USP25 inhibits renal fibrosis by regulating TGFβ-SMAD signaling pathway in Ang II-induced hypertensive mice

doi:10.21203/rs.3.rs-1884837/v1

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USP25 inhibits renal fibrosis by regulating TGFβ-SMAD signaling pathway in Ang II-induced hypertensive mice

https://doi.org/10.21203/rs.3.rs-1884837/v1

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Renal fibrosis is a crucial pathological feature of hypertensive renal disease (HRD). In-depth analysis of the pathogenesis of fibrosis is of great significance for the development of new drugs for the treatment of HRD. USP25 is a deubiquitinase that can regulate the progression of many diseases, but its function in the kidney remains unclear. We found that USP25 was significantly increased in human and mouse HRD kidney tissues. In the HRD model induced by Ang II, USP25^−/− mice showed significant aggravation of renal dysfunction and fibrosis compared with the control mice. Consistently, AAV9-mediated overexpression of USP25 significantly improved renal dysfunction and fibrosis. Mechanistically, USP25 inhibited the TGF-β pathway by reducing SMAD4 k63-linked polyubiquitination, thereby suppressing SMAD2 nuclear translocation. In conclusion, this study demonstrates for the first time that the deubiquitinase USP25 plays an important regulatory role in HRD.

hypertensive renal disease

deubiquitination

USP25

TGF-β

SMADs

Hypertension is a chronic disease that is prevalent worldwide.¹ Hypertensive renal disease (HRD), characterized by progressive renal fibrosis and inflammation associated with hypertension, is a major complication of hypertension.^{2, 3} The pathological manifestations of HRD include increased proteinuria, benign glomerulosclerosis, renal interstitial fibrosis, and inflammatory cell infiltration.^{4, 5} Among them, renal fibrosis is the common pathological basis of various chronic kidney diseases progressing to end-stage renal disease.⁶ Angiotensin II (Ang II) is one of the major pathological factors in the development of hypertensive nephropathy. It can lead to abnormal renal hemodynamics by increasing blood pressure, thus damaging renal function.⁷ It can also directly activate relevant receptor proteins to induce inflammation, fibrosis, and cell apoptosis, promoting renal injury.⁸ It shows that Ang II have a great impact on HRD. Elucidating the pathogenesis of hypertensive nephropathy and finding possible therapeutic targets have important scientific significance and clinical application value for the prevention and treatment of hypertensive nephropathy.

Ubiquitination is a post-translational modification catalyzed by the sequential action of ubiquitin activating enzymes (E1), ubiquitin cross-linking enzymes (E2) and ubiquitin ligases (E3).⁹ Like phosphorylation, ubiquitination is a dynamic and reversible event that can be negatively regulated by deubiquitinating enzymes (DUBs). DUBs can cleave peptide bonds between ubiquitin molecules or between ubiquitin and modified proteins.¹⁰ The human genome encodes nearly 100 DUBs that play critical roles in many diseases, such as autoimmune diseases, infectious diseases, and cancer.¹¹ As important cell signal regulators, the role of DUBs in hypertensive nephropathy has become increasingly prominent. Previously report shows that the deubiquitinase USP4 regulates TGF-β-induced EMT and in vitro migration by directly binding to TβRI in the plasma membrane to enhance its stability,¹² indicating that DUBs affect the function and fate of cells by regulating cellular intracellular signal transduction.

Ubiquitin-specific protease 25 (USP25) belongs to the ubiquitin-specific protease (USP) family and is encoded by a gene located at 21q11.2.¹³ USP25 has been reported to be closely associated with inflammation, immune response and cancer. For example, it has been implicated in endoplasmic reticulum-associated degradation, and acute endoplasmic reticulum (ER) stress regulates the processing of amyloid precursor protein through ubiquitin-dependent degradation of USP25.¹⁴ USP25 is a negative regulator of IL-17-mediated signaling and inflammation, and can modulate TLR4-dependent innate immune responses by deubiquitinating TRAF3, TRAF5, and TRAF6.^{15, 16} Besides, USP25 gene is more than three-fold overexpressed in human breast cancer tissues, and further studies revealed that it is also a putative tumor suppressor in human lung cancer.¹⁷ These results suggest that USP25 is a multifunctional signaling regulator with specific functions in different diseases by acting on multiple cell types and signaling pathways. However, the role of USP25 in kidney diseases has not been reported.

