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

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 (2,3). 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 (4). 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 (5). 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 (6). 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 (7,8). 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 (9). 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 (10,11). RACK1 modulates cellular processes such as migration, adhesion, and differentiation by facilitating signal complex assembly (12). Although RACK1 has been implicated in cancer progression and tissue fibrosis (13,14), 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. We present this article in accordance with the MDAR reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0078/rc).

Methods

Patients

A total of 40 pediatric patients were collected, comprising 10 in the control group (duplex kidney) and 30 in the disease group (UPJO), at Zhangzhou Affiliated Hospital of Fujian Medical University, 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 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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University (No. 2024KYZ357). Written informed consent was obtained from all participants and 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, Santa Clara, USA) following the manufacturer’s instructions. Paraffin-embedded tissue sections were incubated with primary antibodies against human RACK1 (Abcam, Cambridge, USA, 1:200), E-cadherin (Abcam, USA, 1:500), and COL1A1 (Abcam, USA, 1:500) overnight at 4 ℃. 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, Waltham, USA) containing 10% fetal bovine serum (FBS). Cells were maintained at 37 ℃ 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, Cranbury, 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 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 polymerase chain reaction (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 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, Madison, USA), and protein concentrations were measured using the BCA Protein Assay Kit (Pierce, Rockford, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore, Burlington, USA). The membranes were blocked and incubated with specific primary antibodies at 4 ℃ 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, Bethesda, USA). GAPDH was used as the internal control. The following primary antibodies were used in this study: rabbit anti-human RACK1 (Abcam, Cambridge, 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 Kyoto Encyclopedia of Genes and Genomes (KEGG) 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, Santa Clara, USA). Libraries were constructed using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, Ipswich, 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 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, Kumamoto, 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 ℃ 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% FBS. At 0, 24, and 48 hours after wounding, cells were gently rinsed with PBS 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

Demographic and clinical characteristics of control and UPJO groups

A total of 40 pediatric patients were included in the analysis, comprising 10 in the control group (duplex kidney) and 30 in the disease group (UPJO). The mean age at surgery was 3.43±3.16 years in the control group and 4.30±4.53 years in the UPJO group, with no significant difference between groups (P=0.58). Similarly, body weight was comparable between groups (14.17±8.51 vs. 16.16±10.92 kg, P=0.60). A significant difference was observed in sex distribution between groups (P=0.04), with a higher proportion of males in the UPJO group (76.7%) compared to the control group (40.0%). The affected side distribution was similar between groups (P=0.25). Preoperative Society for Fetal Urology (SFU) grading differed significantly between groups (P<0.001). All patients in the control group had SFU grades 0–2 (90%) or grade 3 (10%), while all UPJO group patients had moderate to severe hydronephrosis (grade 3: 30.0%, grade 4: 70.0%). Consistently, the preoperative renal pelvis anteroposterior diameter was significantly larger in the disease group (29.09±12.10 mm) compared to controls (5.55±8.86 mm, P<0.001), with a mean difference of 23.54 mm [95% confidence interval (CI): 16.25–30.83 mm]. Renal cortical thickness was significantly reduced in the disease group (7.07±4.16 mm) vs. controls (10.00±2.58 mm, P=0.041), representing a mean reduction of 2.93 mm (95% CI: −5.72 to −0.14 mm) (Table 1).

RACK1 is highly expressed in UPJO tissues and is associated with changes in EMT and fibrosis markers

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 (Figure 1).

Figure 1 Histological features of UPJ tissues from control and UPJO patients. H&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. H&E, hematoxylin and eosin; UPJ, ureteropelvic junction; UPJO, ureteropelvic junction obstruction.

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 (Figure 2).

Figure 2 Immunohistochemical 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. RACK1, receptor for activated C kinase 1; UPJO, ureteropelvic junction obstruction.

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 (Figure 3, P<0.05 for all comparisons).

Figure 3 mRNA 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 ± standard deviation. *, P<0.05; **, P<0.01; ****, P<0.0001. EMT, epithelial-mesenchymal transition; RACK1, receptor for activated C kinase 1; RT-qPCR, real-time quantitative polymerase chain reaction; UPJ, ureteropelvic junction; UPJO, ureteropelvic junction obstruction.

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) (Figure 4).

