Urine-derived exosomes and their role in modulating uroepithelial cells to prevent hypospadias.

PURPOSE: Urethral hypospadias, a common congenital malformation in males, is closely linked with disruptions in uroepithelial cell (UEC) processes. Evidence exists reporting that urine-derived exosomes (Urine-Exos) enhance UEC proliferation and regeneration, suggesting a potential role in preventing hypospadias. However, the specific influence of Urine-Exos on urethral hypospadias and the molecular mechanisms involved are not fully understood. This study focuses on investigating the capability of Urine-Exos to mitigate urethral hypospadias and aims to uncover the underlying molecular mechanisms. METHODS: Bioinformatics analysis was performed to identify key gene targets in Urine-Exos potentially involved in hypospadias. Subsequent in vitro and in vivo experiments were conducted to validate the regulatory effects of Urine-Exos on hypospadias. RESULTS: Bioinformatics screening revealed syndecan-1 (SDC1) as a potential pivotal gene for the prevention of hypospadias. In vitro experiments demonstrated that Urine-Exos enhanced the proliferation and migration of UECs by transferring SDC1 and inhibiting cell apoptosis. Notably, Urine-Exos upregulated ß-catenin expression through SDC1 transfer, further promoting UEC proliferation and migration. These findings were confirmed in a congenital hypospadias rat model induced by di(2-ethylhexyl) phthalate (DEHP). CONCLUSION: This study reveals the therapeutic potential of Urine-Exos in hypospadias, mediated by the SDC1/ß-catenin axis. Urine-Exos promote UEC proliferation and migration, thereby inhibiting the progression of hypospadias. These findings offer new insights and potential therapeutic targets for the management of congenital malformations.

Molecular effects of intermittent stress on primary feline uroepithelial cell culture as an in vitro model of feline idiopathic cystitis.

Introduction: The most common cause of feline lower urinary tract disease (FLUDT) is feline idiopathic cystitis (FIC), which is a complex multifactorial disease with symptoms including stranguria, dysuria, hematuria, and pain during urination. The development of these symptoms is often triggered by stress, and in case of chronic stress, these symptoms will many times return. One of the most important stress hormones in the pathogenesis of FIC is norepinephrine (NE), as persistently elevated level of this hormone can be measured in the blood of cats with FIC. However, it is not well understood if recurrently elevated level of NE has any direct effect on urinary bladder, therefore the aim of this study was to investigate the molecular effects of intermittent NE exposure on feline primary uroepithelial cell culture. Methods: Primary uroepithelial cells were gained from the mucosa of the bladder of a euthanized cat, and were cultured for 6 days, then they were exposed to 10, 100, and 1,000 µM NE treatment for 3 × 1 h, including a 1 h long regeneration period between exposures. Results: NE was able to trigger pro-inflammatory response and oxidative stress in the uroepithelial cells by increasing the level of stromal cell derived factor 1 (SDF-1) and H2O2 in cell culture media. In addition, NE increased the permeability of the uroepithelium, since decreased glycosaminoglycan (GAG) concentration, tight junction protein claudin-4 content, and TER values were measured after the NE treatments. Discussion: Based on these results it can be concluded that recurrent stress mimicked by 3×1 h NE treatment has a direct molecular effect on the uroepithelial cells, which leads to inflammatory response, oxidative stress and decreased barrier function of the uroepithelium. Therefore, intermittent release of NE may have an important role in the pathogenesis of FIC and the results of this study may contribute to a better understanding of the development of this illness.

SLC1A5 regulates cell proliferation and self-renewal through ß-catenin pathway mediated by redox signaling in arsenic-treated uroepithelial cells.

Arsenic exposure increases the risk of bladder cancer in humans, but its underlying mechanisms remain elusive. The alanine, serine, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is frequently overexpressed in cancer cells. The aim of this study was to evaluate the effects of arsenic on SLC1A5, and to determine the role of SLC1A5 in the proliferation and self-renewal of uroepithelial cells. F344 rats were exposed to 87 mg/L NaAsO2 or 200 mg/L DMAV for 12 weeks. The SV-40 immortalized human uroepithelial (SV-HUC-1) cells were cultured in medium containing 0.5 µM NaAsO2 for 40 weeks. Arsenic increased the expression levels of SLC1A5 and ß-catenin both in vivo and in vitro. SLC1A5 promoted cell proliferation and self-renewal by activating ß-catenin, which in turn was dependent on maintaining GSH/ROS homeostasis. Our results suggest that SLC1A5 is a potential therapeutic target for arsenic-induced proliferation and self-renewal of uroepithelial cells.

Low-dose arsenite causes overexpression of EGF, TGFα, and HSP90 through Trx1-TXNIP-NLRP3 axis mediated signaling pathways in the human bladder epithelial cells.

