Elsevier

European Urology

Volume 55, Issue 5, May 2009, Pages 1213-1223
European Urology

Voiding Dysfunction
Molecular Mechanisms Related to Parturition-Induced Stress Urinary Incontinence

https://doi.org/10.1016/j.eururo.2008.02.027Get rights and content

Abstract

Background

The molecular mechanisms underlying stress urinary incontinence (SUI) at the tissue level are poorly understood.

Objective

To study genetic and molecular alterations in the urethras of animals with experimentally induced SUI.

Design, setting, and participants

Cohort analysis of primiparous 2-month-old female Sprague-Dawley rats with experimentally induced SUI versus those who did not develop SUI in a university research laboratory setting.

Intervention

Rats underwent intravaginal balloon dilation within 24 hours of parturition followed by bilateral ovariectomy one week later. Transvesical cystometry was performed 12 weeks after parturition. Rats were classified as continent (C) or incontinent (I) according to the results of cystometry.

Measurements

The expression of over 22,000 genes in urethral tissue from the two groups was assessed with the use of an oligo microarray. The expression of relevant genes was confirmed by real-time polymerase chain reaction. Protein expression of small mothers against decapentaplegic 2 (Smad2), one of the differentially expressed genes, was extensively studied by immunohistochemistry and Western blot analysis. Regulation of Smad2 activity by transforming growth factor-β (TGF-β) was assessed in cultured urethral smooth muscle cells (USMCs).

Results and limitations

After intervention, 14 (58.3%) rats remained continent and 10 (41.7%) became incontinent. There were significant differences in the expression of 42 urethral genes between continent and incontinent rats. The expression of genes involved in the TGF cellular signaling pathway (Smad2), collagen breakdown (matrix metalloproteinase 13 [Mmp13]), and smooth muscle inhibition (regulator of G-protein signaling 2 [Rgs2]) was significantly increased in the incontinent group. SMAD2 protein expression was significantly upregulated in the incontinent rats. In cultured USMCs, SMAD2 phosphorylation and nuclear translocation increased after Tgf-β treatment.

Conclusions

Genes important in inflammation, collagen breakdown, and smooth muscle inhibition are upregulated in the urethras of female rats with parturition-associated incontinence.

Introduction

Stress urinary incontinence (SUI) is a prevalent urological problem that is most common in women. It had been reported that up to 50% of women older than 60 yr have symptoms of stress-induced or urge urinary incontinence [1], [2]. Although progress has been made in the treatment of SUI [3], our understanding of the molecular mechanisms underlying the condition is poor.

Chen et al [4] studied gene expression in the periurethral connective tissue of the vagina in human women with SUI; they found that genes related to elastin metabolism were upregulated compared with healthy controls. Chen et al further demonstrated that expression of matrix metalloproteinase 1 (MMP1) messenger RNA was increased in human female SUI patients, suggesting that increased collagen breakdown may be an etiological factor in SUI via breakdown of pelvic ligamentous tissues [5].

In light of the limited availability of human tissue for study, animal models are an important adjunct in improving our understanding of SUI [6], [7]. In 1999, our laboratory developed a rat model of parturition-associated female SUI in which female rats were subjected to intravaginal balloon dilation (similar to birth trauma) after pregnancy and vaginal delivery of pups, followed by ovariectomy 1 wk later to simulate menopause. These investigations indicated that the final common pathway for SUI seems to be neuronal and muscular (smooth and striated) changes in the urethra and pelvic floor. Our prior studies have included functional, anatomical, and histological assessment of these model animals and have indicated that our model system approximates the human condition of SUI [6], [7]. As a follow-up to our initial studies, we investigated molecular changes in the urethra of rats with parturition-associated SUI by means of oligo microarray, real-time polymerase chain reaction (PCR), Western blot analysis, and cell culture techniques.

Section snippets

Animals and overview

Twenty-four, 2-month-old, female primiparous pregnant (at gestational day 16) Sprague-Dawley rats weighing approximately 230–280 g were obtained from a commercial vendor. They were housed at a constant 16 °C room temperature and 47% humidity, with a 12-h light-dark cycle. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of California at San Francisco.

Delivery, balloon dilation and ovariectomy treatment, and transvesical cystometry

Creation of the animal model and transvesical cystometry were performed as previously

Rates of incontinence in subject rats

On the basis of cystometric criteria, there were a total of 14 (58.3%) continent rats and 10 (41.7%) stress incontinent rats. None of the rats manifested non-voiding contractions suggestive of bladder overactivity. Eleven urethras (6 from normal and 5 from incontinent animals) were randomly selected for protein isolation and Western blot analysis. The other 13 urethras (8 from normal and 5 from incontinent animals) were used for microarray, real-time PCR, and immunohistochemistry analyses.

Long-term, age-related, labor-induced changes in urethral gene expression

Gene

Discussion

Genes related to the metabolism of collagen, such as Mmp2 and tissue inhibitor of metalloproteinase, are expressed differently in vaginal tissue from women with SUI compared with normal women [4], [5]. However, assessment of gene expression in vaginal and periurethral tissues may not be representative of gene expression in the urethra. In light of the paucity of human urethral tissue available for analysis, our animal model of labor-induced stress incontinence represents a reasonable proxy for

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