods. Body weight was measured prior to STZ injection, at Weeks 1 and 7, weekly during the induction period, before each treatment administration, and at necropsy.

Laboratory evaluations

Fasting blood glucose (FBG) levels were measured after a 6-h fast. Blood was collected from the tail vein using a sterile blade, and glucose levels were measuredwith a glucometer (CareSens) at Weeks 8 and 10, and prior to euthanasia.

Urine samples were collected before euthanasia and analyzed for urinary albumin (ALB) using an ELISA kit (Abcam).

For serum chemistry analysis, blood was drawn from the caudal vena cava of anesthetized mice (via isoflurane inhalation) without anticoagulants and transferred to serum separation tubes (BD Biosciences), and centrifuged at 3,000 rpm for 10 min at 4°C. Separated serum was analyzed for blood urea nitrogen (BUN), ALB, and creatinine.

Necropsy

At the end of the study, surviving mice were weighed and euthanized via isoflurane exposure followed by exsanguination. A comprehensive examination was conducted, examining the carcass and musculoskeletal system, external surfaces and orifices, the cranial cavity and external surfaces of the brain, and thoracic, abdominal, and pelvic cavities including all associated organs and tissues. Kidneys were excised and weighed, with only the unaffected kidney measured in cases of gross abnormalities. After weighing, kidneys were fixed in 10% neutral buffered formalin for histological analysis.

Histopathologic analysis

Kidneys from all mice were fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned at 3 µm, and stained with hematoxylin and eosin, Masson's trichrome, and periodic acid-Schiff reagent. Histopathologic lesions were examined microscopically, and semi-quantitatively graded on a scale of 0–4 (0: no abnormality; 1: ≤ 25%; 2: 25%–50%; 3: 50%–75%; 4: ≥ 75%) based on severity of tubular degeneration, tubular necrosis, interstitial fibrosis, interstitial inflammatory cell infiltration, mesangial proliferation and mesangiolysis, assessed by affected tissue area or glomerular involvement. For morphometric analysis, ten glomeruli per mouse were randomly selected and measured for diameter and area using ImageJ software.

Immunohistochemical analysis

Immunohistochemical staining of kidney tissue sections was conducted using the Ventana BenchMark XT automated system (Roche). The UltraView DAB IHC Detection Kit (Ventana Medical Systems Inc.) was used for reagent application in the automated system. Slides were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted. Staining conditions were optimized to establish a protocol ensuring specific target staining patterns with minimal background.

All stained slides were scanned using a Motic system. Scanned images were processed using QuPath software (version 0.3.2) to quantify glomerular staining area. Antibodies used included CD31 (Abcam, ab182981, dilution 1:1,000), platelet-derived growth factor receptor beta (PDGFR-β) (Abcam, ab32570, dilution 1:500), and VE-cadherin (Abcam, ab33168, dilution 1:400).

Statistical analysis

Data are presented as mean ± standard error of the mean. Statistical analyses were performed using IBM SPSS Statistics v27. Homogeneity of group variances was assessed with Levene's test, and significant differences between groups were determined with one-way ANOVA. When both variance homogeneity and significant differences were confirmed, Scheffé's multiple comparison test was used for post hoc analysis. Conversely, in cases of variance non-homogeneous variances, Dunnett's T3 test was used. A significance level of p ≤ 0.05 was established.

RESULTS

Mortality and clinical observation

All mice survived until study completion. No treatment-related clinical signs were observed in 4E2, dapagliflozin, or their combination groups.

Body weight and kidney weights

The normal control group exhibited consistent weight gain throughout the study. In contrast, the DN groups exhibited a statistically significant reduction in body weight gain starting from Week 8. However, administration of 4E2, dapagliflozin, or their combination did not significantly alter body weight (Fig. 1B).

The DN groups also exhibited higher relative kidney weights compared to the normal control group. A significant reduction in both absolute and relative kidney weights was observed in the 4E2 group (p ≤ 0.05 vs. vehicle group). The absolute kidney weight was slightly reduced in the 4E2 + dapagliflozin group, compared to 4E2 group. However, no significant changes in kidney weight were observed in the dapagliflozin group compared to vehicle group (Table 1).