In our work, the role of USP25 in HRD has been discussed. The results showed that USP25 reduced the expression of genes related to renal fibrosis and renal injury by inhibiting TGF-β-induced ubiquitination of SMAD4, thereby alleviating HRD. Using control and USP25 knockout mesangial cells, we confirmed in vitro that USP25 inhibited TGF-β-induced SMAD signaling. These results collectively suggest that USP25 is involved in the pathogenesis of HRD and may be a potential therapeutic target for HRD.

2.1. Cell culture and treatment

The mouse glomerular mesangial cells SV40 MES-13 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM (Gibco, C11995500BT) containing 4.5-g L^− 1 glucose in an incubator at 37°C and 5% CO₂. The medium was supplemented with 1/4 DMEM F12 medium, 10% fetal bovine serum (Meilunbio, PWL001), 100 U·ml^− 1 penicillin and 100 mg·ml^− 1 streptomycin (Sbjbio, Nan Jing, BC-CE -007). To establish USP25 knockout cell lines, SV40 MES-13 were transduced with lentivirus expressing CRISPER/Cas9 and USP25 gRNA (sequence: CACCGAGTGCACAC AGGTTTACTGG) or lentivirus expressing empty vector in the presence of polybrene (Sigma Aldrich). 72 hours after transduction, blasticidin S (5 µg/ml, YEASEN) was added to screen for single cell clones. Knockout efficiency was proved by Western blotting.

2.2. Animal experiments

USP25^−/− mice were donated by Professor Yuan Jian's research group from Shanghai Jiao Tong University. USP25^−/− mice and littermate wild-type mice were raised in the Experimental Animal Center of Wenzhou Medical University. A 12-h light/12-h dark cycle was performed and fed standard rodent chow and tap water. All animal care and experimental procedures were approved by the Animal Policy and Welfare Committee of Wenzhou Medical University (Approval file number: wydw2020-0137).

Ang II-induced hypertensive renal fibrosis model: Male USP25^−/− mice aged 6–8 weeks and wild-type C57BL/6J mice were infused with Ang II (1000 ng/kg/min) or normal saline by osmotic pump for 4 weeks. Mice were randomly divided into 4 groups: WT-Ctrl, WT-Ang II, USP25^−/−-Ctrl, USP25^−/−- Ang II. With 6 mice per group, blood pressure and body weight were monitored every 3 days. Spontaneous urine was collected 6 hours before the end of modeling, and the mice were sacrificed after modeling to obtain serum and kidney tissue samples. Creatinine, urinary albumin and blood urea nitrogen were detected using commercial kits (Nanjing Jiancheng Bioengineering Institute, China). An ELISA kit (EK-Bioscience, Shanghai) was used to measure Ang II protein levels in tissue lysates.

A model of acute renal fibrosis induced by unilateral ureteral ligation operation (UUO): Male USP25^−/− mice aged 6–8 weeks and wild-type C57BL/6J mice were subjected to general anesthesia by intraperitoneal injection of pentobarbital (50 mg/kg). The left ureter was exposed through the left incision, ligated at two points with 4 − 0 silk, and cut between the two ligation points. The sham operation group was not ligated. Mice were randomly divided into 4 groups: WT-Ctrl, WT-UUO, USP25^−/−-Ctrl, USP25^−/−- UUO. 7 days after modeling, the mice were sacrificed to obtain kidney tissue samples.

