Hypotonicity‐Induced WNK1 Phosphorylation Prevents H₂O₂‐Induced Apoptosis in Vascular Smooth Muscle Cells

ABSTRACT

Vascular smooth muscle cells (VSMCs) are essential for maintaining arterial wall integrity, with their associated apoptosis critically involved in cardiovascular diseases. Intracellular chloride anion (Cl⁻) dysregulation is closely linked to apoptosis. However, the underlying mechanism via chloride‐sensitive kinases remains unclear. Here, we show that hypotonic stimulation activates volume‐regulated chloride channels (VRCCs) and induces the phosphorylation of the chloride‐sensitive kinase WNK1. Activated WNK1 protects VSMCs against H₂O₂‐induced apoptosis by maintaining the Bcl‐2/Bax balance, preserving mitochondrial membrane potential and inhibiting caspase‐9/3 activation. These findings uncover a novel protective role of WNK1 in VRCC‐mediated Cl⁻ signalling and provide new perspectives for targeting chloride‐sensitive kinases in the treatment of hypertension and related cardiovascular diseases.

Plain Language Summary

Vascular smooth muscle cells (VSMCs) employ a chloride‐mediated mechanism to counteract stress and prevent cell death. Upon hypotonic stimulation, volume‐regulated chloride channels activate the chloride‐sensitive kinase WNK1. This activation restores the balance of key mitochondrial proteins, preserves cellular energy integrity and ultimately inhibits the apoptosis cascade. This discovery elucidates a novel ion‐signalling defence mechanism critical for maintaining vascular wall integrity. It positions the chloride–WNK1 axis as a promising therapeutic target for mitigating hypertension and related cardiovascular pathologies.

1. Introduction

The balance between proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is essential for maintaining vascular homeostasis and remodelling. Although previous studies have mainly focused on cation signalling pathways involving Na⁺, K⁺ and Ca²⁺, accumulating evidence indicates that the chloride anion (Cl⁻) acts as an active signalling molecule rather than a passive counterion in regulating vascular cell function [1, 2, 3]. Fluctuations in intracellular Cl⁻ concentration influence essential cellular processes, including membrane potential maintenance, cell volume regulation, migration, proliferation and apoptosis [4, 5].

At the cellular level, the movement of Cl⁻ across the plasma membrane is mainly mediated by various chloride channels, among which the volume‐regulated chloride channel (VRCC) plays a predominant role in VSMCs. Activation of the VRCC under hypotonic stress induces Cl⁻ efflux and initiates a regulatory volume decrease (RVD), thereby preserving cellular morphology and function [6, 7, 8]. Hypotonic activation of VRCC reduces intracellular Cl⁻ concentration, exerting dual effects on VSMCs: promoting proliferation via the PI3K–AKT signalling pathway and attenuating apoptosis by suppressing mitochondrial cytochrome c release, followed by inhibition of caspase activation [9, 10]. These findings highlight the bidirectional regulatory role of Cl⁻ signalling in vascular remodelling.

WNK1, as a critical intracellular Cl⁻ sensor, has its activity tightly regulated by cytosolic Cl⁻ concentration. In normal conditions, intracellular Cl⁻ directly binds to WNK1, resulting in autophosphorylation prohibition, whereas a reduction in intracellular Cl⁻ concentration relieves Cl⁻ binding to WNK1, leading to WNK1 activation [11, 12, 13, 14].

Our previous study demonstrated that hypotonic activation of VRCC induces Cl⁻ efflux, activates WNK1 through Thr60 phosphorylation and promotes VSMC proliferation via the PI3K–AKT pathway [15, 16, 17]. Preliminary data further indicated that hypotonic stimulation protects VSMCs from apoptosis induced by H₂O₂ and modulates mitochondrial apoptotic proteins [18]. Although these findings implicate chloride signalling and WNK1 in the regulation of apoptosis, it remains unknown whether WNK1 is an essential and direct mediator that links VRCC activation to the suppression of mitochondrial apoptosis in VSMCs under oxidative stress.

