Different Expression Of Calpains And Calpastatin In The Laterourethral Tissues Of Women With And Without Stress Urinary Incontinence

IntroductionFemale stress urinary incontinence (SUI) is a common disease that involves leakage of urine during coughing, sneezing, or other increases in intraabdominal pressure, which is associated most often with pelvic organ prolapse(POP). It is defined as "a complaint that the involuntary leakage of urine on effort or exertion, or on sneezing or coughing" and become a common problem in the female population, especially in ageing countries. It is estimated that SUI affects 40% of women, with> 300 000 anti-incontinence procedures performed annually in the USA, while 1/3 of them seek for another because of recurrence. A report revealed that 46.5% women of 18 or over suffer from urinary incontinence in Beijing, while 59.6% of which afflicted by SUI.Although the etiology of SUI is still poorly understood, in general, it is likely to be caused by a combination of anatomical support abnormalities and intrinsic urethral sphincter deficiency. The main risk factors include age, pregnancy, childbirth, obesity and so on. Moreover, high weight index, constipation, chronic coughing, smoking, family history and the level of estrogen may be partly responsible for SUI. The factors above lead to mechanical failure of urinary control, arousing a low pressure reservoir of the urethral sphincter and the involuntary leakage of urine. As well, SUI also can becaused by physical injuries.The fascial and muscular components within the pelvic floor create a dynamic support mechanism that facilitates storage and voiding of urine and faeces. It is reported that 73% old women have ineffective muscle contraction. Hale DS et al also found that some of smooth muscle from women with SUI appeared to be atrophy, apomorphosis or apoptosis. Boreham et al discovered that women with POP had collagen degradation and elastic proteins decrease. In addition, the risk factors, such as vaginal delivery and birth trauma may cause pelvic floor injuries. As a support of urethra and bladder, the laterourethral tissues connect to the endopelvic fascia of the anterior vaginal wall and is sensitive to mechanical force. As it is known to all, calpain does participate in the degradation of smooth muscle, collagen and other matrix of the connective tissue, so it may play a role in cause of SUI.Calpain is a calcium (Ca²⁺)-dependent cysteine protease. There are two classes of calpains: one (comprising calpains 1, 2, 5, 7, 10, 13, and 15) is ubiquitous in cytosol; the other (comprising calpains 3, 6, 8, 9, 11, and 12) occurs only or mainly in certain tissues. The two ubiquitous isoforms, micro-calpain (μ-calpain or calpain-1) and milli-calpain (m-calpain or calpain-2), are different in their sensitivity to Ca²⁺. In vitro analysis has shown that the Ca²⁺ concentration required for optimal activity is 5-50μmol for calpain-1 and 0.2-1mmol for calpain-2. Each of them consists of two different polypeptide subunits. The larger subunit (80 KD) has catalytic activity, whereas the smaller, 30kD, subunit has a regulatory function. More than 50 endogenous and exogenous inhibitors of the calpains have been described. They include cellular and extracellular proteins and drugs such as iodoacetate, iodoacetamide, and N-ethylmaleimide, which are inhibitors of cysteine proteases. The specific endogenous inhibitor of calpain activity is calpastatin, which binds specifically to both isoforms of calpain in a substrate competitive manner. Theintracellular level of calpastatin correlates directly with calpain activation, and the affinity of calpastatin for the activated forms of the calpains is greater than its affinity for the proenzyme.Calpains cover a broad range of physiological functions including proteolysis of molecules involved in cytoskeletal organization, the cell cycle, signal transduction, apoptosis, and protein renewal during growth and tissue regeneration. While being pathologically activated, they would lead to diseases. A number of pathologic conditions have been associated with disturbances of the calpain system. They include type 2 diabetes, cataracts, Duchenne's muscular dystrophy, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis, ischemia, stroke and brain trauma, various platelet syndromes, hypertension, liver dysfunction, and some types of cancer. The application of calpain inhibitors may provide greater specificity and therapeutic potential.In this study, we investigated the expression of calpain-1, calpain-2 and their inhibitors, calpastatin in the human laterourethral tissues to show the potential link between calpain system and SUI, expecting to find a sensitive evaluation index or a potential treatment.Methods and ResultsMethods: The laterourethral tissues of 39 women with SUI and 31 women without SUI were collected to detect the expression of calpains and calpastatin using a semi-quantitative competitive RT-PCR and westblotting. The SUI group were diagnosised by SUI with no hormone treatment. The control group were selected from people with benign gynecological but not hormone releated diseases. Results: (1) There are calpain-1 mRNA and protein expresstions in both groups and the numbers are no differences between the two groups. (2) There are calpain-2mRNA and protein expresstions in both groups, and numbers of mRNA and protein expressions in the group of SUI are more than those in the control group (1.36±0.06 versus 1.16±0.04, p<0.05 and 0.75±0.07 versus 0.56±0.06, p <0.05). (3) There are calpastatin mRNA and protein expresstions in both groups, the mRNA expressions in women with SUI are more than women without SUI (0.85±0.07 versus 0.62±0.06, p <0.05), while the protein expressions are significantly less in them than in women without SUI. (0.72±0.06 versus 1.84±0.09,p <0.01).Conclusions1. Both of calpain-2 mRNA and protein expression are higher in the people with SUI2.The expression of calpastatin mRNA is higher in the people with SUI than the control group, while the protein expression are lower than that in the control group.3. Over expression of calpain-2 and low expression of calpastatin may be play an important role in the cause of SUI, the application of calpastatin may be a new method to treat SUI.