Adeno-associated virus infection in Ang II-induced mice: Adeno-associated virus serotype 9 (AAV-9) has been reported to produce strong expression in renal tissue.¹⁸ We infected mice with AAV-9 encoding USP25 (AAV9-USP25^oe) or an empty vector (AAV9-NC) for overexpression in the kidney. The mouse USP25 gene was cloned into AAV-9 by Genechem Co. LTD (Shanghai, China). USP25^−/− mice and wild-type mice were randomly divided into 3 groups (6 mice in each group) as follows: WT + AAV9-NC + Ang II, USP25^−/− + AAV9-NC + Ang II, USP25^−/− + AAV9-USP25^oe + Ang II. We expressed AAV9 (2 × 10¹¹ virus/ mouse/ month) by tail vein injection 2 months before Ang II stimulation. Urine was collected 6 hours before the end of modeling, and the mice were sacrificed after modeling to obtain serum and kidney tissue samples.

2.3. Histopathological studies

Mouse kidney specimens were fixed in 4% neutral buffered formalin and embedded in paraffin. Routine histological analysis was performed on kidney tissue sections (5 µm) by hematoxylin-eosin staining (Solarbio, G1120). Paraffin sections were also stained with 0.1% Sirius Red (Solarbio, S8060) and 1.3% picric acid-saturated aqueous solution to assess type IV collagen deposition. Tubulointerstitial lesions and fibrosis were assessed with Masson's trichrome staining (Solarbio, G1340-7). The extent of renal injury and fibrosis was analyzed using ImageJ software.

2.4. Immunohistochemical staining

After autoclaving deparaffinized kidney sections in 10 mM citric acid solution (pH 6.5) at 121°C for 2 min for antigen retrieval, peroxidase activity was quenched with 3% H₂O₂, and the tissues were blocked in 5% bovine serum. Sections were incubated with anti-USP25 (Santa Cruz, sc-398414, 1:50) antibody overnight at 4°C. HRP-conjugated secondary antibody (Beyotime Biotechnology, 1:200) and diaminobenzidine (DAB, Solarbio, DA1010) were conjugated for detection of immunoreactivity. Sections were counterstained with hematoxylin. A Nikon microscope imaging system (Nikon Instruments, Japan) was used to capture images.

2.5. Immunofluorescence

Immunofluorescence staining of kidney tissues was performed on 5 µm thick frozen sections. Briefly, sections were fixed in methanol for 10 minutes, blocked with 5% BSA for 1 hour and then blocked with anti-USP25 (Santa Cruz, sc-398414, 1:50), anti-Desmin (Cell Signaling Technology, #5332, 1:200), and anti-SMAD2 (Cell Signaling Technology, #5339, 1:200) overnight. The fluorescence-conjugated secondary antibody (YEASEN) was then incubated at 37°C for 1 hour. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and imaged by Nikon microscope imaging system (Nikon Instruments, Japan).

2.6. Western blot analysis

Kidney tissues and cultured cells were lysed in RIPA lysis buffer (Beyotime Biological, Shanghai, China) supplemented with protease inhibitor PMSF (Solarbio, Beijing). The supernatant was collected by centrifugation at 12,000 g for 10 min at 4°C and the protein concentration was determined by Coomassie Quick Start™ Bradford 1× Dye Reagent. 40–80 µg protein samples were denatured, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes. Western Blot was prepared with anti-USP25 (Abcam, ab187156) 1:2,000, GAPDH (Bioworld, MB001) 1:1,0000, Col-1 (Abcam, ab138492) 1:1,000, α-SMA (Proteintech, 67735-1-Ig) 1:1,000, TGF-β (Abcam, ab215715) 1: 1,000, SMAD2 (Cell Signaling Technology, #5339) 1:1,000, SMAD3 (Cell Signaling Technology, #9523) 1:1,000, SMAD7 (Proteintech, 25840-1- AP) 1:1,000, SMAD4 (Proteintech, 10231-1-AP) 1:1,000, Lamin B1 (Abcam, ab133741) 1:1,000, Ubiquitin (P4D1) (Cell Signaling Technology, #3936) 1:1,000, K48-linkage Specific Polyubiquitin (D9D5) (Cell Signaling Technology, #12805) 1:1,000, K63-linkage Specific Polyubiquitin (D7A11) (Cell Signaling Technology, #12930) 1:1,000, Rabbit IgG (Proteintech, 30000-0-AP), HA -Tag (Proteinech, 51064-2-AP) 1:1,000, and FLAG-Tag (Proteinech, 20543-1-AP) 1:1,000. Imaging in gel by secondary antibody (Beyotime Biotechnology) and NcmECL Ultra (NCM Biotech). The protein bands were captured under the system (BIO-RAD).

