TGF-β1 induces epithelial-mesenchymal transition and fibrosis of ureteral epithelial cells via targeting RACK1 in ureteropelvic junction obstruction

TGF-β1 induces epithelial-mesenchymal transition and fibrosis of ureteral epithelial cells via targeting RACK1 in ureteropelvic junction obstruction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TGF-β1 induces epithelial-mesenchymal transition and fibrosis of ureteral epithelial cells via targeting RACK1 in ureteropelvic junction obstruction Wenyou Chen, Fengguang Ye, Xiaoqiang Lin, Jinrong Chen, Fengbin Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7164773/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ureteropelvic junction obstruction (UPJO) is a frequent cause of congenital hydronephrosis. It involves both structural and functional changes at the ureteropelvic junction (UPJ), where the ureter connects with the renal pelvis. The mechanisms behind tissue remodeling and fibrosis in this condition are not fully understood. This study explores the role of epithelial-mesenchymal transition (EMT) and examines how receptor for activated C kinase 1 (RACK1) may regulate this process in UPJO. Methods Stenotic UPJ tissues were collected from patients with congenital UPJO and analyzed for EMT and RACK1 expression. An in vitro model was established using SV-HUC-1 ureteral epithelial cells treated with TGF-β1 to induce EMT. RACK1 expression was manipulated by siRNA, and changes in EMT-related markers, Smad2/3 and p65 signaling, extracellular matrix proteins, cell proliferation, and migration were evaluated by qRT-PCR, western blotting, and functional assays, respectively. Results EMT features, including increased expression of N-cadherin, β-catenin, Vimentin, and ECM-related proteins (COL1A1, FN1, α-SMA), and decreased E-cadherin expression, were observed in both UPJO tissues and TGF-β1–treated cells. RACK1 was significantly upregulated in stenotic tissues and after TGF-β1 stimulation. RACK1 knockdown suppressed Smad2/3 and p65 activation, inhibited EMT progression, and reduced cell proliferation and migration. Conclusion EMT contributes to tissue remodeling and fibrosis in congenital UPJO. RACK1 promotes TGF-β1–induced EMT through activation of Smad2/3 and p65 signaling. These findings provide new insights into the pathogenesis of UPJO and suggest RACK1 as a potential target for therapeutic intervention. Ureteropelvic junction obstruction Epithelial-mesenchymal transition TGF-β1 RACK1 Fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ureteropelvic junction obstruction (UPJO) is the most common cause of congenital hydronephrosis in children, with an estimated incidence of 1 in 1,500 live births [ 1 ]. It results from impaired urine flow between the renal pelvis and proximal ureter, often due to intrinsic muscular dysplasia or extrinsic vascular compression. If left untreated, UPJO can lead to progressive renal function deterioration, making early diagnosis and timely intervention crucial [ 2 ]. Histologically, stenotic segments of the ureteropelvic junction (UPJ) are characterized by smooth muscle disorganization, increased collagen deposition, and reduced peristalsis, indicative of chronic tissue remodeling and fibrosis [ 3 ]. However, the cellular and molecular mechanisms underlying these pathological changes remain incompletely understood. Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their polarity and adhesion properties while acquiring mesenchymal characteristics, such as enhanced motility and extracellular matrix (ECM) production [ 4 ]. EMT is essential for embryonic development and wound healing, but is also implicated in pathological tissue fibrosis in various organs, including the kidney and urinary tract [ 5 ]. Transforming growth factor-beta 1 (TGF-β1) is a master regulator of EMT and fibrosis, known to activate both canonical Smad2/3 and non-canonical signaling pathways such as NF-κB [ 6 , 7 ]. Elevated expression of TGF-β1 has been observed in the stenotic renal pelvis of UPJO patients, where it contributes to excessive muscle and collagen formation and aberrant scar remodeling [ 8 ]. However, the downstream molecular effectors linking TGF-β1 to EMT in ureteral epithelial cells remain largely unknown. Receptor for activated C kinase 1 (RACK1) is a multifunctional scaffold protein containing WD40 repeats that regulates diverse signaling pathways, including TGF-β, integrin, and NF-κB [ 9 , 10 ]. RACK1 modulates cellular processes such as migration, adhesion, and differentiation by facilitating signal complex assembly [ 11 ]. Although RACK1 has been implicated in cancer progression and tissue fibrosis [ 12 , 13 ], its role in congenital urinary tract malformations such as UPJO has not yet been explored. Whether RACK1 acts as a mediator of TGF-β1-induced EMT and fibrotic remodeling in ureteral epithelial cells is unknown. In this study, we aimed to investigate the expression and function of RACK1 in the pathogenesis of UPJO. We analyzed RACK1 expression in clinical UPJ tissue samples and examined its role in TGF-β1-induced EMT and fibrosis in SV-HUC-1 ureteral epithelial cells. Furthermore, we evaluated the downstream signaling pathways involved and assessed the effects of RACK1 knockdown. Our findings identify RACK1 as a novel regulator of EMT and fibrosis in UPJO and suggest its potential as a therapeutic target for congenital ureteral obstruction. Materials and methods Patients Stenotic tissue samples from the ureteropelvic junction (UPJ) were collected from five pediatric patients with congenital ureteropelvic junction obstruction (UPJO) who underwent pyeloplasty at Zhangzhou Municipal Hospital, Fujian Province, China. The diagnosis of UPJO was based on ultrasonography, radiological imaging, and renal scintigraphy, and was confirmed by histopathological examination of the resected stenotic segments, which showed features such as smooth muscle hypertrophy or hyperplasia and collagen deposition. Control UPJ tissues were obtained from five pediatric patients with renal dysplasia accompanied by duplicated kidneys who underwent nephrectomy. Histological examination confirmed that the UPJ segments in these patients were non-obstructed and structurally normal. All tissue samples were immediately snap-frozen in liquid nitrogen and stored for further analysis. This study was approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University (Zhangzhou, Fujian, China), with approval number 2024KYZ357. Written informed consent was obtained from all participants or their guardians prior to sample collection. Histologic Analysis and Immunohistochemistry For histological analysis, tissue specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 7 µm. Hematoxylin and eosin (H&E) staining was performed using standard protocols to assess general tissue morphology. Masson's trichrome staining was conducted to evaluate collagen fiber deposition. Immunohistochemical staining was performed using the EnVision™+ System-HRP (Dako, USA) following the manufacturer’s instructions. Paraffin-embedded tissue sections were incubated with primary antibodies against human RACK1 (Abcam, USA, 1:200), E-cadherin (Abcam, USA, 1:500), and COL1A1 (Abcam, USA, 1:500) overnight at 4°C. After washing with phosphate-buffered saline (PBS), sections were incubated with HRP-conjugated secondary antibodies provided in the EnVision™+ kit for 30 minutes at room temperature. The signal was developed using diaminobenzidine (DAB) for 5 minutes, followed by counterstaining with hematoxylin. Cell culture V-HUC-1 cells (Simian Virus 40-transformed Human Urothelial Cells) were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in Ham’s F-12K medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. They were seeded in 6-well plates at a density of 1.5 × 10⁵ cells/mL and used for experiments when they reached 70–80% confluence. EMT was induced by treating cells with 10 ng/mL of recombinant human TGF-β1 (PeproTech, USA; Cat# 100 − 21) for 24 hours. All experiments were performed in triplicate and repeated independently at least three times. Small interfering RNA (SiRNA) transfection To reduce RACK1 expression in SV-HUC-1 cells, a specific small interfering RNA (siRNA) targeting RACK1 was synthesized. The sequences were as follows: sense 5′-CUCUGGAUCUCGAGAUAAAdTdT-3′ and antisense 5′-UUUAUCUCGAGAUCCAGAGdTdT-3′. A scrambled siRNA with no homology was used as a negative control. Its sequences were: sense 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and antisense 5′-ACGUGACACGUUCGGAGAAdTdT-3′. Transfection was carried out using Lipofectamine® 2000 (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. Both RACK1-targeting and control siRNAs were obtained from Bioscien Biotechnology Co., Ltd. (Shanghai, China). To assess transfection efficiency, RACK1 expression at both mRNA and protein levels was measured by reverse transcription-quantitative PCR and western blotting. Real-time quantitative PCR (RT-qPCR) Total RNA from clinical tissue samples and treated SV-HUC-1 cells was isolated using TRIzol® reagent (Thermo Fisher Scientific, USA) following the standard protocol. The mRNA expression levels of RACK1, GAPDH, E-cadherin, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the One-Step RT-qPCR Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. GAPDH was used as the internal