Epidemiological studies have demonstrated an increased incidence of bladder cancer in arseniasis- endemic areas; however, the precise molecular mechanisms remain unknown. Our previous results have shown that the protein levels of EGF, TGFα, and HSP90 in arsenite-treated bladder uroepithelial cells increased markedly and contributed to hyperactivation of EGF receptors. The aim of this study was to further explore the regulatory ways underlying overexpression of EGF, TGFα, and HSP90 in these cells. The present results showed that both Trx and GSH systems were stimulated in arsenite-treated cells, and ROS levels in 2 µM arsenite-treated cells did not changed obviously; however, ROS levels in 4 µM arsenite-treated cells increased significantly. By using the antioxidant and specific inhibitors, we found that in 2 µM arsenite-treated cells, JNK/NF-κB signaling pathway was involved in overexpression of EGF and TGFα, and ERK/NF-κB signaling pathway contributed to HSP90 overexpression, however in 4 µM arsenite-treated cells, both ERK/ and JNK/NF-κB signaling pathways were involved in overexpression of EGF, TGFα, and HSP90, and PI3K/AKT/NF-κB signaling pathway contributed to overexpression of EGF and TGFα. Furthermore, our results also showed that the Trx1-TXNIP-NLRP3 axis was activated in arsenite-treated cells, and played a pivotal role in activation of the signaling pathways involved in overexpression of EGF, TGFα, and HSP90. In conclusion, the Trx1-TXNIP-NLRP3 axis might be activated by arsenite-induced redox imbalance in bladder uroepithelial cells, and mediate the activation of signaling pathways involved in overexpression of EGF, TGFα, and HSP90.

Enhanced uropathogenic Escherichia coli-induced infection in uroepithelial cells by sugar through TLR-4 and JAK/STAT1 signaling pathways.

BACKGROUND: Patients with diabetes mellitus (DM) have higher incidence and more severe urinary tract infections (UTIs) for longer duration than those of the patients without DM. It causes more complicated etiologies during uropathogenic Escherichia coli (UPEC) infection. However, studies regarding the molecular mechanism are scarce. METHODS: The present study (1) aimed to verify if sugar influences the process of UPEC-induced cystitis and invasion into the uroepithelial cells and (2) illustrated the mechanism of effects for sugar enhanced the UPEC infection into uroepithelial cells is related to TLR-4-mediated and JAK/STAT1-dependent pathway. RESULTS: The results of the present study indicated that sugar can enhance UPEC infection in uroepithelial cells by up-regulating the transduced circuit between TLR-4-mediated UPEC interaction and JAK/STAT-1 signal pathways. The results of the inhibitor-co-incubating experiments demonstrated that the mechanism involved in the synergistic amplification of TLR-4-mediated UPEC interaction and JAK/STAT1 signaling pathways is responsible for the increased UPEC infection in uroepithelial cells. CONCLUSION: The results also proved that STAT-1 plays a critical role in the regulation of UPEC invasion and infection in the uroepithelial cells, especially those pretreated with glucose. The present study suggests a possible therapeutic approach to preferentially suppress UPEC infection during UTIs in the patients with diabetes.

Role of PI3K/Akt pathway in Benzidine-induced proliferation in SV-40 immortalized human uroepithelial cell.

BACKGROUND: Long term exposure to benzidine has been determined as a cause of urothelial carcinoma. But how it works in the process of cell proliferation that involves in tumor growth is not examined yet. In the current research, the effect of PI3K/Akt on cell proliferation mediated by benzidine was confirmed. METHODS: The immortalized SV-40 human uroepithelial cells (SV-HUC-1) had been subjected to 6 days of benzidine treatment at various contents, then MTT assay, together with subsequent flow cytometry assay were used for observing effects on cell proliferation. Further Western blots were used to detect the expression of total-Akt, phospho-Akt and specific proteins of cell cycle. The Akt, Cyclin D1, PCNA and P21 mRNA levels were detected through RT-PCR. In addition, the blocker-LY294002 was used to cut down the PI3K/Akt signaling pathway. And then those parameters were detected using the same methods as above. RESULTS: Results showed that benzidine acted to induce cell proliferation at low doses (P<0.05 vs. controls) via MTT and flow cytometry assay. The expression of phospho-Akt, Cyclin D1, and PCNA were significantly enhanced compared with that of control (P<0.05; P<0.01), but total-Akt and P21 levels were reduced. Whereas, inhibitor of PI3K/Akt suppressed the proliferating procedure when cells were treated with the blocker (LY294002) and also inhibited the expression of related cycle proteins. CONCLUSIONS: Activated PI3K/Akt signal pathway promotes benzidine-triggered cell proliferation. It may shed light on the molecular mechanisms that the activated PI3K/Akt pathway promotes benzidine-triggered cell proliferation and intervention of its target.

sEcad and EGF Levels Increased in Urine of Non-ferrous Metal Workers and Medium of Uroepithelial Cell Line Treated by Arsenic.

