CYFIP2 deficiency ameliorates renal interstitial fibrosis through attenuating tubular senescence in hypertensive nephropathy

Abstract

Tubulointerstitial fibrosis is the central pathological feature of hypertensive nephropathy, with cellular senescence being a key driver. Therefore, identifying therapeutic targets in senescent renal tubular epithelial cells is clinically important. The cytoplasmic FMR1-interacting protein (CYFIP) family, which comprises two evolutionarily conserved members, CYFIP1 and CYFIP2, plays crucial roles in neurological regulation. CYFIP2, a key member, is implicated in cytoskeletal dynamics and apoptosis within the nervous system; however, its renal expression pattern and function remain undefined. This study revealed that CYFIP2 expression was significantly upregulated in the renal cortex, particularly in the proximal tubules, of DOCA/salt-induced hypertensive mice, and was positively correlated with the extent of fibrosis. Consistently, CYFIP2 was highly expressed in the renal tubules of patients with hypertensive nephropathy, where its level inversely correlated with the estimated glomerular filtration rate (eGFR). Tubule-specific deletion of CYFIP2 attenuated hypertension-induced cellular senescence (reduced SA-β-gal, p53/p21, and SASP; increased Klotho) and mitigated renal dysfunction, collagen deposition, and epithelial‒mesenchymal transition (EMT). In vitro, CYFIP2 silencing alleviated TGF-β1-induced senescence and fibrosis in HK-2 cells. Mechanistically, CYFIP2 and p53 formed a positive feedback loop that promoted fibrosis by inhibiting the Hippo pathway and enhancing YAP nuclear translocation. The p53 agonist Nutlin-3a reversed the protective effect of CYFIP2 knockout, while the inhibitor Pifithrin-α mimicked this effect. These findings underscore the pivotal role of the CYFIP2/p53-Hippo/YAP axis in hypertensive renal injury, and identify CYFIP2 as a potential therapeutic target.

CYFIP2/p53-Hippo signaling drives tubular senescence and renal fibrosis in hypertensive nephropathy.

Introduction

Hypertension is a primary etiological factor in chronic kidney disease (CKD). Its renal complication, hypertensive nephropathy, ranks as the second leading cause of end-stage renal disease (ESRD) [1, 2]. Excessive dietary salt intake, a well-established environmental risk factor for cardiovascular disorders, has been strongly linked to the pathogenesis of hypertension [3]. In individuals with salt-sensitive hypertension, the kidneys exhibit heightened vulnerability to injury. Consequently, a substantial proportion of these patients progress to renal failure, for which dialysis and kidney transplantation are life-sustaining interventions [4]. Therefore, elucidating the underlying mechanisms of this disease is both critically important and clinically imperative.

Salt-sensitive hypertensive nephropathy is characterized by inflammatory cell infiltration, proinflammatory mediator release, and tubulointerstitial fibrosis (TIF). The mechanism underlying renal injury involves an impaired renal capacity for sodium handling and the consequent disruption of renal microenvironmental homeostasis, such as inflammatory responses and oxidative stress. These alterations directly target renal tubular epithelial cells, leading to TIF [5]. Therefore, employing relevant models to study TIF and elucidate its mechanisms is crucial for developing effective therapeutic strategies. At the core of renal fibrosis is the excessive deposition of extracellular matrix (ECM), which is driven primarily by activated myofibroblasts [6]. In addition to classical inflammatory responses and epithelial-mesenchymal transition (EMT), cellular senescence, defined as a state of stable cell cycle arrest, has increasingly been recognized as a key driver of fibrosis [7, 8]. Numerous studies have demonstrated a strong association between renal ageing and TIF [9–11]. Renal tubular epithelial cells are not only injured in CKD but also actively contribute to disease progression. Under CKD conditions, these cells are prone to enter a senescent state, which subsequently promotes fibrosis. Senescent cells establish a local proinflammatory and profibrotic microenvironment by secreting factors such as TGF-β and CTGF through the senescence-associated secretory phenotype (SASP). This process sustains fibroblast activation, exacerbates ECM deposition, and ultimately leads to TIF [12]. Consequently, targeting cellular senescence in renal tubular epithelial cells may disrupt the fibrotic cascade at an upstream stage, offering a novel therapeutic strategy for treating and delaying the progression of hypertensive nephropathy.

The cytoplasmic FMR1-interacting protein (CYFIP) family comprises highly evolutionarily conserved proteins that were initially identified as binding partners of fragile X mental retardation protein (FMRP), an mRNA-binding protein whose absence causes fragile X syndrome. The CYFIP family consists of two members, CYFIP1 and CYFIP2, which play crucial roles in neuronal development, actin cytoskeleton dynamics, and mRNA regulation. Dysregulation of their functions is closely associated with various neurological disorders [13, 14]. Notably, the two family members exhibit distinct tissue expression patterns: CYFIP1 is widely expressed in most human tissues, whereas CYFIP2 is highly abundant in the kidney, brain, and lymph nodes, suggesting a potential specialized function in renal physiology and pathology [15]. As a key member of this family, CYFIP2 was initially characterized as a p53-induced protein. It not only participates in p53-dependent apoptotic pathways but also plays significant regulatory roles in the development and progression of various tumors [16–18]. Recent studies have revealed that CYFIP2 is involved in critical biological processes such as WAVE regulatory complex-mediated actin remodelling, maintenance of mitochondrial homeostasis, and regulation of cellular stress responses, highlighting its importance in maintaining cellular structural and functional balance [19, 20]. Although a retrospective analysis based on data from The Cancer Genome Atlas (TCGA) suggested that CYFIP2 expression is associated with a poor prognosis in renal clear cell carcinoma, experimental and clinical evidence confirming the role of CYFIP2 in the kidneys is lacking [21].

In this study, we identified a critical pathogenic role of CYFIP2 in hypertensive nephropathy. CYFIP2 was found to be significantly upregulated in the kidney, particularly in proximal tubules, of deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mice, thus promoting renal injury in this model. Importantly, the tubule-specific deletion of CYFIP2 markedly alleviated DOCA-induced renal senescence and fibrosis. Mechanistically, CYFIP2 inhibited the Hippo pathway by promoting p53 expression both in vivo and in vitro. This study aims to systematically elucidate how this signalling module mediates renal pathological remodelling.