Accordingly, the present study directly tested the hypothesis that WNK1 phosphorylation mediates the anti‐apoptotic effect of hypotonicity. We focused on mitochondrial apoptotic events including the Bcl‐2/Bax balance, mitochondrial membrane potential (MMP), cytochrome c release and caspase‐9/3 activation to define the molecular mechanism by which WNK1 governs VSMC survival and vascular homeostasis under oxidative stress.

2. Material and Methods

The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [19].

2.1. Cell Culture

A10 VSMCs were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A10 cells were cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) supplemented with 10% newborn calf serum (NCS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Low‐passage cells (typically < 20 passages after thawing) were used for all experiments.

2.2. Cell Viability Assay

Cell viability was measured using the Cell Counting Kit‐8 (CCK‐8, Dojindo Molecular Technologies, MD, Japan), which contains 2‐(2‐methoxy‐4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H‐tetrazolium monosodium salt (WST‐8). This compound is reduced by dehydrogenases in living cells to form water‐soluble orange formazan. The absorbance of formazan at 450 nm is directly proportional to the number of viable cells and can be used to assess cell viability. Cells were seeded in 96‐well plates at a density of 1–2 × 10⁴ cells per well, with six replicates for each experimental condition. After cells adhered, the medium was replaced with serum‐free medium for synchronization, and cells were incubated for 24 h. Following synchronization, appropriate treatments (e.g., interference or plasmid transfection) were applied according to the experimental design. After treatment, the medium was aspirated, and the cells were washed twice with PBS. Fresh medium containing 10% (v/v) CCK‐8 reagent (10 μL CCK‐8 in 100 μL medium per well) was added to each well. The plates were incubated at 37°C in the dark for 1–4 h, and the optical density (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: cell viability (%) = [(OD of the treatment group − OD of the blank group)/(OD of the control group − OD of the blank group)] × 100%.

2.3. Quantification of Apoptosis by Flow Cytometry

Apoptosis was detected by flow cytometry using Annexin V‐APC/7‐AAD double staining (KeyGen Biotech, Nanjing, China). Annexin V binds phosphatidylserine (PS), which flips to the outer cell membrane during early apoptosis. Because PS exposure also occurs during necrosis, 7‐AAD is used in combination to differentiate between live, early apoptotic (Annexin V⁺/7‐AAD⁻) and late apoptotic or necrotic cells (Annexin V⁺/7‐AAD⁺), based on membrane integrity.

For the experimental procedure, cells were first harvested by digestion with trypsin (without EDTA), followed by centrifugation at 2000 rpm for 5 min at 4°C. After washing twice with ice‐cold PBS and discarding the supernatant, the cell pellet was resuspended in 500 μL of binding buffer. Next, 5 μL Annexin V‐APC and 5 μL 7‐AAD were added to the suspension, and the mixture was gently mixed and incubated in the dark at room temperature for 10 min. Samples were analysed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA), at excitation wavelengths of 633 nm and 546 nm, within 1 h of staining. The flow cytometry scatter plot was interpreted as follows: Cells in the lower left quadrant represent live cells (Annexin V⁻/7‐AAD⁻), the lower right quadrant corresponds to early apoptotic cells (Annexin V⁺/7‐AAD⁻), and the upper right quadrant contains late apoptotic or necrotic cells (Annexin V⁺/7‐AAD⁺).

2.4. MMP Test

MMP was assessed using the JC‐1 assay (Beyotime Institute of Biotechnology, Shanghai, China). Cells were seeded at a density of 5 × 10⁵ cells/mL in culture dishes and transfected for WNK1 overexpression or knockdown after reaching 70% confluency. Six hours after transfection, the cells were transferred to a hypotonic medium and incubated for 48 h. MMP was measured using JC‐1 assay kit. Cells were digested with trypsin (without EDTA), and 1 × 10⁵–6 × 10⁵ cells were resuspended in 0.5 mL culture medium. An equal volume (0.5 mL) of JC‐1 staining solution was added, and the suspension was gently mixed and incubated at 37°C for 20 min. During incubation, the JC‐1 staining buffer (1X) was prepared by diluting the 5X buffer stock solution with distilled water in a 1:4 ratio and kept on ice. After incubation, cells were centrifuged at 600 × g at 4°C for 3–4 min, the supernatant was carefully discarded, and the cells were washed twice with 1X JC‐1 staining buffer. The final cell pellet was resuspended in an appropriate volume of JC‐1 staining buffer (1X) for flow cytometric analysis.