Table 1.

Terminal body and kidney weights in the normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups

Group	Terminal body weight (g)	Absolute kidney weight (g)	Relative kidney weight (%)
Normal control	25.66 ± 0.79	0.425 ± 0.013	1.658 ± 0.035
DN + vehicle	21.34 ± 0.63#	0.483 ± 0.028	2.279 ± 0.151#
DN + 4E2	21.71 ± 0.32#	0.415 ± 0.015*	1.912 ± 0.060#,*
DN + dapagliflozin	21.12 ± 0.44#	0.458 ± 0.018	2.172 ± 0.087#
DN + 4E2 + dapagliflozin	21.03 ± 0.57#	0.412 ± 0.011*,%	1.972 ± 0.071#

Data are presented as mean ± SEM. DN, diabetic nephropathy. n = 10; ^#p ≤ 0.01 vs. normal control; *p ≤ 0.05 vs. vehicle group; ^%p ≤ 0.05 vs. dapagliflozin group.

Laboratory evaluations

Table 2 summarizes the results of the laboratory evaluations. FBG levels were within the normal range in the normal control group but were elevated in the DN groups. During the treatment period, FBG levels decreased in the dapagliflozin and combination groups (p ≤ 0.01 vs. vehicle group) but remained unchanged in the 4E2 group.

Table 2.

Laboratory evaluation results for FBG, serum BUN, serum albumin, and urinary albumin in normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups

Group	FBG			Serum BUN (n = 10, mg/dl)	Serum ALB (n = 10, g/dl)	Urinary ALB
	Week 8 (n = 10, mg/dl)	Week 10 (n = 10, mg/dl)	Week 12 (n = 10, mg/dl)			Spot urine (n = 10, µg/ml)	24 h urine (n = 5, µg/ml)
Normal control	210.20 ± 4.21	205.20 ± 4.25	193.90 ± 10.64	25.54 ± 1.87	1.78 ± 0.05	16.16 ± 0.79	19.11 ± 1.60
DN + vehicle	425.50 ± 15.83##	563.10 ± 11.38##	531.30 ± 16.46##	60.64 ± 10.85#	1.46 ± 0.05#	53.29 ± 12.74#	48.34 ± 9.12#
DN + 4E2	458.90 ± 19.51##	532.90 ± 12.42##	512.60 ± 11.80##	35.96 ± 2.54#,*	1.60 ± 0.03#,*	16.24 ± 2.67**	13.34 ± 2.08**
DN + dapagliflozin	433.00 ± 21.72##	480.90 ± 20.35##, **	408.50 ± 23.08##,**	37.48 ± 3.55#	1.56 ± 0.04#	24.37 ± 2.67#	29.75 ± 1.98#
DN + 4E2 + dapagliflozin	475.40 ± 23.57##	462.20 ± 26.12##,**,$	432.50 ± 11.12##,**,$$	42.36 ± 3.13#	1.50 ± 0.04#	13.85 ± 1.86**,%	12.11 ± 2.81**,%

Data are presented as mean ± SEM. DN, diabetic nephropathy; FBG, fasting blood glucose; BUN, blood urea nitrogen; ALB, albumin. ^#p ≤ 0.05 vs. normal control; ^##p ≤ 0.01 vs. normal control; *p ≤ 0.05 vs. vehicle group; **p ≤ 0.01 vs. vehicle group; ^$p ≤ 0.05 vs. 4E2 group; ^$$p ≤ 0.01 vs. 4E2 group; ^%p ≤ 0.01 vs. dapagliflozin group.

Serum BUN levels were significantly higher in the DN groups, with the vehicle group exhibiting the highest levels. However, serum BUN levels were significantly lower in the 4E2 group compared to the vehicle group (p ≤ 0.05 vs. vehicle group). In the dapagliflozin and combination groups, serum BUN levels showed a decreasing trend but did not reach statistical significance.