control, and relative gene expression was calculated using the 2^−ΔΔCt method. The primer sequences were as follows: RACK1 forward 5′-AGATAAGACCATCATCAT-3′ and reverse 5′-AGATAACCACATCACTAA-3′; GAPDH forward 5′-TGTGAGGGAGATGCTCAGTG-3′ and reverse 5′-TGTTCCTACCCCCAATGTGT-3′; E-cadherin forward 5′-ACTGTGAAGGGACGGTCAAC-3′ and reverse 5′-GGAGCAGCAGGATCAGAATC-3′; N-cadherin forward 5′-CAGGGTGGACGTCATTGTAG-3′ and reverse 5′-AGGGTCTCCACCACTGATTC-3′;β-catenin forward 5′-GACCACAAGCAGAGTGCTGA-3′ and reverse 5′-CTTGCATTCCACCAGCTTCT-3′; Vimentin forward 5′-TGAAGGAAGAGATGGCTCGT − 3′ and reverse 5′-TCCAGCAGCTTCCTGTAGGT-3′; COL1A1 forward 5′-GCTCCTCTTAGGGGCCACT-3′ and reverse 5′-CCACGTCTCACCATTGGGG-3′; and FN1 forward 5′-GGCCACACCTACAACCAGTA-3′ and reverse 5′-TCGTCTCTGTCAGCTTGCAC-3′. Primer sequences for E-cadherin, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were synthesized and verified by Sangon Biotech Co., Ltd. (Shanghai, China). Western blot analysis Western blot was performed according to standard procedures. Briefly, proteins from tissue and cell lysates were extracted using RIPA buffer (Promega, USA), and protein concentrations were measured using the BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore, USA). The membranes were blocked and incubated with specific primary antibodies at 4°C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Signals were visualized using the ECL Western Blotting Substrate (Promega, USA), and band intensities were quantified using ImageJ software (NIH, USA). GAPDH was used as the internal control. The following primary antibodies were used in this study: rabbit anti-human RACK1 (Abcam, USA, 1:500), mouse anti-human GAPDH (Abcam, USA, 1:3,000), rabbit anti-human E-cadherin (Abcam, USA, 1:500), rabbit anti-human N-cadherin (Abcam, USA, 1:500), rabbit anti-human β-catenin (Abcam, USA, 1:500), rabbit anti-human Vimentin (Abcam, USA, 1:500), rabbit anti-human COL1A1 (Abcam, USA, 1:500), rabbit anti-human FN1 (Abcam, USA, 1:500), rabbit anti-human Smad2/3 (Abcam, USA, 1:500), rabbit anti-human phospho-Smad2/3 (Abcam, USA, 1:500), rabbit anti-human p65 (Abcam, USA, 1:500), and mouse anti-human phospho-p65 (Abcam, USA, 1:500). RNA sequencing and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis Total RNA was extracted from SV-HUC-1 cells under three different treatment conditions: untreated control (WT), TGF-β1-treated, and TGF-β1 combined with RACK1 knockdown (TGF-β1 + siRACK1). RNA integrity and concentration were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), and sequencing was performed on the Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) were identified using the DESeq2 package in R. Genes with |log2(fold change)| >1 and adjusted P-value < 0.05 were considered significant. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was conducted using the ClusterProfiler package to explore the biological pathways affected by TGF-β1 stimulation and RACK1 knockdown, respectively. Cell proliferation assay Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer’s instructions. SV-HUC-1 cells were seeded into 96-well plates at a density of 3 × 10³ cells per well in 100 µL of complete medium. After the indicated treatments, 10 µL of CCK-8 solution was added to each well at 0, 24, and 48 hours, followed by incubation at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader. Each group was tested in five replicate wells, and all experiments were repeated at least three times independently. Wound-healing assay The migration ability of SV-HUC-1 cells was assessed using a wound-healing assay. Briefly, 1 × 10⁵ cells were seeded into each well of 6-well plates and treated as described above. After 24 hours of treatment, a linear wound was manually created by scraping the cell monolayer with a 1 mL pipette tip, and the medium was replaced with fresh Ham’s F-12K medium containing 1% fetal bovine serum. At 0, 24, and 48 hours after wounding, cells were gently rinsed with phosphate-buffered saline and images were captured under a microscope. Statistical analysis All data were expressed as the mean ± standard deviation based on at least three independent experiments. Statistical analysis was performed using Student’s t-test or one-way analysis of variance. A P value less than 0.05 was considered statistically significant compared with the corresponding control group. Results 1. RACK1 is highly expressed in UPJO tissues and correlates with EMT and fibrosis features Histological staining showed typical pathological alterations in stenotic UPJ tissues from UPJO patients. H&E staining revealed pronounced thickening of the muscularis and disrupted tissue architecture, while Masson’s trichrome staining demonstrated extensive collagen fiber deposition in the UPJO group compared with structurally normal control tissues (Fig. 1 ). Immunohistochemistry further confirmed the histological findings. Compared with controls, UPJO tissues exhibited marked upregulation of RACK1 and COL1A1 expression, accompanied by reduced expression of the epithelial marker E-cadherin (Fig. 2 ). RT-qPCR analysis of five matched pairs of clinical samples showed that mRNA levels of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were significantly higher in UPJO tissues compared to controls, while E-cadherin was significantly lower (Fig. 3 , P < 0.05 for all comparisons). To verify these results at the protein level, we performed Western blot analysis on three paired samples. The densitometric quantification showed consistent trends with the mRNA data: RACK1 and mesenchymal/fibrotic markers (N-cadherin, β-catenin, Vimentin, COL1A1, FN1, α-SMA) were significantly upregulated in UPJO tissues, while E-cadherin expression was decreased. Statistical analysis confirmed the differences were significant (P < 0.05) (Fig. 4 ). 2. TGF-β1 stimulation induces RACK1 expression and promotes EMT and fibrosis in SV-HUC-1 cells To explore whether TGF-β1 mediates EMT and fibrosis in ureteral epithelial cells via RACK1, we stimulated SV-HUC-1 cells with recombinant TGF-β1 (10 ng/mL) for 24 hours. Transcriptome sequencing and KEGG enrichment analysis showed significant activation of pathways related to the TGF-β signaling cascades (Fig. 5 A). RT-qPCR results revealed that TGF-β1 stimulation significantly increased the mRNA expression of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1, while reducing E-cadherin expression (Fig. 6 ). Western blot analysis confirmed that protein levels of RACK1 and mesenchymal markers were elevated, and E-cadherin was suppressed (Fig. 7 ). Furthermore, TGF-β1 also activated downstream Smad2/3 and p65 signaling, as indicated by increased phosphorylation levels of p-Smad2/3 and p-p65 (Fig. 7 ). 3. RACK1 knockdown attenuates TGF-β1-induced EMT and fibrotic responses in SV-HUC-1 cells To investigate the role of RACK1 in TGF-β1-induced EMT and fibrosis, we transfected SV-HUC-1 cells with siRNA targeting RACK1. The knockdown efficiency was validated by RT-qPCR and Western blot, which showed significant reduction in RACK1 expression. Upon RACK1 knockdown, the TGF-β1-induced upregulation of mesenchymal and fibrotic markers (N-cadherin, β-catenin, Vimentin, COL1A1, FN1, α-SMA) was markedly reversed. In parallel, the loss of E-cadherin expression caused by TGF-β1 was partially restored (Fig. 6 – 7 ). Importantly, RACK1 silencing also suppressed phosphorylation of Smad2/3 and p65, indicating that RACK1 mediates TGF-β1 signal transduction through both the Smad-dependent and NF-κB pathways (Fig. 7 ). KEGG analysis further supported these findings, showing suppressed inflammation-related pathway in the siRACK1 + TGF-β1 group compared to the TGF-β1-only group (Fig. 5 B). 4. RACK1 promotes TGF-β1-induced proliferation and migration of ureteral epithelial cells To examine the functional consequences of RACK1 knockdown, we assessed cell proliferation and migration. CCK-8 assays showed that TGF-β1 significantly enhanced proliferation of SV-HUC-1 cells, which was notably inhibited after RACK1 knockdown (Fig. 8 A). Similarly, wound-healing assays revealed that TGF-β1 accelerated cell migration, while silencing RACK1 significantly impaired this response at 24 and 48 hours post-wounding (Fig. 8 B). Discussion This study confirmed that EMT occurs in the stenotic segments of the ureteropelvic junction in patients with congenital UPJO. In our in vitro model, we further showed that TGF-β1 stimulation induces EMT in ureteral epithelial cells and increases extracellular matrix production. More importantly, we identified RACK1 as a key regulator in this process for the first time. Silencing RACK1 significantly reduced the expression of mesenchymal and fibrotic markers, and also inhibited the activation of Smad2/3 and p65 signaling. These results suggest that RACK1 plays an essential role in the TGF-β1–driven EMT pathway. Together, our findings offer a new explanation for tissue remodeling and fibrosis in UPJO. Although EMT has been reported in fibrotic diseases of the urinary system [ 14 , 15 ], its role in congenital UPJO has not been well investigated. Previous molecular and transcriptomic studies on UPJO have identified abnormal expression of genes related to fibrosis (such as TGF-β1 and actin alpha 2) [ 16 , 17 ], inflammation (such as Interleukin 6) [ 18 ], and hypoxia (such as endothelin-1) [ 19 ]. However, no studies