2.5. Preparation of Mitochondrial and Cytosolic Fractions

Intact mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific Inc., Rockford, IL, USA), following the manufacturer's protocol. Prior to extraction, protease inhibitors were added to Reagents A and C. Cells were harvested by centrifugation at 850 × g for 2 min to collect approximately 2 × 10⁷ cells. The pellet was resuspended in 800 μL Reagent A, vortexed for 5 s, and incubated on ice for 2 min. Next, 10 μL Reagent B was added, and the mixture was vortexed at maximum speed for 5 s, followed by a 5‐min incubation on ice with vortexing every minute. Subsequently, 800 μL Reagent C was added, and the tube was gently inverted several times before centrifugation at 700 × g for 10 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 3000 × g for 15 min at 4°C to obtain the cytosolic fraction. The pellet containing the isolated mitochondria was washed and resuspended in 500 μL Reagent C, followed by centrifugation at 12 000 × g for 5 min. The resulting pellet was resuspended in 20 μL of mitochondrial lysis buffer, briefly vortexed, and centrifuged at high speed for 2 min. The supernatant collected at this stage contained mitochondrial proteins that were subsequently used for western blot analysis. Cox IV (Beyotime Institute of Biotechnology) served as a loading control for the mitochondrial fraction.

2.6. Western Blot

Western blot analysis was performed as previously described. Protein lysates were collected and resolved using SDS‐PAGE. After transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA), the membrane was blocked in TBST (1 mM CaCl₂, 136 mM NaCl, 2.5 mM KCl, 25 mM Tris–HCl, and 0.1% (v/v) Tween 20) containing 5% nonfat dry milk. The blot was then incubated with specific primary antibodies, followed by incubation with corresponding secondary antibodies. The primary antibodies against caspase‐3, caspase‐9, Bcl‐2, Bax, and cytochrome c were purchased from Cell Signaling Technology (1:1000; Boston, MA, USA). Secondary antibodies, including anti‐mouse and anti‐rabbit HRP‐linked antibodies, were obtained from Cell Signaling Technology (1:1000). Loading controls included α‐tubulin (1:4000; Santa Cruz, CA, USA), β‐actin (1:4000; Cell Signaling Technology), and Cox‐IV (1:1000; Cell Signaling Technology). Protein bands were visualized using the Bio‐Rad Molecular Imager ChemiDoc XRS+ System (Bio‐Rad, Richmond, CA, USA), and band density was analysed with Bio‐Rad Quantity One 1D analysis software (Bio‐Rad, Hercules, CA, USA).

2.7. Statistical Analysis

Statistical analyses were performed using GraphPad Prism software (Version 10.0). All data are presented as mean ± SEM from at least three independent experiments. Sample sizes (n values) for each experiment are shown in the corresponding figure legends. For experiments with a single variable (e.g., different treatment groups under the same condition), one‐way analysis of variance (ANOVA) was used, followed by the Bonferroni post hoc test to correct for multiple pairwise comparisons. For experiments involving two variables (e.g., treatment type and WNK1 expression status), two‐way ANOVA was applied, with a Bonferroni post hoc test to adjust for multiple comparisons. Statistical significance was set at p < 0.05.

3. Results

3.1. Hypotonic Stimulation Attenuates H₂O₂‐Induced Apoptosis Through Elevation of WNK1 Phosphorylation

Given our previous observation that oxidative stress induces apoptosis and elevates intracellular Cl⁻ concentration [18], we first examined whether H₂O₂ directly regulates WNK1 activity. As shown in Figure 1A, western blot analysis revealed that H₂O₂ stimulation led to a time‐dependent decrease in WNK1 phosphorylation under hypotonic conditions, with a significant reduction detectable at 1 min, whereas total WNK1 protein levels remained unchanged. We further verified that hypotonic stress alone promotes WNK1 phosphorylation (Figure S1A), consistent with our previous study [15]. Next, we investigated whether hypotonic stress affects H₂O₂‐induced apoptosis in VSMCs. Flow cytometry analysis (Figure 1B) showed that hypotonic treatment attenuated H₂O₂‐induced apoptosis in a time‐dependent manner. Compared with the H₂O₂ control group, the apoptotic rates were reduced to 19.04 ± 0.65% at 12 h and further to 15.47 ± 0.46% at 48 h.