Materials and methods

Human renal biopsy samples

The use of human samples in this research was sanctioned by the Institutional Ethical Review Boards (SZRJJ: NO. 2021-417), and signed informed consent was obtained from the patients. Renal biopsy samples were obtained from Shandong Provincial Hospital. Control samples were obtained from individuals without hypertension or renal disease who underwent tumour nephrectomies. Individuals with hypertension and CKD in the absence of other illnesses that may cause kidney disease were classified as having HTN. The investigations were conducted in accordance with the principles of the Declaration of Helsinki.

Animal studies

All animal experiments were conducted in accordance with the guidelines issued by the Animal Care and Use Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University and were approved by this committee (NO.2024-144).

Different groups were allocated in a randomized manner, and investigators were blinded to the allocation of different groups when doing surgeries and outcome evaluations. All the mice (3–5 mice per cage) were housed under standard laboratory SPF conditions with ad libitum access to water and a standard laboratory chow diet. The water and cages were autoclaved. Cages with standard corncob bedding were changed three times a week. For all of the in vivo experiments, littermate control mice were used, with 6 mice per experimental group unless otherwise specified. The mouse genotype did not cause visible changes in initial weight, health, or immune status. Considering that estrogen has effects on hypertension and cardiovascular disease, only male mice were used in this study to avoid confusion and exclude potential effects of sex differences. The number of mice used for the experiments is indicated for each experiment in the figure legends. All experimental animals were kept under barrier conditions under constant veterinary supervision and did not display signs of distress or pathological changes that warranted veterinary intervention.

Generation of tubular-specific CYFIP2 knockout mice

Floxed CYFIP2 mice (C57BL/6JSmoc-CYFIP2^{em1(flox)Smoc}, Shanghai Model Organisms Center, Inc, Shanghai, China) were hybridized with transgenic mice expressing Cre-recombinase under the cadherin 16 promoter (B6.Cg-Tg(Cdh16-cre)91Igr/J, Jackson Laboratory) to generate tubule-specific CYFIP2 knockout mice 1 (Cdh16-Cre⁺/ CYFIP2^fl/fl; Cre⁺/CYFIP2^fl/fl). Age-matched mice without Cre (Cdh16-Cre^-/CYFIP2^fl/fl) were used as controls. Mouse genotyping was performed using genomic DNA isolated from mouse tails by PCR. The specific primers used in this study are listed in Supplemental Table 3. Flox genotyping produced 236 bp and 295 bp fragments for the wild type and mutant, respectively. The wild type mice yielded only a 236 bp band; the homozygous (CYFIP2^fl/fl) mice yielded only a 295 bp band; and the heterozygous (CYFIP2^fl/+) mice yielded both bands. Cre positive (Cre⁺) mice yielded a 420 bp band, but Cre negative (Cre^-) mice yielded no band.

DOCA/salt-induced hypertension in mice

All of the mice underwent unilateral left nephrectomy (UNx) via a flank incision under isoflurane anesthesia at 8 to 10 weeks of age. The mice were randomly assigned to the DOCA/Salt and sham groups a week later. Continuous 21-day release pellets (DOCA, 50 mg) were implanted subcutaneously under isoflurane anesthesia. The drinking water for the UNx DOCA/salt groups was replaced with 1.0% NaCl 1 day later. At the end of the study, blood and 24 h urine samples were collected for biochemical analysis. Urinary albumin and creatinine levels were measured by immunoturbidimetric and enzymatic methods, respectively, using an automated analyzer. Simultaneously, the mice were euthanized, and the kidney samples were harvested. DOCA/salt-treated mice whose systolic blood pressure (SBP) was greater than 130 mmHg were included in the hypertensive group. The number of mice used for the experiments is indicated for each experiment in the figure legends.

Cell culture and treatments

Human tubule epithelial cells (HK-2 cells) and normal rat kidney fibroblasts (NRK-49F cells) were obtained from the American Type Culture Collection (ATCC). HK-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% foetal bovine serum (FBS) and penicillin/streptomycin. NRK-49F cells were cultured in DMEM supplemented with 10% FBS. All the cells were cultured at 37 °C with 5% CO₂. The following stimuli were used in this study: (1) TGF-β1 (a final concentration of 10 ng/mL in culture medium). (2) Nutlin-3a (a final concentration of 10^-5 mol/L in culture medium). (3) Pifithrin-α (a final concentration of 10^-5 mol/L in culture medium).

RNA interference in vitro

Short interfering RNAs for CYFIP2 or an equivalent scramble control (or negative control siRNA) were designed and synthesized by Gene Chem Co., Ltd. (Shanghai, China). Lipofectamine 3000 reagent was used to transfect siRNA into cells following the manufacturer’s protocol. The sequences of the siRNA oligonucleotides used were as follows: negative control (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′) and si-CYFIP2 (sense:  5′-AGAUAGAAGCUGAGGUGAATT-3′; antisense: 5'-UUCACCUCAGCUUCUAUCUTT-3′).

Statistics

The data are expressed as the mean ± SEM of at least three biological replicates. Statistical analyses were performed with GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA). The Kolmogorov‒Smirnov test was used to assess the normality assumption of the data distribution. For normally distributed data, a two-tailed Student’s t test was used to analyze the differences between two groups. For non-normally distributed data, the Mann‒Whitney rank sum test was used to analyze the differences between two groups. One-way ANOVA followed by post hoc Tukey’s test was used to analyze differences between multiple groups with one variable. Two-way ANOVA followed by post hoc Tukey’s test was used to compare multiple groups with more than one variable. Spearman correlation analysis was performed to assess the coefficient (r) and P-value. Linear regression was performed to depict the linear relationship among variables. Spearman correlation analysis and linear regression were performed with GraphPad Prism. All the statistical details regarding the p values and n values can be found in the main and supplementary figures and figure legends. P < 0.05 was considered to indicate statistical significance. Different groups of mice were allocated in a randomized manner, and investigators were blinded to the allocation of different groups when surgeries and outcome evaluations were performed. No animals were excluded from the study on the basis of animal well-being at the beginning of the study.

