Ubiquitin-specific protease 11 facilitates the activation and proliferation of renal interstitial fibroblasts through epidermal growth factor receptor signaling pathways

Abstract

Chronic kidney disease is a worldwide health problem, and its incidence is on the rise. It has previously been shown that ubiquitin-specific protease 11 (USP11) promotes partial epithelial-to-mesenchymal transition in uric acid-stimulated tubular epithelial cells during renal fibrosis. However, its specific function and mechanism in renal interstitial fibroblasts is still unknown. In this study, we found that uric acid could also upregulate the USP11 levels in cultured fibroblasts (NRK-49F) under dose gradient and time gradient, with an approximately 3–4 fold increase. Genetic and pharmacological depletion of USP11 blocked activation of renal interstitial fibroblasts leading to an approximately 70–90% reduction in α-SMA and collagen I expressions. USP11 increased the phosphorylation level of epidermal growth factor receptor, thereby activating its downstream signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/protein kinase B and mitogen-activated protein kinase /extracellular regulated protein kinases, which further activated nuclear transcription factors, Snail and Slug. Moreover, USP11 could also affect the proliferation and migration of NRK49F, by upregulating the expression levels of proliferating cell nuclear antigen and cyclin E. Thus, our study offers comprehensive evidence that USP11 is a promising target for kidney fibrosis and that inhibiting USP11 could be an effective strategy for treating chronic kideny disease.

Introduction

Chronic kidney disease (CKD) is a progressive condition characterized by a gradual decline in kidney function over time. It affects millions of people worldwide and is a significant public health concern due to its association with increased morbidity, mortality, and healthcare costs. End-stage renal disease (ESRD) represents the final stage of CKD, where kidney function is critically impaired, requiring either dialysis or kidney transplantation to sustain life. Over the past 20 years, the global mortality associated with CKD has increased by 82.3%. Particularly concerning is the fact that the 2-year survival rate for patients with CKD stage 4 and 5 is only 62%, with an annual mortality rate exceeding 20%, which is ten times higher than that of the general population [1,2]. By 2040, CKD is projected to become the fifth leading cause of death worldwide, resulting in 2.2 to 4 million deaths annually [1]. The increasing global prevalence underscores the need for improved prevention, early detection, and treatment strategies.

Renal interstitial fibrosis is one of the important pathological changes in CKD, which gradually leads to the loss of kidney function. This process refers to the excessive accumulation of extracellular matrix (ECM) proteins in renal interstitial tissue, leading to thickening and scar formation [3,4]. The activation of renal interstitial fibroblasts (RIF) plays a crucial role in kidney fibrosis. Fibroblasts are the main cell type in the renal interstitium, responsible for synthesizing and maintaining ECM. Under normal circumstances, these cells help maintain the structure and function of kidney tissue. However, in cases of CKD or kidney injury, fibroblasts become activated and transform into myofibroblasts [5]. Excessive matrix accumulation caused by fibroblast activation gradually replaces normal renal tubular tissue, leading to a gradual decline in renal function [3,4]. Overall, the activation of fibroblasts is the core mechanism in the process of kidney fibrosis, directly affecting the progression of the disease and the damage to renal function.

Uric acid (UA) has been increasingly recognized as an important pathogenic factor in the progression of CKD. Sustained hyperuricemia contributes to the development of hyperuricemic nephropathy and is closely associated with renal tubular injury and interstitial fibrosis. Previous studies have demonstrated that elevated UA levels can induce epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells by activating profibrotic signaling pathways, thereby promoting renal fibrogenesis [6,7]. Meanwhile, prior researches have also demonstrated that uric acid stimulation can activate renal interstitial fibroblasts [8]. Therefore, UA stimulation is widely used as an in vitro model to mimic CKD-related tubular injury, EMT processes and renal interstitial fibroblasts activation.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor located on the cell membrane that binds to ligands such as epidermal growth factor (EGF), whose abnormal activation contributes to multiple fibrotic diseases, including liver fibrosis [9], pulmonary fibrosis [10], cardiac fibrosis [11] and kidney fibrosis [12]. Our previous research has confirmed that activation of EGFR signaling pathway promotes the occurrence and development of kidney fibrosis in both unilateral ureteral obstruction (UUO) and hyperuricemic nephropathy (HN) murine models. In cultured renal interstitial fibroblasts (NRK-49F cell line), exposure to TGF-β1 up-regulates EGFR and its downstream signaling pathways, and ultimately leads to the activation of NRK-49F [13]. Genetic or pharmacologic blockade of EGFR effectively inhibits kidney fibrosis and improves renal function [13,14]. So, we point out that inhibition of EGFR may have therapeutic potential for fibrotic kidney disease [13,14]. Meanwhile, both of PI3K/AKT [15,16] and MEK/ERK [17,18], two downstream signaling pathways of EGFR, have been proven to be involved in the process of kidney fibrosis. Furthermore, multiple reports indicate that nuclear transcription factors, Snail and Slug, complete crucial signal transduction in NRK49F activation, proliferation and migration during kidney fibrosis [19–21].