FIGURE 1.

Effects of H₂O₂ on WNK1 phosphorylation and hypotonic inhibition of H₂O₂‐induced apoptosis. (A) (i) Representative western blots of p‐WNK1 and total WNK1. Tubulin served as loading control. (ii) Densitometric quantification of p‐WNK1. p‐WNK1 levels were quantified by densitometry and normalized to tubulin. p‐WNK1 levels decreased significantly within 1 min of H₂O₂ treatment and reached the lowest level at 2 min (*p < 0.05 vs. control; n = 6). Total WNK1 protein expression remained unchanged. (B) (i) Representative flow cytometry plots (Annexin V‐APC/7‐AAD). Apoptotic cells were defined as Annexin V⁺/7‐AAD⁻ (lower right quadrant). (ii) Quantification of apoptotic cells over time. Hypotonic treatment reduced the H₂O₂‐induced apoptosis rate, with the lowest rate observed at 48 h (*p < 0.05 vs. control; n = 6). Based on these results, 48 h was used as the optimal hypotonic stimulation duration in subsequent experiments. Data are presented as mean ± SEM. n represents the number of independent experiments.

Taken together, these findings indicate that oxidative stress rapidly suppresses WNK1 activity, whereas hypotonic activation of VRCC promotes WNK1 phosphorylation and protects VSMCs from H₂O₂‐induced apoptosis. WNK1 may therefore serve as a key mediator linking VRCC activation to anti‐apoptotic signalling. To test this hypothesis, we next manipulated WNK1 expression and activity.

3.2. WNK1 Phosphorylation Mediates the Anti‐Apoptotic Effect of Hypotonic Stimulation

To determine the role of WNK1 in hypotonicity‐induced protection, we performed WNK1 overexpression and knockdown experiments (Figure S1B,C). In hypotonic conditions, H₂O₂‐induced apoptosis was reduced in a WNK1‐dependent manner. As shown in Figure 2A,B, H₂O₂ markedly decreased cell survival, whereas hypotonic treatment improved cell viability, which was further enhanced by WNK1 overexpression and markedly attenuated by WNK1 knockdown.

FIGURE 2.

Role of WNK1 in hypotonicity‐mediated protection against H₂O₂‐induced apoptosis. (A) Cell viability was determined by the CCK‐8 assay. H₂O₂ significantly reduced cell survival under isotonic conditions, which was reversed by hypotonic treatment. WNK1 overexpression further enhanced cell survival (n = 6). (B) WNK1 knockdown abolished the protective effect of hypotonicity on cell survival against H₂O₂‐induced apoptosis (n = 6). *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂.

Consistent with these findings, flow cytometry confirmed that hypotonic treatment significantly reduced H₂O₂‐induced apoptosis. WNK1 overexpression further augmented the anti‐apoptotic effect of hypotonic stimulation, whereas WNK1 knockdown reversed this effect. These results demonstrate that WNK1 is required for the protective effect of hypotonicity against H₂O₂‐induced apoptosis (Figure 3A,B).

FIGURE 3.

Flow cytometry analysis of WNK1 in hypotonic protection against H₂O₂‐induced apoptosis in A10 cells. (A) Effect of WNK1 overexpression on early apoptosis. (i) H₂O₂ elevated early apoptosis, which was attenuated by hypotonic treatment. WNK1 overexpression further decreased early apoptosis (n = 6). (ii) Quantification of early apoptosis. (B) Effect of WNK1 knockdown on early apoptosis. (i) WNK1 knockdown reduced hypotonic protection and increased early apoptosis (n = 5). (ii) Quantification of early apoptosis for WNK1 knockdown. *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂. Data are presented as mean ± SEM. n represents the number of independent experiments.