Results

CYFIP2 expression was significantly upregulated in the kidneys of DOCA/salt-induced hypertensive mice

To investigate the role of CYFIP2 in the pathogenesis of salt-sensitive hypertensive nephropathy, this study employed a DOCA/salt-induced hypertensive mouse model. This model effectively recapitulates hypertension-associated renal pathological alterations and fibrotic progression observed in humans, thus making it suitable for mechanistic exploration in the present research [22]. After 21 days of DOCA/salt treatment, CYFIP2 expression was significantly upregulated at both the mRNA and protein levels in the kidneys of hypertensive mice (Fig. 1a, b). Immunohistochemical (IHC) staining further confirmed that CYFIP2 expression was markedly increased in the renal tubules after DOCA/salt-induced hypertension (Fig. 1c). Consistent with the induction of hypertension, significant renal interstitial fibrosis was observed in DOCA/salt-treated mice by Western blot and IHC analyses (Fig. S1c, d). Notably, the level of CYFIP2 was positively correlated with fibronectin and α-SMA staining in hypertensive mice, suggesting that CYFIP2 may play a significant role in renal fibrosis (Fig. 1d, e). To more precisely determine the segment-specific expression of CYFIP2 in the kidneys, we performed double immunostaining for CYFIP2 and various tubular markers, including lotus tetragonolobus lectin (LTL) for proximal tubules, calbindin D28k for distal convoluted tubules, and aquaporin 3 (AQP3) for collecting ducts. Our results revealed that CYFIP2 expression was predominantly increased in the proximal tubules of hypertensive mice, while no significant changes were observed in other tubular segments (Fig. 1f). Similarly, in vitro, elevated CYFIP2 expression was induced in HK-2 cells following treatment with TGF-β1 (Figs. 1g and S1a, b). Furthermore, compared with renal tissues obtained from patients undergoing tumour nephrectomy without underlying renal disease, CYFIP2 expression was significantly upregulated in the tubules of patients with biopsy-proven hypertensive renal injury (Fig. 1h and Supplementary Table S1). Notably, we observed that the level of CYFIP2 was negatively correlated with the estimated glomerular filtration rate (eGFR) (Fig. 1i).

Fig. 1. The level of CYFIP2 was significantly upregulated in the kidney from DOCA/salt-induced hypertensive mice and in HK-2 cells with TGF-β1 treatment.

a qRT-PCR analysis of CYFIP2 expression in the kidneys of DOCA/salt-induced hypertensive mice. ***P < 0.001 versus sham-operated WT mice (n = 6). b Western blot analysis of the relative protein levels of CYFIP2 in the kidney of DOCA/salt-induced hypertensive mice. ***P < 0.001 versus sham-operated WT mice (n = 6). c Representative images of IHC staining of CYFIP2 in the kidney of DOCA/salt-induced hypertensive mice. Scale bar: 50 μm. ***P < 0.001 versus sham-operated WT mice (n = 11). Correlation between CYFIP2 expression and the degree of α-SMA (d) or fibronectin (e) staining in DOCA/salt-induced hypertensive mice. (n = 11). f Representative immunofluorescent images showing the colocalization of CYFIP2 with LTL, AQP3, and Calbindin-D28k in the kidney of DOCA/salt-induced hypertensive mice. ***P < 0.001 versus sham-operated WT mice, NS versus DOCA/salt-induced WT mice (n = 6). g Western blot analysis of CYFIP2 expression in HK-2 cells with TGF-β1 treatment for 24 h (n = 6). ***P < 0.001 versus the corresponding control. h Representative photomicrographs of CYFIP2 IHC staining in human renal cortical tissue from normal subjects (n = 6) and patients with hypertensive nephropathy (n = 9). Scale bar = 50 μm. ***P < 0.001 versus normal. i Correlation between the CYFIP2 in the kidney and eGFR in patients. (n = 15).

Tubule-specific deletion of CYFIP2 attenuated renal tubulointerstitial fibrosis and EMT in DOCA/salt-induced hypertensive mice

On the basis of the marked upregulation of CYFIP2 in the proximal tubule region, tubule-specific CYFIP2 knockout mice were generated using the Cre-LoxP recombination system to better elucidate the role of CYFIP2 in proximal tubular epithelial cells (Fig. 2a). Cdh16-Cre mice were crossed with CYFIP2^fl/fl mice to generate Cdh16-Cre/CYFIP2^fl/fl mice (Cre⁺/CYFIP2^fl/fl), which were confirmed by tail genotyping (Fig. 2b). All mice were viable and fertile. QRT-PCR and Western blot analyses confirmed a reduction in CYFIP2 in isolated renal tubules (Fig. 2c, d). Moreover, no physiological alterations, including in body weight, kidney weight, or heart rate, were observed in the Cre⁺/CYFIP2^fl/fl mice (Supplementary Table S2). In the DOCA/salt-induced hypertension model, a comparable elevation in blood pressure was observed in both wild-type and Cre⁺/CYFIP2^fl/fl mice, with no significant intergroup difference, suggesting that tubule-specific CYFIP2 deletion did not influence the development of hypertension (Fig. S2). Notably, however, tubule-specific CYFIP2 deletion significantly ameliorated renal functional impairment induced by hypertension, as reflected by a marked decrease in the urinary albumin-to-creatinine ratio (UACR) (Fig. 2e). Furthermore, no obvious changes in renal architecture were detected, indicating that tubule-specific deletion of CYFIP2 did not induce phenotypic changes under basal conditions (Fig. 2f). In addition, PAS staining demonstrated that tubule-specific CYFIP2 deletion substantially alleviated the degree of renal tubular injury (Fig. 2f). Given that tubulointerstitial fibrosis is a hallmark of hypertension-induced CKD, total collagen deposition in mouse kidneys was quantified using Masson’s trichrome and Sirius red staining. Compared with control mice, hypertensive mice exhibited a significant increase in renal collagen content, which was attenuated by tubule-specific CYFIP2 deletion (Fig. 2f). The antifibrotic effect of tubule-specific CYFIP2 deletion under hypertensive conditions was further supported by the downregulation of fibrosis-related markers, including Collagen I, Fibronectin, CTGF, and α-SMA (Fig. 2f, g). Since EMT is widely recognized as a key driver of renal fibrosis, we investigated whether CYFIP2 participated in partial EMT during hypertensive renal fibrosis. In renal tissues from hypertensive mice, a reduction in the expression of the epithelial marker E-cadherin and increases in the expression of the mesenchymal marker vimentin and the transcription factors Snail1 and Slug were observed compared with those in control mice. These alterations were effectively reversed by tubule-specific CYFIP2 deletion (Fig. 3a, b). Collectively, these findings demonstrated that tubule-specific CYFIP2 deletion significantly ameliorated hypertension-induced partial EMT and consequently attenuated the development of renal fibrosis.