have directly confirmed the presence or function of EMT in this context, either at the tissue or cellular level. By combining clinical tissue analysis with in vitro experiments, our study is the first to demonstrate that EMT may underlie the structural changes seen in the stenotic segment of UPJO. TGF-β1 is a central regulator of EMT and fibrotic responses. It promotes transcriptional reprogramming, myofibroblast activation, and collagen deposition through both Smad-dependent and non-canonical signaling pathways [ 20 , 21 ]. Previous studies have shown that TGF-β1 expression is elevated in models of ureteral obstruction and contributes to renal interstitial fibrosis [ 22 ]. In UPJO, increased levels of TGF-β1 have also been reported in stenotic tissues [ 8 , 17 ], but these findings were mostly based on histological staining or transcriptome analysis, lacking functional validation. In our study, TGF-β1 treatment in vitro led to downregulation of E-cadherin and upregulation of mesenchymal markers including N-cadherin, β-catenin, and Vimentin, as well as fibrosis-related molecules such as COL1A1, FN1, and α-SMA. These changes closely mirrored the expression patterns seen in UPJO tissue, confirming the functional role of TGF-β1 in inducing EMT and fibrosis in ureteral epithelial cells. More importantly, this study identified RACK1 as a key effector linking TGF-β1 signaling to the EMT process in ureteral epithelial cells. RACK1 is a multifunctional scaffold protein that regulates various biological processes through its role in assembling and coordinating signaling complexes. It has been previously implicated in liver fibrosis [ 12 , 23 ]. In the respiratory system, inhibition of RACK1 expression was shown to reduce bronchial epithelial cell migration and reverse TGF-β1–induced EMT [ 24 , 25 ]. In the kidney, silencing RACK1 has been reported to alleviate renal fibrosis by blocking the TGF-β1/Smad3 pathway in epithelial cells [ 26 ]. Our study found that RACK1 is highly expressed in UPJO tissues and is also upregulated in ureteral epithelial cells following TGF-β1 stimulation. When RACK1 was knocked down, the activation of Smad2/3 and p65 was suppressed, and the progression of EMT was inhibited, indicating that RACK1 functions as a key adaptor in the TGF-β1 signaling pathway. Functional assays further showed that silencing RACK1 reduced cell proliferation and migration under TGF-β1 treatment. These findings indicate that RACK1 not only promotes EMT but also contributes to the abnormal growth and motility of epithelial cells, which may aggravate luminal narrowing and peristaltic dysfunction in UPJO. This study has some limitations. The number of clinical tissue samples was limited, which may affect the generalizability of the findings. All in vitro experiments were conducted using a single immortalized cell line, which cannot fully replicate the complex cellular environment of the developing ureteropelvic junction. In addition, although we demonstrated the involvement of RACK1 in the TGF-β1/EMT pathway, the upstream regulation of RACK1 itself and its potential interactions with other signaling molecules remain unclear. Future studies using animal models, organoid systems, or patient-derived cells will help to confirm these mechanisms and evaluate whether targeting RACK1 can be translated into therapeutic benefit in congenital UPJO. In summary, this study demonstrates that epithelial-mesenchymal transition (EMT) occurs in the stenotic ureteropelvic junction of patients with congenital UPJO and may contribute to tissue remodeling and fibrosis. TGF-β1 was shown to induce EMT and extracellular matrix production in ureteral epithelial cells, consistent with molecular changes observed in clinical specimens. We further found that RACK1 participates in this process by promoting Smad2/3 and p65 activation and facilitating EMT progression. Silencing RACK1 suppressed mesenchymal markers, reduced cell proliferation and migration, and attenuated TGF-β1 signaling activity. These findings help clarify the molecular mechanisms underlying UPJO and suggest that RACK1 may be a potential regulatory target in the treatment of congenital ureteral fibrosis. Declarations Conflict of Interest The authors declare that they have no competing interests. Funding This work was supported by Natural Science Foundation of Fujian Province (Grant No. 2023J011825) and Zhangzhou Science and Technology Program (Grant No. ZZ2023J31). Author Contribution Wenyou Chen, Fengguang Ye, and Xiaoqiang Lin contributed equally to this work and share first authorship. Wenyou Chen, Fengguang Ye, and Xiaoqiang Lin performed the majority of the experiments and data analysis. Jinrong Chen and Fengbin Yang assisted with experimental validation. Yanhui Wang and Songbin Lin contributed to tissue sample collection and pathological evaluation. Weicheng Huang and Ziwei Jian provided technical guidance and critically revised the manuscript. Chaoming Zhou designed and supervised the study and is the final corresponding author. All authors read and approved the final manuscript. References Isali I, McClellan P, Wong TR et al (2022) A systematic review of underlying genetic factors associated with ureteropelvic junction obstruction in stenotic human tissue. J Pediatr Urol 18(5):629–641. 10.1016/j.jpurol.2022.07.022 Klein J, Gonzalez J, Miravete M et al (2011) Congenital ureteropelvic junction obstruction: human disease and animal models. International journal of experimental pathology, 92(3): pp. 168 – 92. 10.1111/j.1365-2613.2010.00727.x Krajewski W, Wojciechowska J, Dembowski J et al (2017) Hydronephrosis in the course of ureteropelvic junction obstruction: An underestimated problem? Current opinions on the pathogenesis, diagnosis and treatment. Advances in clinical and experimental medicine: official organ Wroclaw Medical University. 26(5):857–864. 10.17219/acem/59509 Manfioletti G, Fedele M (2023) Epithelial-Mesenchymal Transition (EMT). Int J Mol Sci 24(14). 10.3390/ijms241411386 Shi Y, Tao M, Chen H et al (2023) Ubiquitin-specific protease 11 promotes partial epithelial-to-mesenchymal transition by deubiquitinating the epidermal growth factor receptor during kidney fibrosis. Kidney Int 103(3):544–564. 10.1016/j.kint.2022.11.027 Deng Z, Fan T, Xiao C et al (2024) TGF-β signaling in health, disease, and therapeutics. Signal Transduct Target therapy 9(1):61. 10.1038/s41392-024-01764-w Turini S, Bergandi L, Gazzano E et al (2019) Epithelial to Mesenchymal Transition in Human Mesothelial Cells Exposed to Asbestos Fibers: Role of TGF-β as Mediator of Malignant Mesothelioma Development or Metastasis via EMT Event. Int J Mol Sci 20(1). 10.3390/ijms20010150 Yang Y, Zhou X, Gao H et al (2003) The expression of epidermal growth factor and transforming growth factor-beta1 in the stenotic tissue of congenital pelvi-ureteric junction obstruction in children. J Pediatr Surg 38(11):1656–1660. 10.1016/s0022-3468(03)00577-3 Dan H, Liu S, Liu J et al (2020) RACK1 promotes cancer progression by increasing the M2/M1 macrophage ratio via the NF-κB pathway in oral squamous cell carcinoma. Mol Oncol 14(4):795–807. 10.1002/1878-0261.12644 Yoshino Y, Chiba N (2022) Roles of RACK1 in centrosome regulation and carcinogenesis. Cell Signal 90:110207. 10.1016/j.cellsig.2021.110207 Duff D, Long A (2017) Roles for RACK1 in cancer cell migration and invasion. Cell Signal 35:250–255. 10.1016/j.cellsig.2017.03.005 Jia D, Duan F, Peng P et al (2013) Up-regulation of RACK1 by TGF-β1 promotes hepatic fibrosis in mice. PLoS ONE 8(3):e60115. 10.1371/journal.pone.0060115 Bao Q, Wang A, Hong W et al (2024) The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition. Cell communication signaling: CCS 22(1):247. 10.1186/s12964-024-01603-z Xu Z, Jia K, Wang H et al (2021) METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease. Cell Death Dis 12(1):32. 10.1038/s41419-020-03312-0 Yamashita N, Kusaba T, Nakata T et al (2020) Intratubular epithelial-mesenchymal transition and tubular atrophy after kidney injury in mice. Am J Physiol Renal Physiol 319(4):F579–F591. 10.1152/ajprenal.00108.2020 Knerr I, Dittrich K, Miller J et al (2001) Alteration of neuronal and endothelial nitric oxide synthase and neuropeptide Y in congenital ureteropelvic junction obstruction. Urol Res 29(2):134–140. 10.1007/s002400000165 Yang Y, Hou Y, Wang C-L et al (2006) Renal expression of epidermal growth factor and transforming growth factor-beta1 in children with congenital hydronephrosis. Urology 67(4) 817 – 21; discussion 821-2. 10.1016/j.urology.2005.10.062 Ruiz-Deya G, Sikka SC, Thomas R et al (2002) Potential role for the nuclear transcription factor NF-kappa B in the pathogenesis of ureteropelvic junction obstruction. J Endourol 16(8):611–615. 10.1089/089277902320913323 Knerr I, Nyul Z, Miller J et al (2001) Increased endothelin-1 and decreased adrenomedullin gene expression in the stenotic tissue of congenital pelvi-ureteric junction obstruction in children. BJU Int 87(7):667–671. 10.1046/j.1464-410x.2001.02173.x Hong Q, Cai H, Zhang L et al (2022) Modulation of transforming growth factor-β-induced kidney fibrosis by leucine-rich ⍺-2 glycoprotein-1. Kidney international, 101(2): pp. 299–314. 10.1016/j.kint.2021.10.023 Khongpiroon C, Buakaew W, Brindley PJ et al (2025) Effect of 3-HBI on Liver Fibrosis via the TGF-β/SMAD2/3 Pathway on the Human Hepatic Stellate Cell Model. Int J Mol Sci 26(13). 