To determine whether the kinase activity of WNK1 depends on its phosphorylation at the T60 site, we used the mutant plasmid WNK1(T60A). As shown in Supplemental Figure S2A,B, the overexpression of WNK1(T60A) did not increase p‐WNK1 levels under hypotonic conditions, indicating that T60 is critical for WNK1 phosphorylation. Moreover, WNK1(T60A) did not enhance cell survival in the CCK‐8 assay (Figure S2C). These results demonstrate that WNK1 phosphorylation at T60 is essential for its protective effect against H₂O₂‐induced apoptosis.

3.3. WNK1 Regulates Bcl‐2/Bax Ratio in Hypotonic Protection Against H₂O₂‐Induced Apoptosis

The Bcl‐2 family is a key regulator of mitochondrial apoptosis, comprising anti‐apoptotic proteins such as Bcl‐2, which preserve mitochondrial integrity, and pro‐apoptotic proteins such as Bax, which facilitate cytochrome c release and caspase activation. Because the relative balance between these opposing factors determines mitochondrial outer membrane permeabilization, the Bcl‐2/Bax ratio is widely used as an indicator of apoptotic susceptibility [20, 21].

As shown in Figure 4A, exposure to H₂O₂ markedly reduced the Bcl‐2/Bax ratio compared with isotonic controls, consistent with enhanced apoptotic signalling. Hypotonic treatment partially restored this balance, thereby alleviating H₂O₂‐induced apoptosis. Overexpression of WNK1 further amplified this protective effect, whereas silencing of WNK1 abolished the benefit of hypotonicity, leading to a pronounced decrease in the Bcl‐2/Bax ratio (Figure 4B).

FIGURE 4.

Effect of WNK1 overexpression and knockdown on the Bcl‐2/Bax ratio in hypotonic protection against H₂O₂‐induced apoptosis. (A) Effects of WNK1 overexpression on Bcl‐2 and Bax expression. (i) H₂O₂ decreased Bcl‐2 expression and increased Bax expression. Hypotonicity restored Bcl‐2 levels and inhibited Bax expression, and WNK1 overexpression further enhanced these effects. (ii) Densitometric analysis of Bcl‐2 expression (n = 5 for non‐H₂O₂; n = 9 for H₂O₂ groups). (iii) Densitometric analysis of Bax expression (n = 4 for non‐H₂O₂; n = 7 for H₂O₂ groups). (iv) Densitometric analysis of the Bcl‐2/Bax ratio (n = 4 for non‐H₂O₂; n = 7 for H₂O₂ groups). (B) Effect of WNK1 knockdown on Bcl‐2 and Bax expression. (i) Western blot analysis showed that WNK1 knockdown reversed the protective effect of hypotonicity by suppressing Bcl‐2 expression and enhancing Bax expression. (ii) Densitometric analysis of Bcl‐2 expression (n = 6 for non‐H₂O₂; n = 8 for H₂O₂ groups). (iii) Densitometric analysis of Bax expression (n = 4 for non‐H₂O₂; n = 9 for H₂O₂ groups). (iv) Densitometric analysis of the Bcl‐2/Bax ratio (n = 5 for non‐H₂O₂; n = 7 for H₂O₂ groups). *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂. Data are presented as mean ± SEM. n represents the number of independent experiments.

To determine the contributions of Bcl‐2 and Bax, each protein was quantified separately. H₂O₂ treatment decreased Bcl‐2 expression and increased Bax expression. Hypotonic stimulation and WNK1 overexpression partially restored Bcl‐2 levels (by approximately 1.4‐fold) and suppressed Bax expression (by approximately 1.3‐fold).

These findings suggest that WNK1 may preserve mitochondrial integrity and attenuate oxidative injury by modulating the balance between pro‐ and anti‐apoptotic proteins, thereby linking chloride‐sensitive kinase signalling to mitochondrial apoptotic pathways.

3.4. Effect of WNK1 on MMP and Cytochrome c Release in Hypotonic Protection Against H₂O₂‐Induced Apoptosis

During early apoptosis, the MMP decreases. MMP is a critical indicator of mitochondrial integrity during apoptosis, and changes in MMP can be detected using JC‐1, a cationic dye. When MMP is high, JC‐1 forms aggregates in the mitochondrial matrix, emitting red fluorescence; when MMP is low, it remains as a monomer, emitting green fluorescence. Thus, the red‐to‐green fluorescence shift provides a sensitive indicator of MMP alterations.