Ubiquitin-specific protease 11 (USP11) is a deubiquitinase enzyme that plays a crucial role in regulating protein stability and degradation by removing ubiquitin chains from proteins [22]. USP11 is involved in key biological processes such as DNA repair, cell cycle regulation, signal transduction, and associated with various diseases, including cancer, chronic inflammation and fibrotic diseases [22]. Our recent study has reported the first relation between USP11 and kidney fibrosis that USP11 is highly upregulated in the kidneys from patients with CKD and causes kidney fibrosis in both HN- and folic acid (FA)- induced mouse models [23]. USP11 deletion has anti-fibrotic and anti- inflammatory kidney effects with the main mechanism of partial epithelial-to-mesenchymal transition in tubular epithelial cells (TECs) [23]. Lu et al. have also confirmed the promoting kidney fibrosis effect of USP11 via renal tubular cell senescence [24] and renal tubular cell pyroptosis [25]. However, these studies all focus on the role of USP11 in TECs, and its exact function and mechanism in RIF has not yet been reported.

In the current study, we emphasize that USP11 has emerged as a potential regulator of renal interstitial fibrosis. Its activity may influence key EGFR signaling pathways involved in RIF activation, proliferation, and migration. These findings suggest that USP11 could serve as a novel therapeutic target for patients with CKD, potentially complementing existing treatments such as EGFR inhibitors. To our knowledge, this study provides the first evidence supporting a role for USP11 in the activation of RIF during kidney fibrosis, highlighting its translational relevance.

Materials and methods

Antibodies and reagents

Antibodies to p-EGFR (#3777), p-ERK1/2 (#4370), ERK1/2 (#4695), p-Akt (#4060), Akt (#9272), p-MEK1/2 (#9154), MEK1/2 (#8727), Snail (#3879), Cyclin E (#20808) were purchased from Cell Signaling Technology (Dancers, MA). Antibodies to GAPDH (sc-32233) and PCNA (sc-56) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to USP11 (ab109232) and Slug (ab27568) were purchased from Abcam (Cambridge, MA). Antibodies to p-PI3K (AF3241) and PI3K (AF6241) were purchased from Affinity (Affinity Biosciences, United States). Antibody to collagen I (GB11022) was purchased from Servicebio (Wuhan, China). Antibody to EGFR (A11351) was purchased from Abclonal (Wuhan, China). Antibodies to Flag (AF2852), Myc (AF2864) and the Cell Counting Kit-8 (CCK-8) proliferation assay kit was purchased from Beyotime Biotechnology (Haimen, China). MTX, Cycloheximide (CHX), Mitomycin C and gefitinib were purchased from Selleckchem (Houston, TX). FITC and Texas Red for immunofluorescent staining were purchased from Thermo Fisher Scientific (Waltham, MA). EGF protein was purchased from R&D systems (Minneapolis, MN). USP11 siRNA, USP11-Flag and EGFR-Myc tagged expression plasmids were purchased from GenePharma (Shanghai, China). USP11-pcDNA 3.0 plasmid was purchased from Genewiz (Shanghai, China). Lipofectamine 3000 was purchased from Invitrogen (Grand Island, NY). Antibody to α-SMA (A2547), uric acid, secondary antibodies for Western blot, and all other chemicals were purchased from Sigma (St. Louis, MO).

Cell culture and treatment

Rat renal interstitial fibroblast (NRK49F) cells were obtained from ATCC (Manassas, VA). NRK49F cells were cultured and propagated in DMEM with F-12 containing 10% FBS, and 1% penicillin and streptomycin in an atmosphere of 5% CO2-95% air at 37 °C. The newly recovered cells were propagated for at least three generations before they were used in experiments. According to the manufacturer’s instructions, UA was dissolved in 1 M NaOH to prepare a stock solution. The solution was then sterile-filtered and adjusted to physiological pH (7.4) before being diluted to the working concentrations for cell treatment. Firstly, we stimulated NRK49F with UA in dose-dependent manner (0, 200, 400, 800 μM) and in time-dependent manner (0, 12, 24, 36 h). The immunoblot results showed that stimulation with uric acid dose dependently and time dependently increased expression of α-SMA and collagen I, with the maximum induction at a concentration of 800 μM for 36 h. On this basis, we selected 800 μM of uric acid (36 h) to examine the effect of USP11 on uric acid-induced RIF activation. NRK49F cells were starved for 24 h with DMEM containing 0.5% FBS before they were exposed to uric acid in the presence or absence of MTX (1, 5, and 10 μM). To fully demonstrate the relationship between USP11 and EGFR signaling, we stimulated starved NRK49F cells with EGF (5 ng/ml) in the presence or absence of USP11 siRNA or treated with gefitinib (1 nM and 5 nM), a highly selective EGFR inhibitor in the presence of USP11 pcDNA 3.0 plasmid for an additional 36 h. At 36 h after the treatment, cells were harvested for immunoblot analysis. All of the in vitro experiments were repeated for at least three times.