2.7. Immunoprecipitation

Whole cell or tissue lysates were generated as described in “Western Blotting”. Lysates were incubated with anti-SMAD2, anti-SMAD4, or anti-SMAD7 overnight at 4°C with continuous rotation. Immune complexes were captured by incubating with sepharose beads (Beyotime Biological, Shanghai, China) for 2 h at 4°C. The beads were washed 5 times with PBS buffer. Proteins were then eluted with SDS loading buffer at 100°C. Immunoblotting was performed to detect proteins.

2.8. Real-time quantitative polymerase chain reaction (RT-qPCR) assay

Messenger ribonucleic acid (mRNA) levels were examined by RT-qPCR assay. Total ribonucleic acid (RNA) from cells or tissues was extracted using TRIzol kit (TAKARA, Beijing), respectively. 1 mg of RNA was reverse transcribed using HiScript® III All-in-one RT SuperMix (Vazyme, R333-01) reagent. RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) and QuantStudio 3 Real-Time PCR System (Bio-Rad). The relative amount of each gene was normalized to the amount of β-actin. The ΔΔ^−Ct algorithm was used for RT-qPCR analysis. Primer sequences are shown in Table S1.

2.9. Human kidney samples

Archival frozen human kidney tissues were obtained from the First Affiliated Hospital of Wenzhou Medical University. The study was approved by the Ethical Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics Number: LCYJLS-2018‐157) and completed according to the Declaration of Helsinki. The samples were obtained from subjects with and without hypertension. These tissues had been obtained at the time of nephrectomy for conventional renal carcinoma. However, nontumoral specimens were used in this study. Frozen tissue section was obtained for immunofluorescence staining.

2.10. Transcriptome sequencing and analysis

Whole genome analysis of fresh mouse kidney tissues was performed in Lianchuan bio technologies (Hangzhou, Zhejiang). Double terminal sequencing was performed using Illumina novaseq 6000 (California, United States). Bioinformatics analysis of RNA sequencing data was also performed by Lianchuan Bio Technologies (Hangzhou, Zhejiang). We used publicly available transcriptome data at www.ncbi.nlm.nih.gov/geo/. Data related to renal fibrosis were retrieved from the Gene Expression Comprehensive Database. Three datasets GSE153010,¹⁹ GSE149915²⁰ and GSE125015²¹ were selected.

2.11. Statistical analysis

Statistical analysis was performed using GraphPad Prism 8 software (GraphPad). Student’s t-test was used to compare two groups of data, and one-way analysis of variance (ANOVA) was used to compare data of more than two groups. P value of < 0.05 was considered statistically significant. Data are presented as mean ± SEM.

3.1 USP25 expression is elevated in human hypertensive kidneys and experimental models

To investigate the role of USP25 in hypertensive kidney injury, we examined archived renal biopsy frozen section samples from normotensive and hypertensive human subjects. The samples were obtained at the time of nephrectomy for kidney cancer. The nontumoral specimens were used in this study. We first performed USP25 immunofluorescence staining on tissue sections, and our results showed increased USP25 expression in renal tissues from hypertensive subjects (Fig. 1a). In order to clarify the changes of USP25 in hypertensive nephropathy, we subcutaneously implanted Ang II micropump (1000 ng/kg/min) in wild-type (WT) mice to induce hypertension. The mice were sacrificed 4 weeks after modeling, and the kidneys were harvested for analysis. Western Blot data showed that the expression of USP25 was significantly increased in hypertensive nephropathy (Fig. 1b-c). Consistently, the mRNA levels of USP25 were also significantly increased after modeling (Fig. 1d). Immunohistochemistry analysis also showed that USP25 was upregulated in Ang II-induced hypertensive nephropathy (Fig. 1e). In order to further study the distribution of USP25 in the kidney, the above-mentioned kidney tissue sections were taken for immunofluorescence staining. The results shows that USP25 was mostly expressed in the Desmin-positive mesangial cells (Fig. 1f), rather than in Cadherin-16-positive tubular epithelial cells and WT1-positive podocytes (Supplementary Figure S1). Figure 1f also showed that hypertension led to proliferation of mesangial cells and further upregulation of USP25 expression in mesangium (Fig. 1f). In addition, we analyzed publicly available transcriptomic data on the website and found that USP25 gene expression was elevated in the kidneys of modeled mice (GSE153010, GSE149915, and GSE125015) compared with the respective controls (Fig. 1g), which is consistent with our own findings. The above results demonstrate USP25 is likely to play a role in HRD.