As shown in Figure 5, H₂O₂ stimulation markedly reduced MMP compared with isotonic controls, indicating mitochondrial dysfunction. Hypotonic treatment partially preserved MMP under oxidative stress; this effect was further enhanced by WNK1 overexpression, whereas WNK1 knockdown significantly attenuated this protective response. These results demonstrate that WNK1 is essential for sustaining MMP under apoptotic stress.

FIGURE 5.

Effect of WNK1 on mitochondrial membrane potential (MMP) in hypotonic protection against H₂O₂‐induced apoptosis. (A) Effect of WNK1 overexpression on MMP (JC‐1 staining). (i) H₂O₂ significantly decreased MMP (Δψm), which was restored by hypotonic treatment. WNK1 overexpression further elevated MMP (n = 5 for non‐H₂O₂; n = 5 for H₂O₂ groups). (B) Effect of WNK1 knockdown on MMP. (i) WNK1 knockdown diminished the protective effect of hypotonicity on MMP in H₂O₂‐treated cells. (ii) Quantification of MMP (n = 5 for non‐H₂O₂; n = 6 for H₂O₂ groups). *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂. Data are presented as mean ± SEM. n represents the number of independent experiments.

Consistently, Figure 6 shows that H₂O₂ induced a pronounced release of cytochrome c from the mitochondria into the cytosol. Hypotonic conditions attenuated this effect, whereas WNK1 overexpression enhanced the inhibition of cytochrome c release. In contrast, WNK1 knockdown abrogated the protective effect of hypotonic treatment, resulting in increased cytosolic cytochrome c accumulation. Collectively, these findings indicate that WNK1 preserves mitochondrial integrity by maintaining MMP and restricting cytochrome c release, thereby contributing to the protective effects of hypotonicity against H₂O₂‐induced apoptosis.

FIGURE 6.

Effect of WNK1 on cytochrome C release in hypotonic protection against H₂O₂‐induced apoptosis. (A,B) Effect of WNK1 overexpression on cytochrome C release. (i) Western blot analysis showed that H₂O₂ promoted cytochrome C release from mitochondria to the cytoplasm. Hypotonicity reduced this release, and WNK1 overexpression further enhanced the protective effect. (ii) Quantification of cytochrome C (n = 4). (C,D) Effect of WNK1 knockdown on cytochrome C release. (i) Western blot analysis showed that WNK1 knockdown diminished the inhibitory effect of hypotonicity on H₂O₂‐induced cytochrome C release. (ii) Quantification of cytochrome C (n = 4). *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂. Data are presented as mean ± SEM. n represents the number of independent experiments.

3.5. Effect of WNK1 on Caspase‐9 and Caspase‐3 in H₂O₂‐Induced Apoptotic Protection Under Hypotonic Conditions

Caspase‐9 activation is a hallmark of the mitochondrial apoptotic pathway and serves as a critical initiator that subsequently activates caspase‐3 to execute apoptosis. To evaluate the role of WNK1 in modulating these key apoptotic effectors under hypotonic conditions, we performed western blot analysis for cleaved caspase‐9 and cleaved caspase‐3.

As shown in Figure 7A,B, H₂O₂ stimulation led to pronounced activation of caspase‐9, indicating mitochondrial‐dependent apoptotic progression. Hypotonic treatment significantly attenuated this activation, and this protective effect was further enhanced by WNK1 overexpression. In contrast, WNK1 knockdown markedly reduced the protective efficacy against hypotonicity by restoring caspase‐9 cleavage.

FIGURE 7.