Transfection of plasmid or siRNA

The plasmid USP11-pcDNA 3.0 was constructed by inserting a PCR-cloned USP11-gene cDNA into pcDNA3.0 vector (Genewiz, Shanghai, China) and was then verified by sequencing. And the USP11 siRNA, USP11-Flag and EGFR-Myc tagged expression plasmids were synthesis by GenePharma (Shanghai, China). Transfection of plasmid and siRNA was performed according to the manufacturer’s protocol, respectively. Briefly, NRK49F cells were seeded to 70-80% confluence in the antibiotic-free medium and grown followed by transfection with USP11 siRNA (60 pmol) or plasmids using Lipofectamine 3000 (CA, USA). In parallel, scrambled siRNA (60 pmol) or plasmid was used as a control for off-target changes in NRK49F cells. After transfection, the medium was changed to DMEM with F12 containing 0.5% FBS for starvation and then cells were incubated with or without uric acid (800 μM) for an additional 36 h before being harvested for analysis. Immunoblotting analysis showed that siRNA transfection reduced USP11 expression by approximately 70%-90%, whereas overexpression increased USP11 levels by approximately 50%-60%, indicating that the transfection efficiency was sufficient for subsequent experiments. All of the in vitro experiments were repeated for at least three times.

Co-immunoprecipitation (co-IP)

NRK49F cells grown in 10 cm dishes were washed with PBS, then lysed on ice for 30 min in 1 mL cold low‐stringency lysis buffer. Lysates were centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was collected and incubated with Flag antibody, Myc antibody or IgG, followed by addition of 20 µL A/G PLUS‐Agarose beads (SC‐2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA) with rotation at 4 °C overnight. Subsequently, the beads were washed five times with a 1 mL low‐stringency lysis buffer between each wash at 2500 g for 5 min at 4 °C. The wash buffer was discarded, and 50 µL of 1× SDS‐PAGE loading buffer was added to the immune complexes, which were then denatured at 100 °C for 10 min. Subsequently, the samples were processed for western blot analysis as described above.

CHX chase assays

For CHX chase assays, cells were transfected with Scramble siRNA or USP11 siRNA, followed by stimulation with uric acid. Subsequently, CHX (10 µg mL⁻¹) was added to inhibit de novo protein synthesis. Cells were harvested at 0, 6, 12, and 24 h after CHX treatment. Protein levels were determined by immunoblotting and quantified by densitometric analysis. All of the in vitro experiments were repeated for at least three times.

Cell lysis

Following the aforementioned cellular treatments, cells were washed with cold PBS. Cells were then collected and lysed in 120 μl of lysis buffer, which consisted of 20 mM Tris HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM beta-glycerophosphate, 1 mM MgCl₂, 1% Trton X-100, 1 mM sodium orthovanadate, 10 μg/ml protease inhibitors, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. The cell lysates were then sonicated on ice for 12 s, followed by centrifugation at 4 °C at 12,000 r.Pm for 15 min. Protein concentrations of the samples were then determined with use of a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) using BSA as a standard. The samples were prepared for immunoblotting analysis.

Immunoblotting analysis

Samples were all equilibrated to 20 μg and run on a 4–15% SDS-PAGE gel, transferred to a nitrocellulose membrane, and blocked in 5% nonfat biological grade powdered milk dissolved in 25 mM Tris HCl (pH 7.4), 137 mM NaCl, and 0.1% TWEEN20 (TBST) for 30 min. Blots were washed with TBST and incubated with primary antibody in 5% BSA with TBST for 1 h or overnight. The membranes were then washed three times at 10 min intervals with TBST prior to addition of secondary antibody for 1 h. Blots were developed with BeyoECL Plus Chemiluminescence Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instruction. The densitometry analysis of immunoblot results was conducted by using ImageJ software (National Institutes of Health, Bethesda, MD).

Immunofluorescent staining

After different stimulation and treatment, NRK49F cells were fixed with 4% paraformaldehyde (PFA) for 15 min, typically at room temperature. Then, Triton X-100 was used to make the cell membrane permeable for 60 min, allowing antibodies to enter the cell interior. Cells were incubated with diluted primary antibody, including USP11 (sc-365528, Santa Cruz), Fibronectin (ab2413, Abcam), Vimentin (GB111308, Servicebio), EGFR (A11351, Abclonal), Ki67 (84432-1-RR, Proteintech) and Snail (GB11260, Servicebio), overnight at 4 °C or according to the manufacturer’s recommendations. Cells were washed thrice in PBS and then incubated with secondary antibodies for 1 h at room temperature. Finally, the nuclei were stained with DAPI (1 μg/mL, Abcam) for 15 min at room temperature before visualization. Cells were observed with ZEISS Digital Camera for Fluorescence Microscopy.