3.2 USP25 deficiency aggravates Ang II-induced renal injury and renal fibrosis

To investigate the function of USP25 in HRD, we used USP25 knockout (USP25^−/−) mice (Supplementary Figure S2). Knockout of USP25 did not have any effect on the normal growth, health, and longevity of mice. We constructed hypertensive mouse models by subcutaneously implanting Ang II-releasing pumps in WT and USP25^−/− mice. The hypertensive model mice showed increased blood pressure and Ang II levels. However, no sharp differences in blood pressure (Fig. 2a) have been observed. and Ang II levels (Fig. 2b) between hypertensive USP25^−/− mice compared with hypertensive WT mice. Interestingly, kidney weight was significantly increased in USP25^−/− mice in the Ang II-induced hypertension model (Fig. 2c). Serum creatinine, blood urea nitrogen and urinary albumin are important indicators to characterize renal function. We found that compared with the WT hypertensive mice, USP25^−/− hypertensive mice showed higher serum creatinine (Fig. 2d), urea nitrogen (Fig. 2e), and urinary albumin (Fig. 2f) concentrations, indicating that deletion of USP25 exacerbates HRD. Next, we explored the histopathological changes in mouse kidney specimens. Conventional H&E staining showed that Ang II induced glomerular abnormalities, mainly manifested as glomerular cluster disorder and mesangial hyperplasia. Renal fibrosis is an important pathological feature of HRD, and we further investigated the role of USP25 in renal fibrosis. Masson and Sirius Red staining revealed that USP25^−/− hypertensive mice exhibited more interstitial collagen deposition, i.e., more severe renal fibrosis, than WT hypertensive mice (Fig. 2g-2i). Next, we extracted protein and mRNA from mouse kidney tissues, and found that the expression of fibrosis indicators such as Col-1, Col-4, and α-SMA in the kidneys of USP25^−/− hypertensive mice was significantly increased (Fig. 2j and k). The above results indicated that USP25^−/− mice exhibited more severe renal damage and fibrosis after Ang II infusion than WT mice.

3.3 Overexpression of USP25 alleviates Ang II-induced hypertensive renal injury and renal fibrosis

We then examined whether restoring USP25 could attenuate renal impairment and fibrosis in the Ang II-induced hypertension model. Adeno-associated virus serotype 9 (AAV9) has been reported to be strongly expressed in kidney tissue.¹⁸ We next infected mice with AAV encoding USP25 (AAV9-Usp25^oe) and an empty vector (AAV9-NC) for overexpression in kidneys. We found that overexpression of USP25 significantly reduced kidney weight (Fig. 3a) and kidney injury-related indicators (Fig. 3b-d) in USP25^−/− mice. When we examined histological pathological changes, we also found that overexpression of USP25 alleviated glomerular abnormalities and reduced collagen deposition (Fig. 3e). Supplementary Figure S3a and S3b show the statistical data of collagen volume fraction stained with Masson and Sirius red, respectively. Consistently, overexpression of USP25 significantly reduced the protein (Fig. 3f) and mRNA (Fig. 3g) levels of fibrotic factors in kidney tissue samples from USP25^−/− mice, indicating that USP25 overexpression alleviated renal fibrosis. In conclusion, these results suggest that overexpression of USP25 attenuates renal impairment and fibrosis in USP25^−/− hypertensive mice.