10.3390/ijms26136022 Wu C-F, Chiang W-C, Lai C-F et al (2013) Transforming growth factor β-1 stimulates profibrotic epithelial signaling to activate pericyte-myofibroblast transition in obstructive kidney fibrosis. Am J Pathol 182(1):118–131. 10.1016/j.ajpath.2012.09.009 Liu M, Peng P, Wang J et al (2015) RACK1-mediated translation control promotes liver fibrogenesis. Biochem Biophys Res Commun 463(3):255–261. 10.1016/j.bbrc.2015.05.040 Pu Y, Wu Y, Zhou Y et al Azithromycin suppresses TGF-β1-related epithelial-mesenchymal transition in airway epithelial cells via targeting RACK1. Chemico-biological interactions, 2023. 370: p. 110332. 10.1016/j.cbi.2022.110332 Pu Y, Liu Y-Q, Zhou Y et al (2020) Dual role of RACK1 in airway epithelial mesenchymal transition and apoptosis. J Cell Mol Med 24(6):3656–3668. 10.1111/jcmm.15061 Feng J, Xie L, Kong R et al (2017) RACK1 silencing attenuates renal fibrosis by inhibiting TGF-β signaling. Int J Mol Med 40(6):1965–1970. 10.3892/ijmm.2017.3154 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7164773","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504687590,"identity":"7630dd6b-f6c8-4065-85a2-de0fcea3975d","order_by":0,"name":"Wenyou Chen","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenyou","middleName":"","lastName":"Chen","suffix":""},{"id":504687591,"identity":"61b72b71-2144-4e51-b1ef-453c212212df","order_by":1,"name":"Fengguang Ye","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fengguang","middleName":"","lastName":"Ye","suffix":""},{"id":504687592,"identity":"8d5ed704-61c1-4c5b-bf1e-1f3325481a51","order_by":2,"name":"Xiaoqiang Lin","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqiang","middleName":"","lastName":"Lin","suffix":""},{"id":504687593,"identity":"2c497ad9-429c-454f-9ca6-713515ea0f58","order_by":3,"name":"Jinrong Chen","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinrong","middleName":"","lastName":"Chen","suffix":""},{"id":504687594,"identity":"3b38430e-9f80-4861-821e-a96fdac254b7","order_by":4,"name":"Fengbin Yang","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fengbin","middleName":"","lastName":"Yang","suffix":""},{"id":504687595,"identity":"b0b79e65-b658-414b-ae2f-e0d7bdca844b","order_by":5,"name":"Yanhui Wang","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanhui","middleName":"","lastName":"Wang","suffix":""},{"id":504687596,"identity":"a9a0576f-1fcb-4f67-b1fe-6eb9388bbc23","order_by":6,"name":"Songbin Lin","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Songbin","middleName":"","lastName":"Lin","suffix":""},{"id":504687597,"identity":"a65542e0-90df-44d8-819e-af7f4d2bced8","order_by":7,"name":"Weicheng Huang","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weicheng","middleName":"","lastName":"Huang","suffix":""},{"id":504687598,"identity":"e0738844-6ede-4894-aa0e-f555d533b6fd","order_by":8,"name":"Ziwei Jian","email":"","orcid":"","institution":"Zhangzhou Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziwei","middleName":"","lastName":"Jian","suffix":""},{"id":504687599,"identity":"b3661243-012a-47b3-bc5c-9658f9ac90e9","order_by":9,"name":"Chaoming Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3QMQrCMBSA4VeE6BDIJoEOXiFSEEGhV6kIuig4iZMUhE7OYuklPEJKhi7BC3SxOLjGrYODEamjySiYfwh58L4hAXC5frB2zJtri3NVWxDMNXkrNMnTvRWBhuBAdJAN8Yrqek9ESA5SCcDQI11uICADlieiRcvlSayG0E+z6DsJQSKqCWK+JkcMESsNBENxqzXBzF9cBEZWhA9AE6oJ2BIZUHmeM1rOmP5kan4LpkWlNutRSLLpVal63CO+gQDlAB7afUbD+isS6+Oxtdh0uVyuv+0JrO5K9u56Vy0AAAAASUVORK5CYII=","orcid":"","institution":"Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chaoming","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-07-19 13:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7164773/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7164773/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89983783,"identity":"91dc4685-25cf-4da1-bb46-d9af46885589","added_by":"auto","created_at":"2025-08-27 06:34:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3830104,"visible":true,"origin":"","legend":"\u003cp\u003eHistological features of ureteropelvic junction (UPJ) tissues from control and UPJO patients. Hematoxylin and eosin (H\u0026amp;E) staining shows normal architecture in control tissues and marked smooth muscle hyperplasia in UPJO tissues. Masson’s trichrome staining reveals collagen deposition (blue) in stenotic segments from UPJO patients. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/f0640bcf3a2cb9171e5fdd79.png"},{"id":89983790,"identity":"8339fe3f-d5aa-4574-99a7-a1ce072d4bcc","added_by":"auto","created_at":"2025-08-27 06:34:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4539051,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of RACK1, COL1A1, and E-cadherin in control and UPJO tissues. Compared to controls, UPJO tissues exhibited increased expression of RACK1 and COL1A1, and decreased expression of the epithelial marker E-cadherin. Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/d7a1553a3c1047bd7355b240.png"},{"id":89983791,"identity":"b8e3c7ac-ac30-48e2-87c8-bb7a6c85703f","added_by":"auto","created_at":"2025-08-27 06:34:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127006,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression of EMT and fibrosis markers in UPJO and control tissues. RT-qPCR analysis showed significant upregulation of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1, and downregulation of E-cadherin in UPJO tissues (n = 5) compared to controls (n = 5). Results were shown as mean ± SD. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/cdf10aeaea9682e6fa109f19.png"},{"id":89983797,"identity":"0b8ed6fc-69f6-404f-aa12-83a851ba8f18","added_by":"auto","created_at":"2025-08-27 06:34:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320795,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of EMT and fibrosis markers in UPJO and control tissues. Western blotting was performed on stenotic UPJ tissues from three UPJO patients and three control tissues. Quantification of band intensities showed that RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, FN1, and α-SMA protein levels were significantly increased in UPJO tissues, while the epithelial marker E-cadherin was significantly decreased. Statistical analysis was conducted using unpaired t-tests, and results are presented as mean ± SD (n = 3 per group). GAPDH was used as a loading control. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/8394153c93cc3c45f8fcbdeb.png"},{"id":89983784,"identity":"2b0bc294-05d8-4a84-901a-da485578bc18","added_by":"auto","created_at":"2025-08-27 06:34:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":329417,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway analysis of transcriptomic changes in SV-HUC-1 cells. (A) KEGG enrichment analysis of differentially expressed genes (DEGs) in SV-HUC-1 cells treated with TGF-β1 versus untreated control (WT) revealed significant upregulation of signaling pathways related to the TGF-β pathways. (B) Comparison of TGF-β1 + siRACK1 group versus TGF-β1 group showed that knockdown of RACK1 markedly reversed the activation of these pathways, particularly TGF-β/Smad and NF-κB signaling, suggesting that RACK1 is required for the transcriptional response to TGF-β1 stimulation.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/b0f9ca4eea2a47c0574860a5.png"},{"id":89985294,"identity":"e2b515ab-41ed-49c6-ae3a-ad7f8ec296df","added_by":"auto","created_at":"2025-08-27 06:42:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":311669,"visible":true,"origin":"","legend":"\u003cp\u003eRT-qPCR analysis of EMT and fibrosis markers in SV-HUC-1 cells. Compared to untreated cells (WT), TGF-β1 stimulation increased the mRNA levels of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1, and decreased E-cadherin expression. RACK1 knockdown reversed these changes. Data represent mean ± SD from three independent experiments. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/e0259354c0a8f10b4244f653.png"},{"id":89985293,"identity":"f88d5043-0ebc-49e8-8319-53023ec3b525","added_by":"auto","created_at":"2025-08-27 06:42:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":59174,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of EMT and fibrosis-related proteins in SV-HUC-1 cells. TGF-β1 promoted RACK1 and mesenchymal marker expression while reducing E-cadherin. RACK1 knockdown suppressed p-Smad2/3 and p-p65 activation, and reduced fibrotic markers. GAPDH served as the loading control.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/d47e93b0d3cc104b12114463.jpg"},{"id":89983792,"identity":"c8915fe1-6cdd-4097-8752-152e440bce57","added_by":"auto","created_at":"2025-08-27 06:34:31","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142944,"visible":true,"origin":"","legend":"\u003cp\u003eRACK1 knockdown inhibits TGF-β1-induced proliferation and migration of SV-HUC-1 cells. (A) CCK-8 assay shows that TGF-β1 promotes cell proliferation, which is significantly reduced after RACK1 knockdown. (B) Wound-healing assay demonstrates enhanced migration by TGF-β1, which is impaired by siRACK1. Scale bar: 200 µm. Data was shown as mean ±SD. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/7acc7512156ea2ffe4487792.jpg"},{"id":100379845,"identity":"a9ad0a86-ff55-41d1-aacd-e72f4d6c8599","added_by":"auto","created_at":"2026-01-16 09:43:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9889218,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7164773/v1/f459d448-655d-4434-bedb-b882b4567a8a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TGF-β1 induces epithelial-mesenchymal transition and fibrosis of ureteral epithelial cells via targeting RACK1 in ureteropelvic junction obstruction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUreteropelvic junction obstruction (UPJO) is the most common cause of congenital hydronephrosis in children, with an estimated incidence of 1 in 1,500 live births [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It results from impaired urine flow between the renal pelvis and proximal ureter, often due to intrinsic muscular dysplasia or extrinsic vascular compression. If left untreated, UPJO can lead to progressive renal function deterioration, making early diagnosis and timely intervention crucial [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Histologically, stenotic segments of the ureteropelvic junction (UPJ) are characterized by smooth muscle disorganization, increased collagen deposition, and reduced peristalsis, indicative of chronic tissue remodeling and fibrosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the cellular and molecular mechanisms underlying these pathological changes remain incompletely understood.\u003c/p\u003e\u003cp\u003eEpithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their polarity and adhesion properties while acquiring mesenchymal characteristics, such as enhanced motility and extracellular matrix (ECM) production [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. EMT is essential for embryonic development and wound healing, but is also implicated in pathological tissue fibrosis in various organs, including the kidney and urinary tract [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Transforming growth factor-beta 1 (TGF-β1) is a master regulator of EMT and fibrosis, known to activate both canonical Smad2/3 and non-canonical signaling pathways such as NF-κB [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Elevated expression of TGF-β1 has been observed in the stenotic renal pelvis of UPJO patients, where it contributes to excessive muscle and collagen formation and aberrant scar remodeling [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the downstream molecular effectors linking TGF-β1 to EMT in ureteral epithelial cells remain largely unknown.\u003c/p\u003e\u003cp\u003eReceptor for activated C kinase 1 (RACK1) is a multifunctional scaffold protein containing WD40 repeats that regulates diverse signaling pathways, including TGF-β, integrin, and NF-κB [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. RACK1 modulates cellular processes such as migration, adhesion, and differentiation by facilitating signal complex assembly [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although RACK1 has been implicated in cancer progression and tissue fibrosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], its role in congenital urinary tract malformations such as UPJO has not yet been explored. Whether RACK1 acts as a mediator of TGF-β1-induced EMT and fibrotic remodeling in ureteral epithelial cells is unknown.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate the expression and function of RACK1 in the pathogenesis of UPJO. We analyzed RACK1 expression in clinical UPJ tissue samples and examined its role in TGF-β1-induced EMT and fibrosis in SV-HUC-1 ureteral epithelial cells. Furthermore, we evaluated the downstream signaling pathways involved and assessed the effects of RACK1 knockdown. Our findings identify RACK1 as a novel regulator of EMT and fibrosis in UPJO and suggest its potential as a therapeutic target for congenital ureteral obstruction.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003ePatients\u003c/em\u003e\u003c/p\u003e\u003cp\u003eStenotic tissue samples from the ureteropelvic junction (UPJ) were collected from five pediatric patients with congenital ureteropelvic junction obstruction (UPJO) who underwent pyeloplasty at Zhangzhou Municipal Hospital, Fujian Province, China. The diagnosis of UPJO was based on ultrasonography, radiological imaging, and renal scintigraphy, and was confirmed by histopathological examination of the resected stenotic segments, which showed features such as smooth muscle hypertrophy or hyperplasia and collagen deposition. Control UPJ tissues were obtained from five pediatric patients with renal dysplasia accompanied by duplicated kidneys who underwent nephrectomy. Histological examination confirmed that the UPJ segments in these patients were non-obstructed and structurally normal. All tissue samples were immediately snap-frozen in liquid nitrogen and stored for further analysis. This study was approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University (Zhangzhou, Fujian, China), with approval number 2024KYZ357. Written informed consent was obtained from all participants or their guardians prior to sample collection.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHistologic Analysis and Immunohistochemistry\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFor histological analysis, tissue specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 7 \u0026micro;m. Hematoxylin and eosin (H\u0026amp;E) staining was performed using standard protocols to assess general tissue morphology. Masson's trichrome staining was conducted to evaluate collagen fiber deposition. Immunohistochemical staining was performed using the EnVision\u0026trade;+ System-HRP (Dako, USA) following the manufacturer\u0026rsquo;s instructions. Paraffin-embedded tissue sections were incubated with primary antibodies against human RACK1 (Abcam, USA, 1:200), E-cadherin (Abcam, USA, 1:500), and COL1A1 (Abcam, USA, 1:500) overnight at 4\u0026deg;C. After washing with phosphate-buffered saline (PBS), sections were incubated with HRP-conjugated secondary antibodies provided in the EnVision\u0026trade;+ kit for 30 minutes at room temperature. The signal was developed using diaminobenzidine (DAB) for 5 minutes, followed by counterstaining with hematoxylin.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eV-HUC-1 cells (Simian Virus 40-transformed Human Urothelial Cells) were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in Ham\u0026rsquo;s F-12K medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). Cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. They were seeded in 6-well plates at a density of 1.5 \u0026times; 10⁵ cells/mL and used for experiments when they reached 70\u0026ndash;80% confluence. EMT was induced by treating cells with 10 ng/mL of recombinant human TGF-β1 (PeproTech, USA; Cat# 100\u0026thinsp;\u0026minus;\u0026thinsp;21) for 24 hours. All experiments were performed in triplicate and repeated independently at least three times.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSmall interfering RNA (SiRNA) transfection\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo reduce RACK1 expression in SV-HUC-1 cells, a specific small interfering RNA (siRNA) targeting RACK1 was synthesized. The sequences were as follows: sense 5\u0026prime;-CUCUGGAUCUCGAGAUAAAdTdT-3\u0026prime; and antisense 5\u0026prime;-UUUAUCUCGAGAUCCAGAGdTdT-3\u0026prime;. A scrambled siRNA with no homology was used as a negative control. Its sequences were: sense 5\u0026prime;-UUCUCCGAACGUGUCACGUdTdT-3\u0026prime; and antisense 5\u0026prime;-ACGUGACACGUUCGGAGAAdTdT-3\u0026prime;. Transfection was carried out using Lipofectamine\u0026reg; 2000 (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s protocol. Both RACK1-targeting and control siRNAs were obtained from Bioscien Biotechnology Co., Ltd. (Shanghai, China). To assess transfection efficiency, RACK1 expression at both mRNA and protein levels was measured by reverse transcription-quantitative PCR and western blotting.\u003c/p\u003e\u003cp\u003e\u003cem\u003eReal-time quantitative PCR (RT-qPCR)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal RNA from clinical tissue samples and treated SV-HUC-1 cells was isolated using TRIzol\u0026reg; reagent (Thermo Fisher Scientific, USA) following the standard protocol. The mRNA expression levels of RACK1, GAPDH, E-cadherin, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the One-Step RT-qPCR Kit (Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions. GAPDH was used as the internal control, and relative gene expression was calculated using the 2^\u0026minus;ΔΔCt method. The primer sequences were as follows: RACK1 forward 5\u0026prime;-AGATAAGACCATCATCAT-3\u0026prime; and reverse 5\u0026prime;-AGATAACCACATCACTAA-3\u0026prime;; GAPDH forward 5\u0026prime;-TGTGAGGGAGATGCTCAGTG-3\u0026prime; and reverse 5\u0026prime;-TGTTCCTACCCCCAATGTGT-3\u0026prime;; E-cadherin forward 5\u0026prime;-ACTGTGAAGGGACGGTCAAC-3\u0026prime; and reverse 5\u0026prime;-GGAGCAGCAGGATCAGAATC-3\u0026prime;; N-cadherin forward 5\u0026prime;-CAGGGTGGACGTCATTGTAG-3\u0026prime; and reverse 5\u0026prime;-AGGGTCTCCACCACTGATTC-3\u0026prime;;β-catenin forward 5\u0026prime;-GACCACAAGCAGAGTGCTGA-3\u0026prime; and reverse 5\u0026prime;-CTTGCATTCCACCAGCTTCT-3\u0026prime;; Vimentin forward 5\u0026prime;-TGAAGGAAGAGATGGCTCGT \u0026minus;\u0026thinsp;3\u0026prime; and reverse 5\u0026prime;-TCCAGCAGCTTCCTGTAGGT-3\u0026prime;; COL1A1 forward 5\u0026prime;-GCTCCTCTTAGGGGCCACT-3\u0026prime; and reverse 5\u0026prime;-CCACGTCTCACCATTGGGG-3\u0026prime;; and FN1 forward 5\u0026prime;-GGCCACACCTACAACCAGTA-3\u0026prime; and reverse 5\u0026prime;-TCGTCTCTGTCAGCTTGCAC-3\u0026prime;. Primer sequences for E-cadherin, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were synthesized and verified by Sangon Biotech Co., Ltd. (Shanghai, China).