Fig. 2. CYFIP2 deficiency ameliorated kidney injury and tubulointerstitial fibrosis in DOCA/salt-induced hypertensive mice.

a Generation of conditional knockout mice in which CYFIP2 is specifically ablated in tubular cells by using Cre-LoxP recombination system. b The genotype of Cre⁺/CYFIP2^fl/fl mice was confirmed by PCR and tail preparation. c Representative Western blot and quantifications of CYFIP2 expression in isolated tubules from Cre^-/CYFIP2^fl/fl and Cre⁺/CYFIP2^fl/fl mice (n = 6). d qRT-PCR analysis of CYFIP2 expression in isolated tubules from Cre^-/CYFIP2^fl/fl and Cre⁺/CYFIP2^fl/fl mice (n = 6).  ***P< 0.001 versus Cre^-/CYFIP2^fl/fl mice. e The urine albumin-to-creatinine ratio (UACR) in the different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. f PAS staining, Masson´s trichrome staining and Sirius Red staining were performed to assess kidney injury and fibrosis. Photomicrographs and quantifications of α-SMA, FN1, and Collagen-1 staining were performed to assess kidney fibrosis. Scale bar: 50 μm (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. g Western blot analysis of FN1, collagen-1, α-SMA and CTGF expression in the kidney from different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^##P<0.01, ^###P < 0.001 versus DOCA/salt-treated WT mice.

Fig. 3. CYFIP2 deficiency attenuated fibrotic phenotype in DOCA/salt-induced hypertensive mice and TGF-β1-treated HK-2 cells.

a Representative images of IHC staining of E-cadherin and Snail1 in the kidney from different groups of mice. Scale bar: 50 μm (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. b Western blot analysis of Vimentin, Slug, and Snail1 expression in the kidney from different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. c Western blot analysis of FN1, Collagen-1, Snail1, α-SMA, CTGF, Vimentin, Slug, and E-cadherin expression in HK-2 cells after the different treatments (n = 6). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. d Representative immunofluorescent images showing the expression of α-SMA in HK-2 cells after the different treatments. Scale bar: 20 μm (n = 6). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. e qRT-PCR showing the mRNA levels of Snail1, Slug, FN1, α-SMA, and Collagen-1 in HK-2 cells from different groups of mice (n = 6). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA, ^##P < 0.01 versus TGF-β1-treated cells transfected with scramble siRNA.

CYFIP2 silencing attenuated the TGF-β1-induced fibrotic phenotype in HK-2 cells

To validate the effects of CYFIP2 on tubular cell fibrosis and EMT in vitro, CYFIP2 was knocked down using CYFIP2-targeting siRNA (si-CYFIP2) (Fig. S4a, b). Silencing of CYFIP2 effectively reversed the TGF-β1-induced upregulation of fibrotic markers, including Collagen I, Fibronectin, CTGF, and α-SMA, in HK-2 cells (Fig. 3c). Similarly, TGF-β1 treatment induced alterations in the expression of EMT markers, characterized by increased Snail1, Slug, and Vimentin expression, along with decreased E-cadherin expression. These changes were also partially reversed following CYFIP2 silencing (Fig. 3c). Immunofluorescence staining further confirmed that the increase in α-SMA expression induced by TGF-β1 was partially suppressed and that the expression of E-cadherin was partially restored in CYFIP2-silenced cells (Figs. 3d and S4c), suggesting that CYFIP2 plays a critical role in the TGF-β-mediated profibrotic response. Furthermore, qRT‒PCR further confirmed that CYFIP2 silencing partially inhibited the increase in the expression of profibrotic factors induced by TGF-β1 (Fig. 3e). Given the established function of CYFIP2 as a core component of the WAVE regulatory complex in regulating actin cytoskeleton dynamics, and considering that the EMT process itself relies on cytoskeletal remodeling-driven phenotypic switching [23], we further assessed the distribution and polymerization state of filamentous actin (F-actin) using phalloidin staining. These results demonstrated that TGF-β1 treatment led to pronounced aberrant F-actin rearrangement in HK-2 cells, which was significantly suppressed by CYFIP2 silencing (Fig. S4d). In summary, our findings indicated that CYFIP2 plays a significant regulatory role in the mechanism of renal fibrosis in vitro.

Tubule-specific deletion of CYFIP2 attenuated renal tubular senescence in DOCA/salt-induced hypertensive mice

In addition to EMT and fibrosis, cellular senescence has been recognized as a key driver of CKD [24]. To clarify the role of CYFIP2 in renal tubular senescence during hypertensive nephropathy, we systematically evaluated the effects of tubule-specific CYFIP2 deletion on the expression of cellular senescence-related markers in DOCA/salt-induced hypertensive mice. Compared with control mice, CYFIP2 knockout mice presented significantly reduced senescence-associated β-galactosidase (SA-β-gal) activity in the kidney, which was accompanied by decreased p53 expression and increased levels of the anti-ageing protein Klotho (Fig. 4a). Western blot analysis further confirmed that CYFIP2 deficiency decreased the expression of the senescence-associated proteins p53 and p21 but increased the expression of Klotho (Fig. 4b). Furthermore, qRT‒PCR analysis confirmed significantly lower renal mRNA levels of SASP markers (IL-1β, TNF-α, IL-6, and CCL2) in Cre⁺/CYFIP2^fl/fl mice than in control mice (Fig. S3). These findings suggested that DOCA/salt treatment induced renal senescence and that this process could be effectively ameliorated by the tubule-specific deletion of CYFIP2