CCK-8 proliferation assay

The CCK-8 proliferation kit was used according to the manufacturer’s instructions. NRK-49F cells were starved for 24 h with DMEM containing 0.5% FBS and then exposed to uric acid (800 μM) in the presence or absence of USP11 siRNA. After 36 h, the original culture medium was removed, and 100 μL new DMEM/F12 medium containing 10 μL CCK-8 were added to each well in a 96-well plate for 37 °C incubation for an additional 4 h. The final optical density values were read at 450 nm.

Wound healing assay

NRK49F cells were seeded in a 6-well plate and allowed to reach 90% confluence. To specifically assess cell migration, we used Mitomycin C to inhibit cell proliferation. Mitomycin C is a DNA cross-linking agent that effectively block cell proliferation in migration assays [26]. NRK-49F cells were pretreated with Mitomycin C (0.05 μg/mL) for 1–2 h prior to wound scratching, as commonly reported, and all experimental groups were exposed to Mitomycin C under the same conditions. A scratch wound was created on the cell surface using a micropipette tip. Then, cells were washed with phosphate buffer saline (PBS) three times and incubated in UA medium with or without USP11 siRNA transfection. Photomicrographs of migrating cells were taken at 0h and 36h. The width of the wound was measured by ImageJ software (National Institutes of Health, Bethesda, MD). The migratory rate was calculated as (A-B)/A*100%, where A and B reflect the width of the wound at 0h and 36h respectively. Four random and independent fields of view were calculated for each sample.

Statistical analysis

We conducted all the experiments at least three times. Data depicted in graphs represent the means ± SD. An intergroup comparison was made using one-way analysis of variance. Multiple means were compared using Tukey’s test. The differences between the two groups were determined by Student’s T-test. The statistically significant difference between mean values was marked in each graph. p < 0.05 is considered significant. The statistical analyses were conducted by using IBM SPSS Statistics 20.0 (IBM, Armonk, NY).

Results

USP11 is highly upregulated in UA-stimulated RIF

Our previous study has shown that UA increases the expression level of USP11 in cultured renal tubular epithelial cells (HK2) exposed to UA, and USP11 contributes to partial EMT and inflammatory factors release [23]. However, the expression level and exact role of USP11 in RIF under pathological stimulation are unknown. Therefore, we first used UA under dose (0, 200, 400, 800 μM) and time (0, 12, 24, 36 h) gradients to stimulate NRK49F cells. The immunoblot results showed that stimulation with UA dose dependently (Figure 1(A,B)) and time dependently (Figure 1(C,D)) increased expression of USP11 in cultured NRK49F, with the maximum induction at a concentration of 800 μM for 36 h. Meanwhile, UA stimulation caused the activation of RIF with the increased expression of α-SMA and collagen I (Figure 1(A–D)). Collectively, these data indicate that USP11 is highly upregulated in UA-stimulated RIF, and may be related its activation.

Figure 1.

USP11 is highly upregulated in UA-stimulated RIF. In vitro, we stimulated NRK49F with UA in dose-dependent manner (0, 200, 400, 800 μM) and in time-dependent manner (0, 12, 24, 36 hours). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (A, C). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (B, D). Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ^****p < 0.0001.

USP11 contributes to RIF activation in the UA-stimulated NRK49F

To further confirm the role of USP11 in stimulated NRK49F cells, we detected RIF activation through positive or negative regulation of USP11 in vitro. Immunoblot analysis demonstrated that USP11 knockdown via siRNA transfection reduced α-SMA and collagen I expression by approximately 70–90% following UA stimulation (Figure 2(A,B)). Immunofluorescence also presented the decreased number of Fibronectin- and Vimentin-labeled positive cells after transfection with USP11 siRNA (Figure 2(C)). On the other hand, we further used small molecule inhibitor to suppress the expression of USP11, in order to validate the results of siRNA inhibition. Mitoxantrone (MTX) is a chemotherapeutic drug, which is approved by the US Food and Drug Administration to treat various cancers, including breast cancer, acute myeloid leukemia (AML), and multiple sclerosis (MS). Several researches have proven that MTX is capable of impacting USP11 activity and expression [23,24,27,28]. Treatment with MTX (1, 5, and 10 μM) dose dependently decreased USP11, α-SMA and collagen I expression levels in UA-stimulated NRK49F cells (Figure 2(D,E)). And then, we constructed an overexpressed pcDNA 3.0 plasmid for USP11, and validated in cell immunoblotting detection, with the significantly increased expression level of USP11 (Figure 2(F,G)). Overexpression of USP11 by plasmid transfection prominently induced NRK49F activation, characterized by increased α-SMA and collagen I expression (Figure 2(H,I)). These above-mentioned results fully confirm that USP11 contributes to RIF activation in the UA-stimulated NRK49F.

Figure 2.