In contrast, serum ALB levels were higher in the 4E2 group compared to the vehicle and other DN (p ≤ 0.05 vs. vehicle group), groups. The vehicle group exhibited the lowest serum ALB levels, while the dapagliflozin and combination groups showed trended downward without statistical significance.

Elevated urinary ALB levels were noted in the vehicle group. Significant reductions in urinary ALB levels were observed in the 4E2 and combination groups (p ≤ 0.01 vs. vehicle group). The dapagliflozin group showed a decreasing trend but did not reach statistical significance.

Histopathologic analysis

Histologic lesions associated with DN were evaluated in three anatomical regions of the kidney: the tubules, interstitium, and glomeruli. Representative histological images are shown in Fig. 2A. Tubular degeneration and necrosis in the renal tubules were assessed and assigned semi-quantitative scores. Interstitial fibrosis and inflammatory cell infiltration in the renal interstitium were assessed. Lesions assessed in the glomeruli included mesangial proliferation and mesangiolysis. A summary of the overall renal injury scores is presented in Fig. 2B–D.

Fig. 2. Treatment groups demonstrate reduced renal injury across multiple kidney compartments.

(A) Representative histological images of kidney tissue stained with H&E, MT, and PAS showing tubule, interstitium, and glomerulus in the normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups. Scale bar = 20 μM. Renal injury scores in tubule (B), intersitium (C), and glomerulus (D) are presented as mean ± SEM (n = 10; ^#p ≤ 0.05 vs. normal control; ^##p ≤ 0.01 vs. normal control; *p ≤ 0.05 vs. vehicle group; **p ≤ 0.01 vs. vehicle group). H&E, hematoxylin and eosin; MT, Masson’s trichrome; PAS, periodic acid-Schiff; DN, diabetic nephropathy.

The dapagliflozin group showed the highest tubular degeneration score, while the vehicle group exhibited the most severe tubular necrosis. Tubular necrosis was reduced in the 4E2, dapagliflozin, and combination groups (p ≤ 0.05 vs. vehicle group). The lowest inflammatory cell infiltration score was observed in the combination group (p ≤ 0.05 vs. vehicle group). The vehicle group exhibited the highest mesangial proliferation score. Mesangiolysis was observed in both the vehicle and dapagliflozin groups.

The result of morphometric analysis is shown in Fig. 3. In the DN groups, glomerular diameter and area were significantly increased. Compared to the vehicle group, reductions in glomerular diameter were observed in the 4E2, dapagliflozin, and combination groups. Moreover, the 4E2 and combination groups demonstrated significant reductions in glomerular area compared to the dapagliflozin group (p ≤ 0.01 vs. dapagliflozin group). The combination group showed lower glomerular diameter and area compared to the 4E2 and dapagliflozin groups, with significant reductions relative to the dapagliflozin group (p ≤ 0.01 vs. 4E2 and dapagliflozin group).

Fig. 3. Combination therapy effectively reduces diabetic nephropathy (DN)-induced glomerular hypertrophy.

Mean glomerular diameter (A) and area (B) in the normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups. Data are presented as mean ± SEM (n = 10; ^#p ≤ 0.01 vs. normal control; *p ≤ 0.01 vs. vehicle group; ^$p ≤ 0.01 vs. 4E2 group; ^%p ≤ 0.01 vs. dapagliflozin group).

Immunohistochemistry

Immunohistochemical analysis was performed using antibodies against VE-cadherin, PDGFR-β, and CD31 to evaluate the restoration of glomerular vasculature (Fig. 4). Ten glomeruli per mouse were randomly selected for analysis. CD31, an endothelial cell marker involved in angiogenesis and vascular integrity, and VE-cadherin, an endothelial junction protein critical for cell-cell adhesion, were assessed to monitor endothelial health. PDGFR-β, a receptor expressed on vascular smooth muscle cells and implicated in vascular remodeling, was evaluated to examine perivascular changes.