Fig. 4. CYFIP2 deficiency mitigated tubular senescence.

a SA-β-gal, p53 and Klotho stainings were performed to assess senescence in tubular epithelial cells. Scale bar: 50 μm (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. b Western blot analysis of p53, p21, and Klotho expression in the kidney from different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. c Western blot analysis of p53, p21, and Klotho expression in HK-2 cells after the different treatments (n = 6). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. d Representative images and quantitative analysis of SA-β-gal staining in HK-2 cells after the different treatments (n = 6). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. e Experimental scheme for cell treatment: after 24 h treatment of HK-2 cells with TGF-β1, the drug was washed out and the cells continued in culture for 24 h. The conditioned medium (CM) was then collected and added to serum-starved NRK49F cells. f Western blot analysis of α-SMA, Collagen-1, and Vimentin expression in NRK-49F cells incubated with the culture medium of HK-2 cells with different treatments (n = 3). ***P < 0.001 versus CM from untreated HK-2 cells; ^###P < 0.001 versus CM from TGF-β1-treated HK-2 cells.

CYFIP2 contributes to kidney fibrosis by mediating cell crosstalk between senescent tubular epithelial cells and interstitial fibroblasts

To elucidate the relationship between CYFIP2-mediated tubular senescence and tubulointerstitial fibrosis, we investigated the crosstalk between senescent tubular epithelial cells and fibroblasts. In vitro, Western blot analysis revealed that knockdown of CYFIP2 downregulated the expression of the senescence markers p21 and p53 but upregulated the protein level of the anti-ageing protein Klotho (Fig. 4c). This result was consistent with that of SA-β-gal staining, which indicated that silencing CYFIP2 effectively suppressed the increase in cellular senescence induced by TGF-β1 (Fig. 4d). In addition, qRT‒PCR confirmed that CYFIP2 knockdown significantly reduced the mRNA expression and secretion of SASP markers (Fig. S5).

Given that senescent tubular cells may promote fibroblast activation through the secretion of SASP components, we further cultured NRK-49F fibroblasts using conditioned medium (CM) collected from TGF-β1-treated HK-2 cells (Fig. 4e). The results demonstrated that while CM from control cells significantly promoted fibroblast activation, CM from CYFIP2-silenced cells reversed this effect, as indicated by the markedly reduced expression of the fibroblast activation markers α-SMA, vimentin, and FN1 (Fig. 4f). These findings collectively indicated that CYFIP2 drove fibroblast activation by promoting tubular epithelial cell senescence and SASP secretion.

CYFIP2 and p53 form a positive feedback regulatory loop

To elucidate the molecular mechanism through which CYFIP2 regulates cellular senescence, we performed RNA-seq analysis on HK-2 cells subjected to TGF-β1 stimulation and compared the CYFIP2-silenced and control groups. KEGG pathway enrichment analysis revealed that the p53 signalling pathway was among the pathways most significantly regulated by CYFIP2 (Fig. 5a, b). To validate this sequencing result, we further examined p53 expression levels in both in vivo and in vitro models. The results revealed that both total p53 protein and p53 phosphorylation levels were significantly reduced in the renal tissues of CYFIP2 knockout mice (Fig. 5c) and in CYFIP2-silenced HK-2 cells (Fig. S6b), suggesting that CYFIP2 and p53 signalling are closely associated. To further investigate the regulatory relationship between these genes, coimmunoprecipitation (Co-IP) was performed, which demonstrated that stimulation with TGF-β1 markedly increased the binding of CYFIP2 and p53 (Fig. 5d). Analysis of the p53 protein half-life using the protein synthesis inhibitor cycloheximide (CHX) revealed that CYFIP2 silencing accelerated p53 degradation, suggesting that CYFIP2 enhances p53 protein stability (Fig. 5e). Given that the ubiquitin–proteasome pathway is a major route for p53 degradation [25], this hypothesis was further tested in the presence of the proteasome inhibitor MG132. Treatment with MG132 significantly reversed the reduction in p53 protein levels induced by CYFIP2 knockdown (Fig. S6b), indicating that CYFIP2 may regulate p53 stability via the proteasome-mediated degradation pathway. To further verify this, p53 ubiquitination levels were examined, which were found to be significantly increased in CYFIP2-silenced HK-2 cells (Fig. 5f). These results further support that CYFIP2 likely maintains p53 stability by inhibiting its ubiquitination-dependent degradation. We hypothesized that since CYFIP2 has been reported to be a transcriptional target of p53 [16], these genes may form a bidirectional positive feedback loop. Intervention experiments in HK-2 cells revealed that the p53 agonist Nutlin-3a significantly upregulated both the mRNA and protein expression of CYFIP2, whereas the p53 inhibitor Pifithrin-α had the opposite effect (Fig. S6c, d). Taken together, these data demonstrate a bidirectional positive feedback regulation between CYFIP2 and p53: p53 transcriptionally activates CYFIP2 expression, whereas CYFIP2 enhances p53 stability through direct binding and inhibition of its ubiquitination-mediated degradation.