Inorganic arsenic (iAs) is a carcinogen and could increase the risks of bladder, lung, and skin cancer. Mining and smelting of non-ferrous metals are common occupational arsenic exposures. In this study, 125 individuals working in non-ferrous metal smelting plants were separated into two groups according to urinary total arsenic (TAs) levels: group 1, TAs < 100 µg/g Cr; group 2, TAs ≥ 100 µg/g Cr. Demographic characteristics of participants were obtained by questionnaire interview. Levels of E-cadherin soluble ectodomain fragment (sEcad) and epidermal growth factor (EGF) in workers urine were determined by ELISA test. We found that concentrations of sEcad and EGF present in urine were significantly elevated in the high urinary arsenic group 2 compared with the low urinary arsenic group 1. Urinary levels of the shedding of E-cadherin soluble ectodomain fragment (sEcad) and epidermal growth factor (EGF) were positively related to the concentrations of iAs in urine after adjusting for the confounding effects. A positive correlation between sEcad and EGF concentrations in urine was also observed. In order to verify the effects of iAs on sEcad and EGF, the human uroepithelial cell line (SV-HUC-1) was treated with NaAsO2 for 24 h in vitro. sEcad and EGF levels in the 4 µM NaAsO2-treated SV-HUC-1 cell medium significantly increased compared to the control group. In conclusion, urinary levels of sEcad and EGF increased in higher urinary arsenic workers of non-ferrous metal plants and are closely associated with urinary iAs concentration. The results suggested that sEcad and EGF may potentially be preclinical prognostic factors of bladder injury and early detection in arsenic exposure individuals.

Extract of Clinopodium bolivianum protects against E. coli invasion of uroepithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clinopodium bolivianum is a South American plant with anti-inflammatory and anti-infective activities. The increasing antibiotic resistance urges for alternative therapy. Based on its use in traditional medicine, we investigated the effect of C. bolivianum on the ability to defend bladder epithelial cells from E. coli infection. MATERIALS AND METHODS: The extract was analyzed by LC-MS. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli No. 12, its isogenic mutant WE16 csgBA bscA::Cm and CFT073 were used to investigate the effect of C. bolivianum on uroepithelial infection. Bacterial adherence and invasion to cells treated with C. bolivianum were analyzed. Expression of uroplakin 1a, ß1 integrin, caveolin-1, IL-8 and antimicrobial peptides in response to C. bolivianum treatment was assessed using RT-PCR. Protein expression was confirmed by Western blot analysis or ELISA. The antimicrobial effects of C. bolivianum on bacteria and fungus were investigated using minimum inhibitory concentration. Furthermore, the formation of biofilm was investigated with crystal violet assay. RESULTS: C. bolivianum extract consisted of more than 70 different types of phytochemicals including sugars and phenolic compounds. The extract decreased the uroplakin 1a expression and E. coli adhesion and invasion of uroepithelial cells while up-regulated caveolin-1. In uninfected C. bolivianum treated cells, IL-8 was lower than in non-treated cells. In infected cells, however, no difference was observed between treated and non-treated cells. Further, C. bolivianum treatment reduced uropathogenic E. coli (UPEC) biofilms but did not inhibit bacterial growth. CONCLUSIONS: Our results show that C. bolivianum has a protective role on bladder epithelial cells against UPEC infection by decreasing the bacterial adhesion, invasion and biofilm formation.

Effect of vitamin e on uroepithelial cells and changes of urinary sediments in oncology hospital nursing personnel.

INTRODUCTION: Vitamin E is an important natural antioxidant, and its most common and biologically active form is α-tocopherol. The antiproliferative effects of alpha-tocopherol have been previously demonstrated. In this study we investigated the effects of vitamin E on urinary epithelial cells and urinary sediments of nursing from oncology hospital. MATERIAL AND METHODS: Sixty-two female nursing personnel from oncology hospital participated in the study. They received orally 200mg of vitamin E per day for two weeks. Also prior to vitamin E and after vitamin E administration, the uroepithelial cells counts and other components of urinary sediments were carried out. RESULTS: There were significant differences in the epithelial cells count and treatment with vitamin E causing significantly more number of epithelial cells and urinary sediments to be excreted in the urine. DISCUSSION: Vitamin E significantly plays an important role on the excretion of uroepithelial cells and urinary sediments. CONCLUSION: In conclusion we propose that use of vitamin E at nontoxic levels would significantly enhance its antioxidative properties, especially among individuals subjected to prophylaxis of occupational hazards.