USP11 contributes to RIF activation in the UA-stimulated NRK49F. NRK49F cells were seeded to 70–80% confluence in the antibiotic-free medium and grown followed by transfection with USP11 siRNA or USP11-pcDNA 3.0 plasmid. After transfection, the medium was changed to DMEM with F12 containing 0.5% FBS for starvation and then cells were incubated with or without uric acid (800 μM) for an additional 36 hours before being harvested for analysis. Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (A, F, H). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (B, G, I). Representative images and quantitative analysis of immunofluorescence staining for Fibronectin and Vimentin with DAPI nuclear counterstaining in each group (C). Then NRK49F cells were starved for 24 h with DMEM containing 0.5% FBS before they were exposed to uric acid in the presence or absence of MTX (1, 5, and 10 μM). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (D). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (E). Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, ^**** p < 0.0001. Scale bars = 100μm.

USP11 directly interacts with EGFR and stabilizes its protein level

EGFR is one of the main signaling pathways that activate RIF and contribute to kidney fibrosis ultimately [13,14]. We next investigated whether USP11 regulates the EGFR signaling pathway in UA-stimulated NRK49F. As shown in immunofluorescence co-staining, positive USP11 was overlapping with EGFR partly in the nucleus (Figure 3(A)). To address whether there is a direct interaction between USP11 and EGFR, we performed additional co-IP experiments in NRK49F cells. As shown in Figure 3(B,C), Flag-tagged USP11 and Myc-tagged EGFR directly interacted with each other, as confirmed by both forward and reverse co-IP assays. In addition, CHX chase assays were performed to assess EGFR protein stability in Figure 3(D,E). Under UA stimulation, knockdown of USP11 markedly accelerated the degradation of EGFR protein compared with Scramble siRNA, indicating that USP11 contributes to EGFR protein stability. Together, these findings demonstrate that USP11 directly interacts with EGFR to maintain its protein stability.

Figure 3.

USP11 directly interacts with EGFR and stabilizes its protein level. Immunofluorescence co-staining of USP11 and EGFR in the UA-stimulated NRK49F (A). Co-IP assay in NRK49F cells. Flag-tagged USP11 and Myc-tagged EGFR were immunoprecipitated using an anti-Flag/Myc antibody, and the presence of Myc-tagged EGFR and Flag-tagged USP11 in the precipitates was detected by immunoblotting (B, C). Western blot analysis of USP11 and EGFR protein expressions in each group were conducted, with normalization to GAPDH (D). Quantitative analysis of EGFR protein levels in each group (E). Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference, ***p < 0.001. Scale bars = 50μm.

USP11 activates EGFR signaling pathway in the UA-stimulated NRK49F

We next investigated whether USP11 regulated EGFR phosphorylation and the activation of downstream signaling pathways. UA stimulation could activate EGFR signaling molecules with the increased expression of phosphorylated EGFR (p-EGFR), while USP11 siRNA transfection markedly reduced p-EGFR levels to baseline (Figure 4(A,B)). In addition, treatment with MTX also dose dependently decreased p-EGFR level (Figure 4(C,D)). On the contrary, overexpression of USP11 upregulated the expression level of p-EGFR (Figure 4(E,F)).

Figure 4.

USP11 activates EGFR signaling pathway in the UA-stimulated NRK49F. Western blot analysis of p-EGFR and EGFR protein expressions in each group were conducted, with normalization to GAPDH (A, C, E). Quantitative analysis of p-EGFR and EGFR protein levels in each group (B, D, F). Data were expressed as mean ± SD (n = 4 for each group). N.S.: no significant difference. ***p < 0.001, ^****p < 0.0001.

We additionally used EGF to stimulate NRK49F cells in the presence or absence of USP11 siRNA, on the basis of UA stimulation. The immunoblot results showed that EGF could further upregulate EGFR signaling molecules and activate NRK49F cells, while USP11 siRNA transfection blocked EGFR activation and decreased α-SMA and collagen I expression levels to almost the same level as the control group (Figure 5(A–D)). On the other hand, we treated NRK49F cells with gefitinib (1 nM and 5 nM), a highly selective EGFR inhibitor, in the presence of USP11 pcDNA 3.0 plasmid for an additional 36 h. Gefitinib treatment could reduce the levels of α-SMA and collagen I by approximately 60–70% in the presence of USP11 pcDNA 3.0 plasmid transfection, while made no difference on the expression of USP11 (Figure 5(E–H)). Taken together with the previous co-IP and CHX experiments, these results indicate that EGFR is a downstream signaling molecule under USP11, and that USP11 regulates EGFR signaling pathway, resulting in RIF activation.

Figure 5.