Fig. 5. CYFIP2 and p53 form a positive feedback regulatory loop.

a KEGG enrichment analysis of RNA-sequence results. The p53 signalling pathway is the primary pathway impacted by CYFIP2 deficiency in HK-2 cells. b Heat map  showing p53 expression in HK-2 cells after the different treatments (n = 3). c Western blot analysis of p-p53 and p53 expression in the kidney from different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. d Immunoprecipitation of cell lysates showing endogenous CYFIP2 interaction with endogenous p53. Negative control using isotype matched normal IgG (rabbit) was done to check for antibody specificity. e Western blot analysis of p53 expression in HK-2 cells pretreated with TGF-β1 for 24 h then stimulated with CHX for 0 h, 2 h, 4 h, and 8 h. f In vitro ubiquitination assay was conducted to detect the ubiquitination of p53 after the different treatments (n = 3). g Representative images of IHC staining of YAP in the kidney from different groups of mice (n = 6). Scale bar: 50 μm. h Western blot analysis of p-LATS1, LATS1, p-MST1, MST1, p-YAP, and YAP expression in the kidney from different groups of mice (n = 6). ***P < 0.001 versus sham-operated WT mice, ^###P < 0.001 versus DOCA/salt-treated WT mice. i Western blot analysis of p-LATS1, LATS1, p-MST1, MST1, p-YAP, and YAP expression in HK-2 cells after the different treatments (n = 3). ***P < 0.001 versus PBS treated cells transfected with scramble siRNA, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. j Representative immunofluorescent images showing the expression of YAP in HK-2 cells after the different treatments (n = 6). Scale bar: 20 μm.

CYFIP2 deficiency activated the Hippo pathway

Having established the CYFIP2/p53 positive feedback axis, we further investigated its downstream effectors. Our RNA-seq data indicated significant alterations in the Hippo signalling pathway. This, together with our prior observation of marked YAP activation in the DOCA/salt-induced hypertensive mice, led us to focus on this pathway [26]. The Hippo pathway plays a pivotal role in regulating tissue homeostasis, fibrosis, and senescence, and aberrant nuclear translocation of its core effector YAP is a key mechanism driving the progression of kidney disease [27–29]. We therefore hypothesized that the CYFIP2/p53 axis may promote disease progression by suppressing the Hippo pathway and activating YAP. To test this hypothesis, we systematically assessed Hippo pathway activity in both in vivo and in vitro models. Immunohistochemical staining revealed a substantial reduction in YAP nuclear localization in CYFIP2 knockout mice (Fig. 5g). Western blot analysis showed significantly increased phosphorylation levels of LATS1, MST1, and YAP, along with decreased total YAP protein expression, in the kidney tissues of CYFIP2 knockout mice compared with controls (Fig. 5h). In vitro, CYFIP2 knockdown in TGF-β1-treated HK-2 cells effectively reversed the TGF-β1-induced suppression of LATS1, MST1, and YAP phosphorylation (Fig. 5i) and significantly downregulated the mRNA expression of the YAP target genes CTGF and CYR61 (Fig. S7). Immunofluorescence further confirmed that CYFIP2 knockdown inhibited YAP nuclear translocation (Fig. 5j). Collectively, these results demonstrate that CYFIP2 deficiency relieves the inhibition of the Hippo pathway, increases its kinase cascade, promotes YAP phosphorylation and cytoplasmic retention, and ultimately suppresses its profibrotic and prosenescent transcriptional activity.

The CYFIP2/p53 axis is a key regulator in hypertensive renal injury

P53 and the Hippo signalling pathway form a complex regulatory network in kidney disease [30]. To investigate whether p53 mediates the regulatory effect of CYFIP2 on the Hippo pathway, we designed functional rescue experiments targeting the p53 pathway. Using HK-2 cells as an in vitro model and Cre^⁺/CYFIP2^fl/fl mice as an in vivo model, we used the p53-specific agonist Nutlin-3a for intervention. In vitro, HK-2 cells were stimulated with Nutlin-3a (Fig. S8a). Western blot analysis revealed that phosphorylation levels of the core Hippo pathway kinases LATS1 and MST1, as well as the downstream effector YAP, were significantly downregulated (Fig. 6a). Immunofluorescence staining further confirmed that Nutlin-3a intervention effectively reversed the CYFIP2 deficiency-induced blockade of YAP nuclear localization (Fig. S8c). With respect to the animal model, Cre⁺/CYFIP2^fl/fl mice were first subjected to slow-release DOCA pellet implantation to establish a hypertensive renal injury model. From days 1 to 21 postmodelling, the mice were administered 20 mg/kg Nutlin-3a via gavage every 48 h (Fig. 6b). Subsequent analyses indicated that the activation level of the Hippo signalling pathway in mouse renal tissues was significantly suppressed by this agonist (Fig. 6c, d). Further phenotypic analysis showed that, in the animal model, the agonist also reversed the protective effects of CYFIP2 deficiency against renal fibrosis and cellular senescence, manifested as increased renal collagen deposition and upregulated expression of senescence-associated markers (Fig. 6d, e). In vitro, Nutlin-3a treatment attenuated the inhibitory effects of CYFIP2 deficiency on renal tubular epithelial cell fibrosis and cellular senescence (Fig. 6f, S9). To determine whether p53-mediated regulation is involved in the regulatory effect of CYFIP2 on the cytoskeleton, we further validated the effect of Nutlin-3a using phalloidin staining. The results revealed that Nutlin-3a treatment failed to reverse the inhibitory effect of CYFIP2 silencing on TGF-β1-induced F-actin rearrangement (Fig. S4d). This finding suggests that the regulation of the actin cytoskeleton by CYFIP2 may be independent of the p53 signalling pathway. In summary, our results indicated that the CYFIP2/p53-Hippo/YAP signalling axis plays a central regulatory role in hypertension-induced renal injury, whereas the regulation of the cytoskeleton by CYFIP2 involves a p53-independent parallel mechanism, both of which contribute to disease progression.

Fig. 6. The CYFIP2/p53 axis is a key regulator in hypertensive renal injury.

a Western blot analysis of p-LATS1, LATS1, p-MST1, MST1, p-YAP and YAP expression in HK-2 cells after the different treatments (n = 3). ***P < 0.001 versus the corresponding control. ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA, ⁺⁺⁺P < 0.001 versus TGF-β1-treated cells transfected with CYFIP2 siRNA. b Schematic diagram showing Nutlin-3a treatment in DOCA/salt-induced hypertensive mice. c Western blot analysis of p-LATS1, LATS1, p-MST1, MST1, p-YAP and YAP expression in the kidney from different groups of mice (n = 3). ***P < 0.001 versus vehicle-treated mice. d Representative images of immunohistochemical staining for α-SMA, FN1, p53, and YAP, and Masson staining in kidneys from the different groups of mice. Arrows indicate positive staining. Scale bar: 50 μm (n = 3). ***P < 0.001 versus vehicle-treated mice. e Western blot analysis of FN1, Collagen-1, p53, and p21 expression in the kidney from different groups of mice (n = 3). ***P < 0.001 versus vehicle-treated mice. f Western blot analysis of FN1, Collagen-1, α-SMA, CTGF, Vimentin, Slug, p53, p21 and Klotho expression in HK-2 cells after the different treatments (n = 3). ***P < 0.001 versus the corresponding control. ^#P < 0.05, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA, ⁺P < 0.05, ⁺⁺⁺P < 0.001 versus TGF-β1-treated cells transfected with CYFIP2 siRNA.