Figure 4 Protein 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 ± standard deviation (n=3 per group). GAPDH was used as a loading control. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RACK1, receptor for activated C kinase 1; UPJO, ureteropelvic junction obstruction.

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 (Figure 5).

Figure 5 KEGG pathway analysis of transcriptomic changes in SV-HUC-1 cells. (A) KEGG enrichment analysis of 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. COVID-19, coronavirus disease 2019; DEGs, differentially expressed genes; IL-17, interleukin-17; KEGG, Kyoto Encyclopedia of Genes and Genomes; NF, nuclear factor; NOD, nucleotide-binding oligomerization domain; RACK1, receptor for activated C kinase 1; TGF-β1, transforming growth factor-beta 1; TNF, tumor necrosis factor; WT, wild-type.

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 (Figure 6). Western blot analysis confirmed that protein levels of RACK1 and mesenchymal markers were elevated, and E-cadherin was suppressed (Figure 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 (Figure 7).

Figure 6 RT-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 ± standard deviation from three independent experiments. **, P<0.01; ***, P<0.001; ****, P<0.0001. EMT, epithelial-mesenchymal transition; RACK1, receptor for activated C kinase 1; RT-qPCR, real-time quantitative polymerase chain reaction; TGF-β1, transforming growth factor-beta 1; WT, wild-type.

Figure 7 Western 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. EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RACK1, receptor for activated C kinase 1; TGF-β1, transforming growth factor-beta 1.

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 (Figures 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 (Figure 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 (Figure 5).

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 (Figure 8A). 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 (Figure 8B).

Figure 8 RACK1 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 ± standard deviation. **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; NC, negative control; RACK1, receptor for activated C kinase 1; TGF-β1, transforming growth factor-beta 1.

Discussion

This study confirmed that EMT occurs in the stenotic segments of the UPJ 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 ECM 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 (15,16), its role in congenital UPJO has not been well investigated. The search for non-invasive diagnostic and prognostic tools for UPJO has led to significant interest in urinary biomarkers. A systematic review by Paraboschi et al. (17) highlighted several candidate molecules, including proteins related to inflammation, fibrosis, and renal tubular damage, such as NGAL, KIM-1, and MCP-1. Previous molecular and transcriptomic studies on UPJO have identified abnormal expression of genes related to fibrosis (such as TGF-β1 and actin alpha 2) (18,19), inflammation (such as Interleukin 6) (20), and hypoxia (such as endothelin-1) (21). 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 (22,23). Previous studies have shown that TGF-β1 expression is elevated in models of ureteral obstruction and contributes to renal interstitial fibrosis (24). In UPJO, increased levels of TGF-β1 have also been reported in stenotic tissues (9,25), 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. It has been previously implicated in liver fibrosis (13,26). These cross-organ conserved mechanisms suggest that RACK1 may be a “universal regulatory node” in fibrotic diseases, and its role in UPJO is not isolated but a conserved mechanism in the pathological process of fibrosis, providing solid cross-disease evidence for the translation of UPJO therapeutic targets. Currently, intervention strategies targeting RACK1 have shown potential in other fibrotic diseases. In the respiratory system, inhibition of RACK1 expression was shown to reduce bronchial epithelial cell migration and reverse TGF-β1-induced EMT (27,28). In the kidney, silencing RACK1 has been reported to alleviate renal fibrosis by blocking the TGF-β1/Smad3 pathway in epithelial cells (29). 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. Based on the above findings, combined with RACK1’s validated role in other fibrotic diseases, its translational potential as a therapeutic target for UPJO merits in-depth exploration, providing important support for accelerating clinical translation.

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 UPJ. 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.

Conclusions

In summary, this study demonstrates that features of EMT are present in the stenotic UPJ of patients with congenital UPJO and may contribute to tissue remodeling and fibrosis. TGF-β1 was shown to induce EMT and ECM 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.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0078/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0078/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0078/prf

Funding: This work was supported by Natural Science Foundation of Fujian Province (grant No. 2023J011825), and Zhangzhou Science and Technology Program (grant No. ZZ2023J31).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-1-0078/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Zhangzhou Affiliated Hospital of Fujian Medical University (No. 2024KYZ357). Written informed consent was obtained from all participants and their guardians prior to sample collection.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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