USP11 is involved in the activation of EGFR signaling pathway in the UA-stimulated NRK49F. To fully demonstrate the relationship between USP11 and EGFR signaling, we stimulated starved NRK49F cells with EGF (5 ng/ml) in the presence or absence of USP11 siRNA or treated with gefitinib (1 nM and 5 nM), a highly selective EGFR inhibitor in the presence of USP11 pcDNA 3.0 plasmid for an additional 36 hours. Western blot analysis of p-EGFR and EGFR protein expressions in each group were conducted, with normalization to GAPDH (A, E). Quantitative analysis of p-EGFR and EGFR protein levels in each group (B, F). Western blot analysis of USP11, α-SMA and Collagen I protein expressions in each group were conducted, with normalization to GAPDH (C, G). Quantitative analysis of USP11, α-SMA and Collagen I protein levels in each group (D, H). Data were expressed as mean ± SD (n = 4 for each group). N.S.: no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ^****p < 0.0001.

USP11 regulates EGFR downstream signaling pathways, PI3K/AKT and MEK/ERK

Both of PI3K/AKT [15,16] and MEK/ERK [17,18], two downstream signaling pathways of EGFR, have been proven to be involved in the process of kidney fibrosis. Given that, we further verified whether PI3K/AKT and MEK/ERK were involved in the USP11-EGFR signal axis during RIF activation. In the cultured NRK49F cells, UA stimulation could increase p-PI3K and p-Akt expression levels, while inhibition of USP11 with siRNA transfection reduced the phosphorylation of PI3K and Akt to approximately 0.7- and 0.5-fold of control levels, respectively (Figure 6(A,B)). On another downstream signaling pathway, we also confirmed that inhibition of USP11 with siRNA transfection blocked the activation of MEK1/2 and ERK1/2 signaling molecules (Figure 6(C,D)). It was noteworthy that USP11 inhibition had no effect on the total protein expressions of PI3K/AKT and MEK/ERK (Figure 6(A–D)). In addition, we obtained similar results that inhibition of USP11 with MTX treatment also suppressed PI3K/AKT and MEK/ERK activation (Figure 6(E–H)). Collectively, these results suggest that USP11 can regulate EGFR downstream signaling pathways, PI3K/AKT and MEK/ERK, during RIF activation.

Figure 6.

USP11 regulates EGFR downstream signaling pathways, PI3K/AKT and MEK/ERK. Western blot analysis of p-PI3K, PI3K, p-Akt and Akt protein expressions in each group were conducted, with normalization to GAPDH (A, E). Quantitative analysis of p-PI3K, PI3K, p-Akt and Akt protein levels in each group (B, F). Western blot analysis of p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 protein expressions in each group were conducted, with normalization to GAPDH (C, G). Quantitative analysis of p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 protein levels in each group (D, H). Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001, ^****p < 0.0001.

USP11-EGFR signal axis upregulates nuclear transcription factors Snail and Slug during RIF activation

Because nuclear transcription factors are key downstream effectors of growth factor signaling, and involved in the signal transduction in NRK49F activation, proliferation and migration [19–21], we further examined the expression of Snail and Slug with or without USP11 knockdown in cultured NRK49F cells exposed to UA. Our results demonstrated that transfection with USP11 siRNA significantly inhibited UA-induced upregulation of Snail and Slug by nearly 100%, returning expression to baseline levels (Figure 7(A,B)). Immunofluorescence staining similarly showed that USP11 siRNA transfection decreased the number of Snail-positive cells from ∼200 to ∼40, corresponding to a 5-fold reduction compared with the UA stimulation group (Figure 7(C,D)). Treatment with MTX in different dose also had the inhibitory effect on both of the nuclear transcription factors, especially for Snail (Figure 7(E,F)). So, we consider that USP11-EGFR signal axis upregulates nuclear transcription factors Snail and Slug during RIF activation.

Figure 7.

USP11-EGFR signal axis upregulates nuclear transcription factors Snail and Slug during RIF activation. Western blot analysis of Snail and Slug protein expressions in each group were conducted, with normalization to GAPDH (A, E). Quantitative analysis of Snail and Slug protein levels in each group (B, F). Representative images (C) and quantitative analysis (D) of immunofluorescence staining for Snail in each group. Data were expressed as mean±SD (n = 4 for each group). N.S.: no significant difference. *p < 0.05; ^****p < 0.0001. Scale bars = 100μm.

Inhibition of USP11 attenuates the proliferation and migration in the UA-stimulated NRK49F

As we all know that the proliferation and migration ability of activated RIF are usually enhanced, causing adverse kidney fibrosis and loss of renal function [29,30]. In this part, we detected the migration ability of NRK49F through wound healing assay, and evaluated its proliferation level by CCK-8 assay. We also explored the expression levels of PCNA and Cyclin E, two proliferation marker proteins, with immunoblotting assays. The wound healing assay results showed that UA could significantly enhance the migration ability of NRK49F cells, while inhibition of USP11 with siRNA transfection impaired abnormal migration and decreased the migratory rate (Figure 8(A,B)). Mitomycin C was used to block cell proliferation, ensuring that the observed effects reflected changes in migration rather than proliferation. CCK-8 assay proved that USP11 inhibition reduced the level of proliferation in UA-stimulated NRK49F cells (Figure 8(C)). In addition, we performed additional Ki-67 immunofluorescence staining in NRK49F cells, a specific marker of cell proliferation, as shown in Figure 8(D,E). The results demonstrated that USP11 knockdown significantly reduced the number of Ki-67 positive cells. Moreover, both of USP11 siRNA transfection and MTX treatment could decreased the expression levels of PCNA and Cyclin E (Figure 8(F–I)). Taken together, these results illustrate the importance of USP11 in RIF activation and that inhibition of USP11 attenuates the proliferation and migration in the UA-stimulated NRK49F.