Pharmacological inhibition of p53 alleviated TGF-β1-induced senescence and fibrosis in vitro

Building on the established role of the CYFIP2/p53 axis, we next asked whether inhibiting p53 could mimic the protective effects of CYFIP2 loss. We found that the p53 inhibitor Pifithrin-α significantly suppressed p53 expression (Fig. S8b), promoted the phosphorylation of key Hippo pathway proteins (LATS1, MST1, and YAP), and reduced total YAP protein levels (Fig. 7a). Functionally, Pifithrin-α effectively alleviated TGF-β1-induced cellular fibrosis and epithelial-mesenchymal transition (EMT), as indicated by marked reductions in the expression of fibrotic markers (Collagen I, Fibronectin, α-SMA) and EMT-related molecules (CTGF, Vimentin, Slug) (Fig. 7b, e). Concurrently, Pifithrin-α treatment significantly suppressed cellular senescence, evidenced by decreased protein levels of p53 and p21, restored Klotho expression (Fig. 7c), and reduced SA-β-gal activity (Fig. 7e). Immunofluorescence further confirmed that Pifithrin-α attenuated YAP nuclear localization (Fig. 7f). In summary, these results demonstrate that pharmacological inhibition of p53 is sufficient to phenocopy, in vitro, the protective effects mediated by CYFIP2 deficiency. This not only reinforces the critical role of the CYFIP2/p53 axis in renal interstitial fibrosis and senescence but also highlights the therapeutic potential of targeting this axis to mitigate the progression of hypertensive kidney injury.

Fig. 7. Pharmacological inhibition of p53 alleviated TGF-β1-induced senescence and fibrosis in vitro.

a–d Western blot analysis of p-LATS1, LATS1, p-MST1, MST1, p-YAP, YAP, FN1, Collagen-1, p53 and p21 expression in HK-2 cells after the different treatments (n = 3). *P < 0.05, ***P < 0.001 versus the corresponding control. ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. e Representative images and quantitative analysis of SA-β-gal staining in HK-2 cells after the different treatments. Scale bar: 50 μm (n = 3). ***P < 0.001 versus the corresponding control, ^###P < 0.001 versus TGF-β1-treated cells transfected with scramble siRNA. f Representative immunofluorescent images showing the expression of YAP in HK-2 cells after the different treatments. Scale bar: 20 μm (n = 3).

Discussion

Hypertension serves as a critical driver in the onset and progression of chronic kidney disease (CKD) and is closely associated with cellular senescence. They share underlying pathological mechanisms such as inflammation and oxidative stress, collectively promoting the decline of vascular and renal structure and function [31, 32]. The kidneys, being highly reliant on energy metabolism and protein clearance, are particularly susceptible to senescence [33], and progressive CKD exhibits a high degree of similarity to renal aging [34]. CYFIP2, a transcriptional target of p53 involved in apoptosis regulation, also functions as a core component of the WAVE regulatory complex (WRC) and plays significant roles in neural development and various brain disorders [17–19, 35]. Recently, its functions in Alzheimer’s disease, certain cancers, and autoimmune diseases have also been gradually uncovered [36]. The WAVE complex regulates the actin cytoskeleton and forms a fundamental basis for pathological processes in the kidney, including fibroblast activation and epithelial-mesenchymal transition [37]. Furthermore, CYFIP2 can enhance T-cell adhesion, potentially participating in immune-stromal interactions [38]. However, the expression pattern and functional role of CYFIP2 in renal tissues, particularly in the context of hypertension-associated kidney injury, remain unclear.

This study provides the first systematic investigation into the role of CYFIP2 in the context of hypertensive nephropathy. In both the DOCA/salt-induced hypertensive mouse model and human renal biopsy tissues from hypertensive patients, we observed significant upregulation of CYFIP2 in the renal proximal tubules. More importantly, in the DOCA/salt-induced hypertensive model, the marked increase in tubular CYFIP2 was positively correlated with the expression of fibrosis markers fibronectin and α-SMA, further suggesting an important role for CYFIP2 in renal fibrosis. Our findings confirm that renal tubular epithelial cells play a critical role in hypertensive nephropathy. As the major constituent of the kidney, these cells are susceptible to injury and may enter a state of maladaptive repair, acquiring fibroblast-like functions that ultimately contribute to renal fibrosis [39]. To further clarify its function, we generated tubule-specific CYFIP2 deletion mice and demonstrated that CYFIP2 deficiency significantly ameliorated hypertension-induced renal dysfunction, collagen deposition, and the process of epithelial-mesenchymal transition (EMT). In vitro, when TGF-β1-stimulated HK-2 cells were used to mimic a profibrotic microenvironment, silencing CYFIP2 similarly reversed the TGF-β1-induced fibrotic and senescent phenotypes. Collectively, these results indicate that CYFIP2 plays key profibrotic and prosenescent roles in the stress response of renal tubules.