3.4 USP25 participates in TGF-β-mediated fibrosis and SMAD activation

To understand how the deubiquitinating enzyme USP25 is involved in the regulation of renal fibrosis, we performed RNA-sequencing of kidney tissues from the three groups of mice shown in Fig. 2. Hypertensive kidneys were induced by Ang II in all three groups. Using Venn diagram, we screened 100 statistically significant genes that were up-regulated in the USP25^−/− group but down-regulated after overexpression of USP25 (Fig. 4a). Among these genes, a range of fibrosis genes including TGF-β were markedly altered by USP25 (Fig. 4b). TGF-β is a central mediator of renal fibrosis and mainly activates the intracellular SMAD signaling system. Multiple studies have shown that TGF-β-SMAD signaling has multiple functions in regulating cell growth, differentiation, apoptosis, migration and invasion/metastasis, and plays a pivotal role in progressive kidney injury and fibrosis. ²²Therefore, we speculated that USP25 reduces the expression of genes related to renal fibrosis and renal injury by inhibiting the TGF-β pathway, thereby alleviating the occurrence and development of HRD.

To further clarify the role of USP25 in renal cells, we obtained a USP25-knockout mesangial cell line by CRISPR-Cas9 technology (Supplementary Figure S4a). After 36 h of TGF-β stimulation, knockout of USP25 resulted in increased expression of fibrotic factors (Fig. 4c), whereas overexpression of USP25 in USP25^−/− cells reversed this effect (Fig. 4d). SMAD2 is a key downstream mediator responsible for the biological effects of TGF-β. We found that SMAD2 nuclear translocation was significantly increased in USP25 knockout cells after 30 min of TGF-β stimulation (Fig. 4e), while SMAD2 phosphorylation levels were not affected by USP25 (Supplementary Figure S4b), indicating that USP25 may affect the ubiquitination of SMAD2 rather than its phosphorylation. Next, we extracted cytoplasmic nuclei from fresh animal kidney tissues and also demonstrated a significant increase in SMAD2 nuclear translocation in the USP25^−/− hypertensive group compared with the WT hypertensive group (Fig. 4f). Figure 4g and 4h are statistical graphs of SMAD2 nuclear translocation in cells and tissues, respectively. Immunofluorescence further demonstrated that deletion of USP25 promoted nuclear translocation of SMAD2 upon TGF-β stimulation (Fig. 4i). The above results show that USP25 can inhibit TGF-β-mediated fibrosis and SMAD activation.

3.5 USP25 inhibits K63 ubiquitination of SMAD4

Recent studies have shown that the activity of SMAD is tightly regulated by ubiquitination modification.²³ As a DUB, USP25 regulates intracellular signal transduction by changing the ubiquitination state of proteins. SMAD2 has been reported to be regulated by various deubiquitinases and E3 ubiquitin ligases. The above results suggest that the deubiquitinating enzyme USP25 inhibits the nuclear translocation of SMAD2 without affecting the phosphorylation of SMAD2, indicating that USP25 may affect the ubiquitination of SMAD2 or other SMADs regulating SMAD2. As the only universal SMAD in the TGF-β-SMAD signaling pathway, SMAD4 can promote the nuclear translocation of SMAD2. Additionally, SMAD7 is an inhibitory SMAD that regulates SMAD2 through a negative feedback mechanism.²⁴ Thus, we assessed the ability of USP25 to regulate the ubiquitination of SMAD2, SMAD4 and SMAD7. As shown in Fig. 5a-c, interestingly, deletion of USP25 significantly increased the ubiquitination of SMAD4 without affecting the ubiquitination of SMAD2 and SMAD7. These results show that USP25 affects the nuclear translocation of SMAD2 by inhibiting the ubiquitination of SMAD4.