Effect of WNK1 on cleaved caspase‐9 and caspase‐3 expression in hypotonic protection against H₂O₂‐induced apoptosis. (A) Representative western blots (i) and quantification (ii) of cleaved caspase‐9 in WNK1‐overexpressing A10 cells. H₂O₂ increased cleaved caspase‐9 levels; hypotonicity suppressed this increase, and WNK1 overexpression further enhanced the protective effect (n = 4 for non‐H₂O₂; n = 8 for H₂O₂ groups). (B) Quantification of cleaved caspase‐3 in WNK1‐overexpressing cells. H₂O₂ elevated cleaved caspase‐3 levels, which were reduced by hypotonicity and further lowered by WNK1 overexpression (n = 4 for non‐H₂O₂; n = 9 for H₂O₂ groups). (C) Representative western blots and quantification of cleaved caspase‐9 after WNK1 knockdown. Hypotonicity reduced H₂O₂‐induced cleaved caspase‐9 levels, whereas WNK1 knockdown abolished this protection (n = 4 for non‐H₂O₂; n = 7 for H₂O₂ groups). (D) Quantification of cleaved caspase‐3 after WNK1 knockdown. Hypotonicity attenuated H₂O₂‐induced cleaved caspase‐3 activation, which was restored by WNK1 knockdown (n = 4 for non‐H₂O₂; n = 8 for H₂O₂ groups). *p < 0.05 versus isotonic + H₂O₂; #p < 0.05 versus hypotonic + H₂O₂. Data are presented as mean ± SEM. n represents the number of independent experiments.

Similarly, as depicted in Figure 7C,D, cleaved caspase‐3 levels increased following H₂O₂ exposure, reflecting enhanced execution of apoptosis. Hypotonic treatment decreased caspase‐3 activation, which was amplified by WNK1 overexpression. Conversely, WNK1 knockdown reversed this protective effect by promoting caspase‐3 cleavage under hypotonic stress.

Collectively, these results indicate that WNK1 is a pivotal regulator of the mitochondrial apoptotic cascade, modulating both caspase‐9 and caspase‐3 activity. Consistent with its effects on mitochondrial integrity (MMP) and cytochrome c release, WNK1 suppresses downstream caspase activation, thereby contributing to the cytoprotective effects of hypotonic conditions against oxidative stress–induced VSMC apoptosis.

4. Discussion

Over the past few decades, VSMC apoptosis has become a prominent research topic. In healthy blood vessels, apoptosis counterbalances cell differentiation and proliferation during vascular remodelling [21, 22]. Beyond classical cationic regulators such as calcium, emerging evidence highlights the role of anions, particularly intracellular Cl⁻, as critical modulators of cell signalling and apoptosis. Altered Cl⁻ homeostasis has been closely linked to apoptosis initiation, yet the underlying signalling mechanisms remain incompletely defined. The WNK family of protein kinases is known as Cl⁻ sensors and plays a key role in maintaining cell volume and ion homeostasis, which may be a new target for cardiovascular diseases [23, 24, 25]. Our previous study demonstrated that hypotonic challenge activates WNK1 to promote its phosphorylation in turn participating in VSMC proliferation [15]. In the present study, we extended these findings by demonstrating that WNK1 protects VSMCs from oxidative stress‐induced apoptosis. Specifically, VRCC‐mediated Cl⁻ efflux activates WNK1, which preserves mitochondrial integrity by maintaining the Bcl‐2/Bax balance, reducing cytochrome c release and suppressing caspase‐9/3 activation. This anti‐apoptotic pathway operates in parallel with a previously described pro‐proliferative effect, suggesting that WNK1 integrates both survival and growth signals in response to changes in ionic environment.

The dual role of WNK1 in VSMC proliferation and apoptosis raises the question of how this pathway contributes to vascular remodelling in vivo. During hypertension or vascular injury, VSMCs are exposed to mechanical stress, inflammatory cytokines and oxidative stress, all of which can alter intracellular Cl⁻ homeostasis [1, 3]. Our results suggest that the activation of VRCCs and subsequent WNK1 phosphorylation could serve as an endogenous protective mechanism to limit VSMC apoptosis under oxidative stress, thereby preserving vessel integrity. Conversely, dysregulation of this pathway—due to genetic variants in WNK1 or abnormal Cl⁻ transport—might shift the balance towards excessive apoptosis, contributing to medial thinning, aneurysm formation or plaque instability. Indeed, mutations in WNK kinases cause pseudo‐hypoaldosteronism type II (Gordon's syndrome), a form of hypertension [23, 24], and recent studies have implicated WNK1 in vascular inflammation and remodelling [14, 16]. Our findings suggest that WNK1 may influence the long‐term structural remodelling of the arterial wall by modulating mitochondrial apoptosis.