Figure 8.

Inhibition of USP11 attenuates the proliferation and migration in the UA-stimulated NRK49F. Photomicrographs of migrating cells in wound healing assay were taken at 0h and 36h (A). The migratory rate was calculated as (A-B)/A*100%, where A and B reflect the width of the wound at 0h and 36h respectively (B). The CCK-8 proliferation kit was used according to the manufacturer’s instructions and the final optical density values were read at 450 nm (C). Representative images and quantitative analysis of immunofluorescence staining for Ki67 with DAPI nuclear counterstaining in each group (D, E). Western blot analysis of PCNA and Cyclin E protein expressions in each group were conducted, with normalization to GAPDH (F, H). Quantitative analysis of PCNA and Cyclin E protein levels in each group (G, I). Data were expressed as mean±SD (n = 4 for each group). N.S., no significant difference. *p < 0.05, ***p < 0.001, ^****p < 0.0001. Scale bars = 500μm (A) and 50μm (D).

Discussion

Although increasing reports have confirmed that USP11 plays an important role in the EMT, cell senescence and cell pyroptosis of renal TECs [23–25], research on the mechanism pathway of USP11 in kidney fibrosis is still limited. Our findings reveal that USP11 directly binds to EGFR and regulates its protein stability, acting as a critical mediator of fibroblast activation in kidney fibrosis through EGFR phosphorylation and downstream signaling. Genetic and pharmacological depletion of USP11 blocked the activation of NRK49F, and also attenuated its proliferation and migration. This study once again reaffirms the importance of USP11 in the process of kidney fibrosis and provides a new perspective to explore the relationship between them.

TECs undergoing phenotype transformation are one of the main sources of RIF. And fibroblasts can also cause damage to epithelial cells in turn [31]. Fibroblasts alter the microenvironment of TECs by secreting multiple cytokines, chemokines, and extracellular matrix components. For example, inflammatory factors such as TGF-β, IL-1β, TNF-α secreted by fibroblasts not only stimulate damage and death of TECs, but also promote their EMT process [32]. In addition, the activation of fibroblasts also affects the filtration and reabsorption capacity of the kidneys by regulating the function of TECs. It is worth noting that TECs play an important role in the repair process after kidney injury, as they can regulate the activity of fibroblasts by secreting certain factors such as HGF, EGF, etc [33]. However, under sustained damage and inflammatory conditions, activated fibroblasts will form a negative feedback on epithelial cells, ultimately weakening the repair function of TECs. The bidirectional interaction between fibroblasts and TECs further exacerbates kidney fibrosis and injury [31]. Therefore, this study mainly focuses on the role of USP11 in RIF, hoping to restore the function and repair ability of TECs by inhibiting the activation of RIF.

USP11, as a deubiquitinating enzyme, can exert different biological functions by regulating various substrate proteins. A recent study has shown that lymphoid-specific helicase (LSH), a chromatin-remodeling protein, interacts with and is stabilized by USP11 via deubiquitination [34]. And the USP11/LSH/CYP24A1 signaling axis is involved in the process of ferroptosis in colorectal cancer [34]. Jiao et al. have proven that USP11 deubiquitinates OTUD5 and regulates the OTUD5-STING signaling pathway, which further induces the inflammatory response in endothelial cells and exacerbates radiation-induced pneumonitis (RIP) [27]. Wu et al. have proposed that USP11 directly interacts and stabilizes with SREBF1, while blockade of USP11 promotes its degradation through K48-linked deubiquitination [35]. Abnormally elevated levels of USP11 enhance lipogenesis and proliferation in hepatocellular carcinoma cells [35]. Furthermore, USP11 has also been reported to interact with the HIF-1α complex and maintain HIF-1α protein stability by removing ubiquitin, resulting in glycolysis activation through the PDK1 and LDHA pathways [36]. In the process of kidney fibrosis and injury, these biological functions mentioned above, including ferroptosis, inflammatory response in endothelial cells, lipid metabolism and glycolysis, are also involved. Therefore, in addition to the existing reported substrates, USP11 may also promote kidney injury through ubiquitination modification of other substrates. This will be further explored and discussed in our future research.