Given that (i) USP25 is a multifunctional deubiquitination enzyme that can regulate multiple types of ubiquitination modifications, including K48 and K63; (ii) K48-specific ubiquitination is a key signal for proteasome-mediated protein degradation; (iii) K63-specific ubiquitination mainly regulates non-degradative activity. Figure 5b and Supplementary Figure S5 show that knockdown or overexpression of USP25 did not affect the degradation of SMAD4, indicating that USP25 is likely to inhibit the function of SMAD4 by reducing its K63 ubiquitination. As shown in Figs. 5d, deletion of USP25 significantly increased the total ubiquitination and K63 ubiquitination of SMAD4. In contrast, SMAD4 was weakly modified with K48 polyubiquitin chains. On the other hand, overexpression of USP25 in USP25^−/− cells significantly reduced the total and K63 ubiquitination of SMAD4 (Fig. 5e). Next, the findings were further consolidated with animal tissues, and the results showed that USP25 knockout significantly increased the total and K63 ubiquitination of SMAD4 without affecting its K48 ubiquitination (Fig. 5f-h). Overexpression of USP25 reversed this result, which is consistent with the in vitro results described above. Next, we further explored the role of SMAD4 in anti-fibrosis function of USP25 by knocking down SMAD4. As shown in Fig. 5i and 5j, silencing of SMAD4 reversed the inhibitory effect of USP25 on renal fibrosis. Taken together, these data suggest that USP25 inhibits SMAD2 nuclear translocation by reducing the K63 ubiquitination and activity of SMAD4.

3.6 Loss of USP25 exacerbates renal fibrosis induced by UUO

UUO is another important model to study renal fibrosis at home and abroad. The interaction of obstruction and injury after UUO can often lead to ureteral stenosis and fibrosis, which further aggravates urinary tract obstruction, causes abnormal urine transport, and ultimately leads to impaired renal function.²⁵ To further validate the role of USP25 regulating renal fibrosis, we constructed the UUO mouse model by ligating the unilateral ureter in WT and USP25^−/− mice. It was found that mice receiving UUO for 7 days exhibited marked increases in renal volume and weight on the obstructed side (Fig. 6a-c). H&E staining showed that UUO mice had different degrees of calyceal and renal pelvis dilation, and the injury was more obvious after USP25 deletion. At the same time, Masson and Sirius Red staining showed obvious fibrosis in the renal cortex and the cortex-medullary junction of UUO mice, and USP25^−/− UUO mice exhibited more severe tubular damage and fibrotic lesions (Fig. 6d-f). Consistently, the protein and mRNA levels of fibrosis genes were significantly increased in kidney tissue samples from USP25^−/− UUO mice compared with WT UUO mice (Fig. 6g-h). Taken together, the above in vivo results suggest that the deubiquitinating enzyme USP25 can inhibit kidney injury and fibrosis in UUO mice.

In this study, we found that USP25 inhibited hypertension-induced renal dysfunction as well as renal fibrosis. Mechanistically, USP25 inhibited SMAD2 nuclear translocation by inhibiting K63 ubiquitination of SMAD4 in the TGF-β pathway during Ang II-induced HRD, thereby reducing the expression of fibrosis-related genes and the occurrence and development of HRD. The schematic diagram of the main findings is shown in Fig. 7.