Although we established that phosphorylation of WNK1 at Thr60 is essential for its anti‐apoptotic function, several limitations of the present study should be acknowledged. First, our data demonstrate that WNK1 regulates the Bcl‐2/Bax balance, MMP, cytochrome c release and caspase‐9/3 activation during oxidative stress. However, these findings do not establish a direct phosphorylation‐mediated regulatory relationship between WNK1 and these apoptotic proteins. Therefore, the observed changes in mitochondrial apoptotic signalling should be interpreted as downstream events associated with WNK1 activation. Although the direct downstream substrates of WNK1 in this pathway remain to be identified, our previous study suggests that SGK1 represents a plausible intermediate signalling molecule linking WNK1 activation to mitochondrial apoptotic regulation [18]. Also, WNK1 may indirectly affect MMP via ion cotransporters. Given that WNK1 is known to interact with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 and the K⁺/Cl⁻ cotransporter KCC3/4 [11], it is plausible that WNK1‐mediated changes in intracellular ion concentrations could influence mitochondrial function and redox balance. Future studies should employ phosphoproteomics to identify direct WNK1 substrates in VSMCs and use conditional knockout mouse models to assess in vivo remodelling. Additionally, pharmacological modulators of WNK1 activity (e.g., WNK463) can be tested for their ability to prevent VSMC apoptosis and pathological vascular remodelling in animal models.

In conclusion, this study provides a novel perspective on how a chloride‐sensitive kinase integrates ionic and mitochondrial signalling to regulate VSMC survival. By identifying the chloride‐sensitive kinase WNK1 as a key determinant of apoptotic resistance, these findings open new avenues for understanding ion‐mediated vascular biology and may ultimately inform therapeutic strategies for hypertension and related cardiovascular disorders.

Author Contributions

Yajuan Zhang performed the Western Blot and flow cytometry experiments and wrote the manuscript; Wei Lin acquired and analysed the flow cytometry data; Wei Lin and Fengting Lu conducted cell culture and related experiments; Zhujun Liang performed the CCK‐8 assay; Chengcui Huang analysed and interpreted the data and performed the statistical analysis; Guozheng Liang revised the manuscript; Xiaofei Lv conceived and designed the study.

Funding

This work was supported by the National Natural Science Foundation of China (81903598 and 82473916) and the Natural Science Foundation of Jiangsu Province (BK20250354).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: WNK1 is regulated by hypo‐osmotic stress and genetic manipulation. (A) Effect of hypotonic stress on WNK1 phosphorylation. Immunoblotting analysis of WNK1 phosphorylation in response to hypotonic stimulation. A10 cells were exposed to hypotonic medium for the indicated times and the lysates were probed with antibodies against p‐WNK1. Tubulin served as a loading control. (B) Overexpression and (C) knockdown of WNK1. Immunoblots confirm the successful manipulation of WNK1 levels. (B) WNK1 overexpression in cells transfected with a WNK1 plasmid versus the empty vector (vec) or untransfected controls (con). (C) WNK1 knockdown in cells transfected with WNK1‐targeting siRNA versus non‐targeting siRNA (negative) or controls (con). Total WNK1 and tubulin levels are shown.

Figure S2: Effect of WNK1(T60A) mutant on hypotonic protection against H₂O₂‐induced apoptosis. (A) WNK1(T60A) overexpression under hypotonic conditions. (i) Western blot analysis confirmed the elevated WNK1 expression after transfection. (ii) Quantification of total WNK1 (*p < 0.05 vs. hypotonic control; n = 5). (B) The effect of WNK1(T60A) on p‐WNK1 under hypotonic conditions. (i) Overexpression of WNK1(T60A) did not increase p‐WNK1 levels, indicating that T60 is essential for WNK1 activation. (ii) Quantification of p‐WNK1 (*p < 0.05 vs. hypotonic control; n = 6). (C) Cell viability was assessed by the CCK‐8 assay. WNK1(T60A) did not alter the protective effect of hypotonicity against H₂O₂‐induced apoptosis (*p < 0.05 vs. isotonic + H₂O₂; n = 6). Data are presented as mean ± SEM. n represents the number of independent experiments.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.