Notably, senescent renal tubular epithelial cells drive the activation of interstitial fibroblasts and extracellular matrix (ECM) deposition in a paracrine manner through the secretion of the senescence-associated secretory phenotype (SASP), which has been recognized as a central link in the process of renal fibrosis [40]. Accumulating evidence indicates a close association between renal tubular epithelial cell senescence and the progression of renal fibrosis [34]. For example, the aryl hydrocarbon receptor (AHR) accelerates renal aging and fibrosis by inhibiting mitochondrial biogenesis [41]. Histone modification H3K9cr is involved in the process of renal tubular senescence by regulating macrophage activation [37]. In addition, WNT9a has been reported to directly promote senescence in renal tubular epithelial cells, thereby driving the progression of fibrosis [42]. Regarding therapeutic strategies, targeting renal tubular senescence has demonstrated potential value. Studies have shown that mesenchymal stem cell-derived exosomal miR-125b-5p promotes tubular repair by suppressing the p53 signalling pathway [43]. The clearance of senescent renal epithelial cells has also been confirmed to effectively alleviate kidney injury [44]. Moreover, knockout of p21 and p16 genes has been shown to effectively prevent tubular senescence and fibrotic changes in CKD [45, 46]. Collectively, these findings support the potential of tubular senescence-targeted therapeutic strategies in CKD. In this study, we found that CYFIP2 promoted fibroblast activation by facilitating renal tubular epithelial cell senescence and SASP secretion, underscoring the importance of CYFIP2 in driving tubular senescence and renal fibrosis.

Having established the pro-senescent and pro-fibrotic functions of CYFIP2, we further explored its underlying molecular mechanisms. The Hippo signalling pathway is central to regulating tissue homeostasis, fibrosis, and cellular senescence [47]. When activated, the core kinases LATS1/2 mediate the phosphorylation of YAP/TAZ, leading to their cytoplasmic retention and degradation. In contrast, pathway inactivation allows YAP/TAZ to translocate into the nucleus, where they act as transcriptional co-activators by binding to transcription factors such as TEAD, upregulating target genes including CTGF and CYR61, thereby promoting cell proliferation and fibrosis [47]. Aberrant YAP activation is observed in both UUO models and renal tissues from CKD patients [48], and its hyperactivation can induce DNA damage, activate p53, upregulate p21, and trigger p53-dependent cellular senescence, highlighting its critical role in renal aging [49]. The Hippo pathway is a potential therapeutic target: verteporfin (a YAP inhibitor) ameliorates renal fibrosis by disrupting YAP–TEAD interaction [50], and the FXR agonist GW4064 alleviates renal interstitial fibrosis in UUO models by activating MST1/LATS1 to promote YAP phosphorylation [51]. Our in vivo and in vitro experiments identified CYFIP2 as a key regulator of the Hippo pathway: CYFIP2 deletion/silencing (in DOCA/salt-induced hypertensive renal injury or TGF-β1-stimulated HK-2 cells) significantly activated the Hippo kinase cascade, enhancing LATS1/MST1 phosphorylation, promoting YAP phosphorylation, and inhibiting its nuclear translocation. Notably, this regulation relies on close crosstalk between the Hippo pathway and p53 [30]. LATS2 can bind MDM2 to prevent p53 ubiquitination/degradation and enhance its stability [30] and Rac1 GTPase participates in fibrosis via the EGFR-Hippo/YAP/TAZ-p53 axis [52]. These findings outline a complex p53–Hippo regulatory network, but its upstream initiating mechanisms in hypertensive nephropathy remain unclear. Building on this, our study for the first time revealed that the CYFIP2/p53 positive feedback loop served as the key upstream event driving p53-Hippo pathway activation. Specifically, in the context of hypertensive nephropathy, p53 transcriptionally upregulated CYFIP2 expression. CYFIP2, in turn, directly bound to p53 and enhanced its protein stability by inhibiting its ubiquitination-mediated degradation. Functionally, p53 agonist treatment reversed both the CYFIP2 deficiency-induced activation of the Hippo pathway and its associated renal protective effects, while p53 inhibition phenocopied the protective effects observed upon CYFIP2 loss. These results collectively demonstrated that the CYFIP2/p53-Hippo/YAP signalling axis constituted a central regulatory mechanism governing the progression of hypertensive renal injury.

Notably, this study has certain limitations. First, the spatiotemporal specificity of genetic manipulation could be improved. Although the renal tubule-specific knockout model mediated by Cdh16-Cre used in this study could clarify the function of CYFIP2 in proximal tubules, nonspecific recombination may occur during mouse development, preventing precise control of the knockout timeframe. Future studies should employ doxycycline- or tamoxifen-inducible conditional knockout systems to achieve spatiotemporally specific CYFIP2 deletion in adult mice, allowing for a more precise assessment of its role at different stages of hypertensive nephropathy. Second, the renal tubule-specific deletion of CYFIP2 in this study did not significantly affect mouse blood pressure, suggesting that the regulation of blood pressure by CYFIP2 may be specific to the cell type. Subsequent research could further establish CYFIP2-specific knockout mouse models in other key renal cell lineages (such as endothelial cells or podocytes). In combination with various hypertensive animal models, this would allow for a systematic investigation of the function and mechanisms of CYFIP2 in different renal cell types and under different hypertensive pathological contexts.

In summary, to our knowledge, our results demonstrated for the first time the central role of the CYFIP2/p53–Hippo/YAP signalling axis in renal tubular senescence and fibrosis in hypertensive nephropathy. This pathway drives disease progression by coordinating the promotion of both cellular senescence and fibrosis. Consequently, targeting these signalling pathways at multiple levels is a promising novel therapeutic strategy for hypertensive nephropathy.

Author contributions

MCS conducted the in vivo and in vitro experiments, performed data analysis, and wrote the manuscript. FWZ and YZW performed in vivo animal studies. HSL and HRZ performed in vitro experiments. XLR and MBZ analyzed the data. HLL performed the confocal microscopy. CCL, CWS and JCW designed the experiment, interpreted the data, wrote the manuscript, and approved the final version of the manuscript for publication.

Funding

This study was supported by the National Nature Science Foundation of China (82170734) and the Shandong Provincial Natural Science Foundation, China (ZR2023LSW013, ZR202111290287, ZR2022QH109, and ZR2023MH077, ZR2025QC1660).

Data availability

Detailed descriptions of the experimental methods are presented in the Supplemental Material. The major resource table is also included in the Supplemental Material. The data that support the findings of this study are available within the article, in the Supplemental Material, or from the corresponding author upon reasonable request. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in the National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA014573), which are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-026-01793-x.