\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern blot analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWestern blot was performed according to standard procedures. Briefly, proteins from tissue and cell lysates were extracted using RIPA buffer (Promega, USA), and protein concentrations were measured using the BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore, USA). The membranes were blocked and incubated with specific primary antibodies at 4\u0026deg;C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Signals were visualized using the ECL Western Blotting Substrate (Promega, USA), and band intensities were quantified using ImageJ software (NIH, USA). GAPDH was used as the internal control. The following primary antibodies were used in this study: rabbit anti-human RACK1 (Abcam, USA, 1:500), mouse anti-human GAPDH (Abcam, USA, 1:3,000), rabbit anti-human E-cadherin (Abcam, USA, 1:500), rabbit anti-human N-cadherin (Abcam, USA, 1:500), rabbit anti-human β-catenin (Abcam, USA, 1:500), rabbit anti-human Vimentin (Abcam, USA, 1:500), rabbit anti-human COL1A1 (Abcam, USA, 1:500), rabbit anti-human FN1 (Abcam, USA, 1:500), rabbit anti-human Smad2/3 (Abcam, USA, 1:500), rabbit anti-human phospho-Smad2/3 (Abcam, USA, 1:500), rabbit anti-human p65 (Abcam, USA, 1:500), and mouse anti-human phospho-p65 (Abcam, USA, 1:500).\u003c/p\u003e\u003cp\u003e\u003cem\u003eRNA sequencing and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from SV-HUC-1 cells under three different treatment conditions: untreated control (WT), TGF-β1-treated, and TGF-β1 combined with RACK1 knockdown (TGF-β1\u0026thinsp;+\u0026thinsp;siRACK1). RNA integrity and concentration were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Libraries were constructed using the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (NEB, USA), and sequencing was performed on the Illumina NovaSeq 6000 platform.\u003c/p\u003e\u003cp\u003eDifferentially expressed genes (DEGs) were identified using the DESeq2 package in R. Genes with |log2(fold change)| \u0026gt;1 and adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was conducted using the ClusterProfiler package to explore the biological pathways affected by TGF-β1 stimulation and RACK1 knockdown, respectively.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell proliferation assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the manufacturer\u0026rsquo;s instructions. SV-HUC-1 cells were seeded into 96-well plates at a density of 3 \u0026times; 10\u0026sup3; cells per well in 100 \u0026micro;L of complete medium. After the indicated treatments, 10 \u0026micro;L of CCK-8 solution was added to each well at 0, 24, and 48 hours, followed by incubation at 37\u0026deg;C for 2 hours. The absorbance was measured at 450 nm using a microplate reader. Each group was tested in five replicate wells, and all experiments were repeated at least three times independently.\u003c/p\u003e\u003cp\u003e\u003cem\u003eWound-healing assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe migration ability of SV-HUC-1 cells was assessed using a wound-healing assay. Briefly, 1 \u0026times; 10⁵ cells were seeded into each well of 6-well plates and treated as described above. After 24 hours of treatment, a linear wound was manually created by scraping the cell monolayer with a 1 mL pipette tip, and the medium was replaced with fresh Ham\u0026rsquo;s F-12K medium containing 1% fetal bovine serum. At 0, 24, and 48 hours after wounding, cells were gently rinsed with phosphate-buffered saline and images were captured under a microscope.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation based on at least three independent experiments. Statistical analysis was performed using Student\u0026rsquo;s t-test or one-way analysis of variance. A P value less than 0.05 was considered statistically significant compared with the corresponding control group.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003e1. RACK1 is highly expressed in UPJO tissues and correlates with EMT and fibrosis features\u003c/em\u003e\u003c/p\u003e\u003cp\u003eHistological staining showed typical pathological alterations in stenotic UPJ tissues from UPJO patients. H\u0026amp;E staining revealed pronounced thickening of the muscularis and disrupted tissue architecture, while Masson\u0026rsquo;s trichrome staining demonstrated extensive collagen fiber deposition in the UPJO group compared with structurally normal control tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemistry further confirmed the histological findings. Compared with controls, UPJO tissues exhibited marked upregulation of RACK1 and COL1A1 expression, accompanied by reduced expression of the epithelial marker E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRT-qPCR analysis of five matched pairs of clinical samples showed that mRNA levels of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1 were significantly higher in UPJO tissues compared to controls, while E-cadherin was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo verify these results at the protein level, we performed Western blot analysis on three paired samples. The densitometric quantification showed consistent trends with the mRNA data: RACK1 and mesenchymal/fibrotic markers (N-cadherin, β-catenin, Vimentin, COL1A1, FN1, α-SMA) were significantly upregulated in UPJO tissues, while E-cadherin expression was decreased. Statistical analysis confirmed the differences were significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e2. TGF-β1 stimulation induces RACK1 expression and promotes EMT and fibrosis in SV-HUC-1 cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo explore whether TGF-β1 mediates EMT and fibrosis in ureteral epithelial cells via RACK1, we stimulated SV-HUC-1 cells with recombinant TGF-β1 (10 ng/mL) for 24 hours. Transcriptome sequencing and KEGG enrichment analysis showed significant activation of pathways related to the TGF-β signaling cascades (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRT-qPCR results revealed that TGF-β1 stimulation significantly increased the mRNA expression of RACK1, N-cadherin, β-catenin, Vimentin, COL1A1, and FN1, while reducing E-cadherin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Western blot analysis confirmed that protein levels of RACK1 and mesenchymal markers were elevated, and E-cadherin was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Furthermore, TGF-β1 also activated downstream Smad2/3 and p65 signaling, as indicated by increased phosphorylation levels of p-Smad2/3 and p-p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e3. RACK1 knockdown attenuates TGF-β1-induced EMT and fibrotic responses in SV-HUC-1 cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of RACK1 in TGF-β1-induced EMT and fibrosis, we transfected SV-HUC-1 cells with siRNA targeting RACK1. The knockdown efficiency was validated by RT-qPCR and Western blot, which showed significant reduction in RACK1 expression.\u003c/p\u003e\u003cp\u003eUpon RACK1 knockdown, the TGF-β1-induced upregulation of mesenchymal and fibrotic markers (N-cadherin, β-catenin, Vimentin, COL1A1, FN1, α-SMA) was markedly reversed. In parallel, the loss of E-cadherin expression caused by TGF-β1 was partially restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Importantly, RACK1 silencing also suppressed phosphorylation of Smad2/3 and p65, indicating that RACK1 mediates TGF-β1 signal transduction through both the Smad-dependent and NF-κB pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). KEGG analysis further supported these findings, showing suppressed inflammation-related pathway in the siRACK1\u0026thinsp;+\u0026thinsp;TGF-β1 group compared to the TGF-β1-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cem\u003e4. RACK1 promotes TGF-β1-induced proliferation and migration of ureteral epithelial cells\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo examine the functional consequences of RACK1 knockdown, we assessed cell proliferation and migration. CCK-8 assays showed that TGF-β1 significantly enhanced proliferation of SV-HUC-1 cells, which was notably inhibited after RACK1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Similarly, wound-healing assays revealed that TGF-β1 accelerated cell migration, while silencing RACK1 significantly impaired this response at 24 and 48 hours post-wounding (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study confirmed that EMT occurs in the stenotic segments of the ureteropelvic junction in patients with congenital UPJO. In our in vitro model, we further showed that TGF-β1 stimulation induces EMT in ureteral epithelial cells and increases extracellular matrix production. More importantly, we identified RACK1 as a key regulator in this process for the first time. Silencing RACK1 significantly reduced the expression of mesenchymal and fibrotic markers, and also inhibited the activation of Smad2/3 and p65 signaling. These results suggest that RACK1 plays an essential role in the TGF-β1\u0026ndash;driven EMT pathway. Together, our findings offer a new explanation for tissue remodeling and fibrosis in UPJO.