The TGF-β/SMAD signaling pathway is a major driver of renal fibrosis and may serve as a therapeutic target to prevent or ameliorate HRD or UUO fibrosis.²² Numerous studies have demonstrated increased expression of TGF-β during the progression of human kidney diseases.^{26, 27} In addition, many animal experiments have identified TGF-β as a major stimulator of glomerular and tubulointerstitial fibrosis. Mechanistically, TGF-β induces renal fibrosis by activating canonical (SMAD-based) and non-canonical (non-SMAD-based) signaling pathways, resulting in overproduction of extracellular matrix (ECM). The effector SMAD proteins (SMAD2, SMAD3, SMAD4 and SMAD7) play different or even opposite functions during fibrosis and are regulated by ubiquitination. SMAD2 and SMAD3 are key nuclear importing proteins in the TGF-β-SMAD pathway and are regulated by various E3 ubiquitin ligases and deubiquitinases.²⁸ It has been reported that the deubiquitinating enzyme OTUB1 can enhance TGFβ signaling by inhibiting the ubiquitination and degradation of SMAD2/3.²⁹ Another study showed that the E3 ubiquitin ligase Smurf2 inhibits the TGFβ signaling pathway by promoting the degradation of SMAD2.³⁰ As a universal SMAD, SMAD4 is required for the translocation of all TGF-β families into the nucleus, and has also received extensive attention in recent years. For example, the E3 ubiquitin ligase TRIM47 promotes the growth and invasion of colorectal cancer cells by binding to SMAD4, increasing its ubiquitination and degradation.³¹ SMAD7 is an inhibitory SMAD that blocks SMAD2/3 activation by degrading TβRI and SMADs. As a multifunctional deubiquitinase, USP25 can regulate the ubiquitination states of TRAF5 (k63), TRAF6 (k63),¹³ TRAF3 (K48),¹⁵ TRiC³² and HBO1³³ and affect various signaling pathways and diseases, but its role in the kidney has not been reported. Here, we found for the first time that, during kidney diseases, USP25 affects the function of SMAD4 by inhibiting k63 ubiquitination of SMAD4, thereby promoting TGFβ signaling.

Undeniably, our study has certain limitations. First, we did not elucidate the upstream mechanism of how Ang II upregulates USP25. As the main active substance in RAAS, Ang II acts through its receptors AT1 and AT2. In the adult kidney, AT1 receptors account for about 90%. It will be interesting to study the mechanism by which Ang II stimulation up-regulates USP25 expression in a feedback manner. Secondly, we used USP25 knockout mice and performed mechanistic studies in mesangial cells. While our data show that USP25 is mainly expressed in mesangial cells in mouse kidneys, we are fully aware of the importance of analyzing other kidney cell types. We anticipate that other cell types in renal tissues, including epithelial cells and podocytes, may also utilize this signaling pathway. But this possibility needs to be confirmed by further studies.

In summary, our study reveals a potentially important mechanism by which Ang II induces renal dysfunction. As a multifunctional deubiquitinating enzyme, USP25 can affect the TGF-β pathway by inhibiting the ubiquitination of SMAD4, thereby alleviating HRD-induced renal dysfunction and renal fibrosis. At present, there are few studies on the role of deubiquitinase in kidney diseases, and our study sheds light on the important role of DUB in the kidney.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81930108 to G.L., 81900496 to X.W., and 81971143 to X.W.), Zhejiang Provincial Key Scientific Project (2021C03041 to G.L.), and the Qianjiang Talent Program of Zhejiang Province (QJD1902017) to X.W.

Author contributions

This work was performed with the collaboration of all authors. Guang Liang and Xu Wang contributed to the literature search and study design. Guang Liang and Ying Zhao participated in the drafting of the article. Ying Zhao, Xi Chen, Yimin Lin, and Xian Su carried out the experiments. Ying Zhao, Bozhi Ye, Yanghao Chen and Zhongding Li revised the manuscript. Shijie Fan contributed to data collection. All authors have read and agreed the final manuscript

Conflict of Interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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USP25 inhibits renal fibrosis by regulating TGFβ-SMAD signaling pathway in Ang II-induced hypertensive mice

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cell culture and treatment

2.2. Animal experiments

2.3. Histopathological studies

2.4. Immunohistochemical staining

2.5. Immunofluorescence

2.6. Western blot analysis

2.7. Immunoprecipitation

2.8. Real-time quantitative polymerase chain reaction (RT-qPCR) assay

2.9. Human kidney samples

2.10. Transcriptome sequencing and analysis

2.11. Statistical analysis

3. Results

3.1 USP25 expression is elevated in human hypertensive kidneys and experimental models

3.2 USP25 deficiency aggravates Ang II-induced renal injury and renal fibrosis

3.3 Overexpression of USP25 alleviates Ang II-induced hypertensive renal injury and renal fibrosis

3.4 USP25 participates in TGF-β-mediated fibrosis and SMAD activation

3.5 USP25 inhibits K63 ubiquitination of SMAD4

3.6 Loss of USP25 exacerbates renal fibrosis induced by UUO

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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