Fig. 4. Diabetic nephropathy (DN) alters glomerular endothelial and pericyte marker expression.

(A) Representative immunohistochemistry-stained kidney glomerulus from scanned images. Scale bar = 20 μM. Quantification of the positive area for CD31 (B), PDGFR-β (C), VE-cadherin (D) staining in the normal control, vehicle-treated, 4E2-treated, dapagliflozin-treated, and combination groups. Data are presented as mean ± SEM (n = 10, ^#p ≤ 0.01 vs. normal control; *p ≤ 0.05 vs. vehicle group; **p ≤ 0.01 vs. vehicle group; ^$p ≤ 0.01 vs. 4E2 group; ^%p ≤ 0.01 vs. dapagliflozin group). PDGFR-β, platelet-derived growth factor receptor beta; VE-cadherin, vascular endothelial cadherin.

The results revealed that CD31 and VE-cadherin expression was highest in the normal control group but significantly reduced in the vehicle group. Treatment with 4E2, dapagliflozin, or their combination significantly increased their expression compared to the vehicle group (p ≤ 0.01 vs. vehicle group), with the greatest increase observed in the combination group (p < 0.05 vs. 4E2 or dapagliflozin alone).

Conversely, PDGFR-β expression was lowest in the normal control group but significantly elevated in the vehicle group. Treatment with 4E2, dapagliflozin, or their combination reduced PDGFR-β expression relative to the vehicle group (p ≤ 0.05 vs. vehicle group), with the most substantial reduction seen in the combination group (p < 0.05 vs. 4E2 or dapagliflozin alone).

DISCUSSION

This study demonstrates that 4E2 effectively improves renal function and histological outcomes in an STZ-induced DN mouse model. The STZ-induced DN model is widely used in animal studies to investigate DN associated with type 1 diabetes. STZ, an N-acetylglucosamine (GlcNAc) derivative, selectively targets pancreatic β-cells, leading to their destruction and subsequent hyperglycemia. This model replicates key features of DN, including moderate albuminuria and characteristic histological changes. In this study, DN was induced using multiple low-dose STZ injections to reduce organ toxicity. Strain-specific variations in DN progression were observed across inbred mouse strains. Notably, the DBA/2J strain exhibited marked glomerulosclerosis, thickened GBM, and elevated albuminuria, making it an optimal model for this investigation [26].

Treatment with 4E2 significantly decreased kidney weights, BUN levels, and urinary ALB levels while increasing serum ALB concentrations. Histologically, the 4E2-treated group exhibited reduced DN-associated lesions, along with significant decreases in glomerular diameter and area. Immunohistochemical analysis demonstrated that 4E2 and dapagliflozin upregulate CD31 and VE-cadherin—key markers of endothelial health critical for angiogenesis (CD31) and vascular barrier function (VE-cadherin)—thereby stabilizing the glomerular endothelium and reducing DN-associated vascular lesions [27]. These findings suggest that 4E2 and dapagliflozin confer protective effects against DN-induced endothelial damage, as evidenced by the increased expression of these markers.

In contrast to the upregulation of endothelial markers, PDGFR-β—a receptor on vascular smooth muscle cells that drives perivascular remodeling and fibrosis—demonstrated reduced expression following treatment with 4E2 and dapagliflozin. This decrease suggests suppression of DN-associated vascular smooth muscle remodeling, which may mitigate glomerular hypertrophy and extracellular matrix accumulation. Collectively, these results demonstrate that 4E2 and dapagliflozin enhance endothelial junction stability and reduce vascular permeability in DN, as reflected by diminished glomerular lesions [28].

Building on these protective effects, the combination of 4E2 with dapagliflozin—an SGLT2 inhibitor known for its glycemic control and renoprotective properties [29]—resulted in synergistic improvements in kidney function, vascular integrity, and glucose levels. While 4E2 primarily targets vascular stabilization through angiogenesis modulation, dapagliflozin complements this effect by enhancing glycemic control. Together, they provide a dual-benefit approach, reinforcing both vascular and metabolic health, thereby positioning 4E2 as a promising therapeutic candidate for DN.