\u003c/p\u003e\u003cp\u003eAlthough EMT has been reported in fibrotic diseases of the urinary system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], its role in congenital UPJO has not been well investigated. Previous molecular and transcriptomic studies on UPJO have identified abnormal expression of genes related to fibrosis (such as TGF-β1 and actin alpha 2) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], inflammation (such as Interleukin 6) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and hypoxia (such as endothelin-1) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, no studies have directly confirmed the presence or function of EMT in this context, either at the tissue or cellular level. By combining clinical tissue analysis with in vitro experiments, our study is the first to demonstrate that EMT may underlie the structural changes seen in the stenotic segment of UPJO.\u003c/p\u003e\u003cp\u003eTGF-β1 is a central regulator of EMT and fibrotic responses. It promotes transcriptional reprogramming, myofibroblast activation, and collagen deposition through both Smad-dependent and non-canonical signaling pathways [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Previous studies have shown that TGF-β1 expression is elevated in models of ureteral obstruction and contributes to renal interstitial fibrosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In UPJO, increased levels of TGF-β1 have also been reported in stenotic tissues [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], but these findings were mostly based on histological staining or transcriptome analysis, lacking functional validation. In our study, TGF-β1 treatment in vitro led to downregulation of E-cadherin and upregulation of mesenchymal markers including N-cadherin, β-catenin, and Vimentin, as well as fibrosis-related molecules such as COL1A1, FN1, and α-SMA. These changes closely mirrored the expression patterns seen in UPJO tissue, confirming the functional role of TGF-β1 in inducing EMT and fibrosis in ureteral epithelial cells.\u003c/p\u003e\u003cp\u003eMore importantly, this study identified RACK1 as a key effector linking TGF-β1 signaling to the EMT process in ureteral epithelial cells. RACK1 is a multifunctional scaffold protein that regulates various biological processes through its role in assembling and coordinating signaling complexes. It has been previously implicated in liver fibrosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the respiratory system, inhibition of RACK1 expression was shown to reduce bronchial epithelial cell migration and reverse TGF-β1\u0026ndash;induced EMT [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the kidney, silencing RACK1 has been reported to alleviate renal fibrosis by blocking the TGF-β1/Smad3 pathway in epithelial cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our study found that RACK1 is highly expressed in UPJO tissues and is also upregulated in ureteral epithelial cells following TGF-β1 stimulation. When RACK1 was knocked down, the activation of Smad2/3 and p65 was suppressed, and the progression of EMT was inhibited, indicating that RACK1 functions as a key adaptor in the TGF-β1 signaling pathway. Functional assays further showed that silencing RACK1 reduced cell proliferation and migration under TGF-β1 treatment. These findings indicate that RACK1 not only promotes EMT but also contributes to the abnormal growth and motility of epithelial cells, which may aggravate luminal narrowing and peristaltic dysfunction in UPJO.\u003c/p\u003e\u003cp\u003eThis study has some limitations. The number of clinical tissue samples was limited, which may affect the generalizability of the findings. All in vitro experiments were conducted using a single immortalized cell line, which cannot fully replicate the complex cellular environment of the developing ureteropelvic junction. In addition, although we demonstrated the involvement of RACK1 in the TGF-β1/EMT pathway, the upstream regulation of RACK1 itself and its potential interactions with other signaling molecules remain unclear. Future studies using animal models, organoid systems, or patient-derived cells will help to confirm these mechanisms and evaluate whether targeting RACK1 can be translated into therapeutic benefit in congenital UPJO.\u003c/p\u003e\u003cp\u003eIn summary, this study demonstrates that epithelial-mesenchymal transition (EMT) occurs in the stenotic ureteropelvic junction of patients with congenital UPJO and may contribute to tissue remodeling and fibrosis. TGF-β1 was shown to induce EMT and extracellular matrix production in ureteral epithelial cells, consistent with molecular changes observed in clinical specimens. We further found that RACK1 participates in this process by promoting Smad2/3 and p65 activation and facilitating EMT progression. Silencing RACK1 suppressed mesenchymal markers, reduced cell proliferation and migration, and attenuated TGF-β1 signaling activity. These findings help clarify the molecular mechanisms underlying UPJO and suggest that RACK1 may be a potential regulatory target in the treatment of congenital ureteral fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Natural Science Foundation of Fujian Province (Grant No. 2023J011825) and Zhangzhou Science and Technology Program (Grant No. ZZ2023J31).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWenyou Chen, Fengguang Ye, and Xiaoqiang Lin contributed equally to this work and share first authorship. Wenyou Chen, Fengguang Ye, and Xiaoqiang Lin performed the majority of the experiments and data analysis. Jinrong Chen and Fengbin Yang assisted with experimental validation. Yanhui Wang and Songbin Lin contributed to tissue sample collection and pathological evaluation. Weicheng Huang and Ziwei Jian provided technical guidance and critically revised the manuscript. Chaoming Zhou designed and supervised the study and is the final corresponding author. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIsali I, McClellan P, Wong TR et al (2022) A systematic review of underlying genetic factors associated with ureteropelvic junction obstruction in stenotic human tissue. J Pediatr Urol 18(5):629\u0026ndash;641. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jpurol.2022.07.022\u003c/span\u003e\u003cspan address=\"10.1016/j.jpurol.2022.07.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKlein J, Gonzalez J, Miravete M et al (2011) \u003cem\u003eCongenital ureteropelvic junction obstruction: human disease and animal models.\u003c/em\u003e International journal of experimental pathology, 92(3): pp. 168\u0026thinsp;\u0026ndash;\u0026thinsp;92.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2613.2010.00727.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2613.2010.00727.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrajewski W, Wojciechowska J, Dembowski J et al (2017) \u003cem\u003eHydronephrosis in the course of ureteropelvic junction obstruction: An underestimated problem? Current opinions on the pathogenesis, diagnosis and treatment.\u003c/em\u003e Advances in clinical and experimental medicine: official organ Wroclaw Medical University. 26(5):857\u0026ndash;864. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17219/acem/59509\u003c/span\u003e\u003cspan address=\"10.17219/acem/59509\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManfioletti G, Fedele M (2023) Epithelial-Mesenchymal Transition (EMT). Int J Mol Sci 24(14). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms241411386\u003c/span\u003e\u003cspan address=\"10.3390/ijms241411386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi Y, Tao M, Chen H et al (2023) Ubiquitin-specific protease 11 promotes partial epithelial-to-mesenchymal transition by deubiquitinating the epidermal growth factor receptor during kidney fibrosis. Kidney Int 103(3):544\u0026ndash;564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.kint.2022.11.027\u003c/span\u003e\u003cspan address=\"10.1016/j.kint.2022.11.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng Z, Fan T, Xiao C et al (2024) TGF-β signaling in health, disease, and therapeutics. Signal Transduct Target therapy 9(1):61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-024-01764-w\u003c/span\u003e\u003cspan address=\"10.1038/s41392-024-01764-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurini S, Bergandi L, Gazzano E et al (2019) Epithelial to Mesenchymal Transition in Human Mesothelial Cells Exposed to Asbestos Fibers: Role of TGF-β as Mediator of Malignant Mesothelioma Development or Metastasis via EMT Event. Int J Mol Sci 20(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms20010150\u003c/span\u003e\u003cspan address=\"10.3390/ijms20010150\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Zhou X, Gao H et al (2003) The expression of epidermal growth factor and transforming growth factor-beta1 in the stenotic tissue of congenital pelvi-ureteric junction obstruction in children. J Pediatr Surg 38(11):1656\u0026ndash;1660. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0022-3468(03)00577-3\u003c/span\u003e\u003cspan address=\"10.1016/s0022-3468(03)00577-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDan H, Liu S, Liu J et al (2020) RACK1 promotes cancer progression by increasing the M2/M1 macrophage ratio via the NF-κB pathway in oral squamous cell carcinoma. Mol Oncol 14(4):795\u0026ndash;807. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/1878-0261.12644\u003c/span\u003e\u003cspan address=\"10.1002/1878-0261.12644\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshino Y, Chiba N (2022) Roles of RACK1 in centrosome regulation and carcinogenesis. Cell Signal 90:110207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cellsig.2021.110207\u003c/span\u003e\u003cspan address=\"10.1016/j.cellsig.2021.110207\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuff D, Long A (2017) Roles for RACK1 in cancer cell migration and invasion. Cell Signal 35:250\u0026ndash;255. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cellsig.2017.03.005\u003c/span\u003e\u003cspan address=\"10.1016/j.cellsig.2017.03.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia D, Duan F, Peng P et al (2013) Up-regulation of RACK1 by TGF-β1 promotes hepatic fibrosis in mice. PLoS ONE 8(3):e60115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0060115\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0060115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBao Q, Wang A, Hong W et al (2024) The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition. Cell communication signaling: CCS 22(1):247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12964-024-01603-z\u003c/span\u003e\u003cspan address=\"10.1186/s12964-024-01603-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu Z, Jia K, Wang H et al (2021) METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease. Cell Death Dis 12(1):32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-020-03312-0\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-03312-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamashita N, Kusaba T, Nakata T et al (2020) Intratubular epithelial-mesenchymal transition and tubular atrophy after kidney injury in mice. Am J Physiol Renal Physiol 319(4):F579\u0026ndash;F591. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajprenal.00108.2020\u003c/span\u003e\u003cspan address=\"10.1152/ajprenal.00108.2020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKnerr I, Dittrich K, Miller J et al (2001) Alteration of neuronal and endothelial nitric oxide synthase and neuropeptide Y in congenital ureteropelvic junction obstruction. Urol Res 29(2):134\u0026ndash;140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s002400000165\u003c/span\u003e\u003cspan address=\"10.1007/s002400000165\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Hou Y, Wang C-L et al (2006) Renal expression of epidermal growth factor and transforming growth factor-beta1 in children with congenital hydronephrosis. Urology 67(4) 817\u0026thinsp;\u0026ndash;\u0026thinsp;21; discussion 821-2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.urology.2005.10.062\u003c/span\u003e\u003cspan address=\"10.1016/j.urology.2005.10.062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuiz-Deya G, Sikka SC, Thomas R et al (2002) Potential role for the nuclear transcription factor NF-kappa B in the pathogenesis of ureteropelvic junction obstruction. J Endourol 16(8):611\u0026ndash;615. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/089277902320913323\u003c/span\u003e\u003cspan address=\"10.1089/089277902320913323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKnerr I, Nyul Z, Miller J et al (2001) Increased endothelin-1 and decreased adrenomedullin gene expression in the stenotic tissue of congenital pelvi-ureteric junction obstruction in children. BJU Int 87(7):667\u0026ndash;671. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1464-410x.2001.02173.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1464-410x.2001.02173.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong Q, Cai H, Zhang L et al (2022) \u003cem\u003eModulation of transforming growth factor-β-induced kidney fibrosis by leucine-rich ⍺-2 glycoprotein-1.\u003c/em\u003e Kidney international, 101(2): pp. 299\u0026ndash;314.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.kint.2021.10.023\u003c/span\u003e\u003cspan address=\"10.1016/j.kint.2021.10.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhongpiroon C, Buakaew W, Brindley PJ et al (2025) Effect of 3-HBI on Liver Fibrosis via the TGF-β/SMAD2/3 Pathway on the Human Hepatic Stellate Cell Model. Int J Mol Sci 26(13). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms26136022\u003c/span\u003e\u003cspan address=\"10.3390/ijms26136022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu C-F, Chiang W-C, Lai C-F et al (2013) Transforming growth factor β-1 stimulates profibrotic epithelial signaling to activate pericyte-myofibroblast transition in obstructive kidney fibrosis. Am J Pathol 182(1):118\u0026ndash;131. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajpath.2012.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.ajpath.2012.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu M, Peng P, Wang J et al (2015) RACK1-mediated translation control promotes liver fibrogenesis. Biochem Biophys Res Commun 463(3):255\u0026ndash;261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2015.05.040\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2015.05.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePu Y, Wu Y, Zhou Y et al \u003cem\u003eAzithromycin suppresses TGF-β1-related epithelial-mesenchymal transition in airway epithelial cells via targeting RACK1.\u003c/em\u003e Chemico-biological interactions, 2023. 370: p. 110332.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cbi.2022.110332\u003c/span\u003e\u003cspan address=\"10.1016/j.cbi.2022.110332\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePu Y, Liu Y-Q, Zhou Y et al (2020) Dual role of RACK1 in airway epithelial mesenchymal transition and apoptosis. J Cell Mol Med 24(6):3656\u0026ndash;3668. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.15061\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.15061\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng J, Xie L, Kong R et al (2017) RACK1 silencing attenuates renal fibrosis by inhibiting TGF-β signaling. Int J Mol Med 40(6):1965\u0026ndash;1970. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ijmm.2017.3154\u003c/span\u003e\u003cspan address=\"10.3892/ijmm.2017.3154\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ureteropelvic junction obstruction, Epithelial-mesenchymal transition, TGF-β1, RACK1, Fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-7164773/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7164773/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eUreteropelvic junction obstruction (UPJO) is a frequent cause of congenital hydronephrosis. It involves both structural and functional changes at the ureteropelvic junction (UPJ), where the ureter connects with the renal pelvis. The mechanisms behind tissue remodeling and fibrosis in this condition are not fully understood. This study explores the role of epithelial-mesenchymal transition (EMT) and examines how receptor for activated C kinase 1 (RACK1) may regulate this process in UPJO.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eStenotic UPJ tissues were collected from patients with congenital UPJO and analyzed for EMT and RACK1 expression. An in vitro model was established using SV-HUC-1 ureteral epithelial cells treated with TGF-β1 to induce EMT. RACK1 expression was manipulated by siRNA, and changes in EMT-related markers, Smad2/3 and p65 signaling, extracellular matrix proteins, cell proliferation, and migration were evaluated by qRT-PCR, western blotting, and functional assays, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eEMT features, including increased expression of N-cadherin, β-catenin, Vimentin, and ECM-related proteins (COL1A1, FN1, α-SMA), and decreased E-cadherin expression, were observed in both UPJO tissues and TGF-β1\u0026ndash;treated cells. RACK1 was significantly upregulated in stenotic tissues and after TGF-β1 stimulation. RACK1 knockdown suppressed Smad2/3 and p65 activation, inhibited EMT progression, and reduced cell proliferation and migration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eEMT contributes to tissue remodeling and fibrosis in congenital UPJO. RACK1 promotes TGF-β1\u0026ndash;induced EMT through activation of Smad2/3 and p65 signaling. These findings provide new insights into the pathogenesis of UPJO and suggest RACK1 as a potential target for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"TGF-β1 induces epithelial-mesenchymal transition and fibrosis of ureteral epithelial cells via targeting RACK1 in ureteropelvic junction obstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:34:25","doi":"10.21203/rs.3.rs-7164773/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2a72cb0-847f-44f3-8a43-e73caeabfdfa","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-09T12:09:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 06:34:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7164773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7164773","identity":"rs-7164773","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}