In kidney fibrosis, abnormal activation of EGFR signaling can lead to activation and proliferation of RIF, and promote the release of fibrotic factors [13,14]. We found that UA stimulation could activate EGFR signaling molecules with the increased expression of its phosphorylation, while both of USP11 siRNA transfection and MTX treatment markedly reduced its level. On the contrary, overexpression of USP11 upregulated the expression level of p-EGFR. Therefore, USP11 promotes the activation and proliferation of RIF during kidney fibrosis through the EGFR and its downstream signaling pathways. In addition to USP11, there are other ubiquitin-specific regulators that can mediate the ubiquitination level of EGFR and its stability. A recent study has reported the key role of USP18 in breast cancer development by upregulating EGFR and subsequently activating the AKT/Skp2 feedback loop pathway [37]. At the same time, analysis of ubiquitin linkages on activated EGFR in lung cancer showed that E3 ubiquitin ligase positively regulated the stability of EGFR through polyubiquitin binding at K6 and K11 sites [38], and its ubiquitination modification at K48 site was related to breast cancer [39]. However, highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediated the ubiquitin level of EGFR, ultimately causing its endocytosis and lysosomal degradation [40]. In our previous research, we revealed that USP11 deubiquitinated and stabilized EGFR and protected it from proteasome-dependent degradation [23]. Therefore, degradation method of EGFR depends on different ubiquitin modifying enzymes.

Mitoxantrone is a synthetic anthraquinone chemotherapy drug widely used in the treatment of cancer and certain immune disorders. It mainly exerts anti-tumor effects by inhibiting DNA synthesis, preventing DNA repair, and causing DNA strand breaks [41]. Mitoxantrone has been approved by the US Food and Drug Administration (FDA) for the treatment of several different types of diseases, including acute myeloid leukemia (AML), multiple sclerosis (MS), breast cancer and so on [41]. In a randomized, open-label, active-controlled, single-center, phase II clinical trial, mitoxantrone hydrochloride liposome provided a promising alternative for the treatment of advanced breast cancer, with a good balance of efficacy and safety in Chinese patients [42]. Another multicenter phase I study showed that combination therapy of mitoxantrone and cabazitaxel had a good therapeutic effect on patients with chemotherapy-naïve and pretreated metastatic castration-resistant prostate cancer (mCRPC), and their safety had also been confirmed [43]. Considering that mitoxantrone has an impact on cell proliferation, inflammatory reaction and oxidative stress, it may display a certain therapeutic effect on these triggering factors of kidney injury. In our previous research, we proved that pharmacologic inhibition of USP11 with mitoxantrone attenuated pathological lesions and improved kidney function in two mouse CKD models [23]. Combined with the results of this study, it is demonstrated that mitoxantrone has great potential in the treatment of kidney diseases. In the future, there may be more clinical trials to validate the potential application of mitoxantrone in kidney disease and explore its safety, dose optimization, and efficacy.

Although our experiments provide evidence supporting a critical role for USP11 in RIF activation and renal interstitial fibrosis, several limitations of the study should be acknowledged. First, our experiments were conducted exclusively in NRK49F cells, an established renal fibroblast cell line, which may not fully recapitulate the behavior of fibroblasts in vivo. Thus, isolating primary renal fibroblasts and performing mechanistic studies with them is worth considering. Second, the study lacks in vivo validation, and therefore the effects of USP11 on the regulation of the EGFR signaling pathway and RIF activation in an intact organism remain to be confirmed. Addressing these limitations in future studies will be essential to fully elucidate the mechanisms of USP11 in kidney fibrosis and to evaluate its potential as a therapeutic target.

In summary, we mainly focused on the role of USP11 on RIF and revealed that USP11 promoted the activation and proliferation of RIF through regulating EGFR and its downstream signaling molecules, PI3K/AKT and MEK/ERK. Both of USP11 deletion with siRNA transfection and pharmacologic inhibition with MTX effectively inhibited the activation of EGFR signal pathway and nuclear transcription factors, Snail and Slug. Thus, this study further supplements and completes the important role of USP11 in kidney fibrosis, and targeting USP11 could represent a potential strategy to mitigate CKD progression.

Funding Statement

This study was supported by the Shanghai Sailing Program [23YF1434700], the National Nature Science Foundation of China grants [82570815, 82300849, 82070791], the Shanghai Scientific Committee of China [23ZR1452200], the Key Discipline Construction Project of Shanghai Pudong New Area Health Commission [PWZxk2022-05], the New Quality Clinical Specialty Program of High-end Medical Disciplinary Construction in Shanghai Pudong New Area [2025-PWXZ-09], the Outstanding Leaders Training Program of Pudong Health Bureau of Shanghai [PWR12021-02], the Young Medical Talents Training Program of Shanghai Pudong New Area Health Commission [PWRq2024-04], the grant of Shanghai East Hospital [2024-DFTS-002, DFLC2022016], and the Pudong fundation for development of science and technology [PKJ2022-Y56].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The data analyzed during this study are included in this published article. Additional supporting data are available from the corresponding authors upon reasonable request.