To better understand these in vivo effects, we first confirmed 4E2's Tie2 activation in vitro. 4E2 exhibited strong binding affinity for Tie2-overexpressing human and mouse cell lines, and its Tie2-activating properties were validated in human umbilical vein endothelial cells and a vessel leakage assay [23]. Consistent with previous findings identifying 4E2 as a Tie2 activator that promotes pathological vascular normalization, our study further highlights its role in stabilizing the glomerular vasculature and modulating key mechanistic markers, thus laying a foundation for its potential use in DN therapy. These mechanistic insights are highly relevant to DN pathology in patients, where endothelial injury—characterized by elevated von Willebrand factor—leads to basement membrane thickening and advanced lesions [30-32]. Notably, our STZ-induced DN model effectively recapitulates these pathological features, reinforcing the clinical relevance of 4E2’s therapeutic effects.

While our findings highlight 4E2's vascular stabilization, angiogenesis—another key feature of DN—has been targeted by anti-VEGF therapies. Angiogenesis represents a potential therapeutic target in DN. Anti-VEGF therapy, which targets a critical regulator of angiogenesis, has shown promise in several animal studies [33]. VEGF-A, essential for both physiological and pathological angiogenesis, drives abnormal neovascularization in DN. Specifically, hyperglycemia-induced VEGF-A overexpression in podocytes contributes to glomerular structural changes [34]. As a result, inhibitors like SU5416, which block VEGF-A intracellular signaling pathways, have demonstrated efficacy in animal models [35]. However, clinical trials of anti-VEGF agents have revealed adverse effects, such as proteinuria and hypertension, which can worsen DN progression [36]. These renal complications highlight the need to explore alternative targets for regulating angiogenesis in DN.

The receptor tyrosine kinase Tie2 offers an alternative target for preserving vascular integrity [37]. The angiopoietin-Tie2 signaling pathway plays a critical role in regulating endothelial quiescence [38], and its activation has been shown to strengthen vascular barrier function and reduce inflammation [39]. Unlike anti-VEGF therapies, 4E2-mediated Tie2 activation enhances vascular integrity without impairing physiological angiogenesis, suggesting a safer therapeutic profile. However, its precise effects on the glomerular endothelium in DN remain to be elucidated. Our findings indicate that 4E2, via Tie2 activation, stabilizes the glomerular endothelium in DN by downregulating PDGFR-β expression while upregulating CD31 and VE-cadherin expression. To further clarify these mechanisms, future studies should utilize RNA sequencing or phosphoproteomic analysis to explore downstream signaling pathways activated by 4E2 in DN. Supporting this approach, a prior study using a Tie2 activator in an ischemia-reperfusion acute kidney injury mouse model demonstrated preservation of renal function and structure [40]. These results collectively suggest that Tie2 activation may protect renal vasculature against various injuries, including those observed in DN.

While these results are promising, several limitations must be considered. First, the STZ-induced DN model employed here mimics type 1 diabetes, limiting its applicability to type 2 diabetes, which is primarily driven by obesity and insulin resistance. Second, while 4E2 exhibited Tie2 activation in vitro, additional in vivo studies are required to confirm its long-term efficacy and safety. Third, although our findings indicate a synergistic effect between 4E2 and dapagliflozin, the underlying mechanisms of this interaction remain to be elucidated. Finally, the protective effects of 4E2 were assessed solely in male mice, as female mice exhibit greater resistance to STZ-induced hyperglycemia compared to males [41], and their results are often confounded by variability due to the estrous cycle [42]. Future studies should explore potential sex differences in 4E2 efficacy to address this gap.

In conclusion, our findings demonstrate that 4E2, a Tie2 activator, provides functional and structural renal benefits in the STZ-induced DN mouse model. These renoprotective effects were further enhanced when combined with dapagliflozin, an SGLT2 inhibitor. Together, these results underscore the potential of Tie2-targeted therapy as a novel approach to attenuate DN progression.