Phenotypic Modulation Of Bladder Smooth Muscle In Diabetic Rats

BACKGROUND&OBJECTIVEDiabetes mellitus is a kind of chronic metabolic endocrine disease,which is common and frequently encountered disease that severely harm for human life and healthy.The estimated prevalence of diabetes among a representative sample of Chinese adults was 11.6%,meanwhile,over 50% of men and women with diabetes have bladder dysfunction.Diabetic bladder dysfunction refers to a group of bladder symptoms occurring in patients with diabetes mellitus.Bladder dysfunction in patient with diabetic is a common disease of Urology,which has been classically described as decreased bladder sensation,increased bladder capacity and impaired bladder emptying with resultant increased post-void residual volume,however,the obvious clinical urinary tract symptoms includes overactive bladder like frequency and urgency,voiding dysfunction,and urinary retention etc.Clinical studies reported that 22.5% has overactive bladder in type 2 diabetes subjects,and 48.0% of those with overactive bladder had incontinence.At present,bladder dysfunction with diabetes is diagnosed with urodynamics. Management goals include relief of symptoms,prevention and treatment of urinary tract infections,and adequate bladder emptying.In this regard,the management strategies can be groups into three classes:behavioral,pharmacological,and surgical.The pathophysiology of diabetic bladder dysfunction is multifactorial,including disturbances of the detrusor,neuron,urothelium,and urethra. Since the pathogenesis underlying diabetic bladder dysfunction is complicated,the pathogenesis of diabetic bladder dysfunction remain to be further investigated which play important roles in improving diabetic bladder dysfunction management in clinical practice.The wall of bladder included mucosa,muscular layer and adventitia,from the inside to the outside.Muscle layer is composed of smooth muscle cells, known as detrusor which contraction removes urea through urethra by increasing the pressure of bladder.The unique physiology and contractile properties of smooth muscle cells underlie their capacity to regulate arterial tone as well as gastrointestinal and genitourinary function.In contrast to striated muscle cells,smooth muscle cell can be further divided into two type based on structure and function:the contraction type and synthetic type.The major function of the former one is contraction,and that of the latter one is proliferation,migration,secretion and regulation of extracellular proteins,etc.The contraction type smooth muscle cell can transform into synthetic type ones and get to be proliferation when the condition is changed or the smooth muscle cell cultured in vitro are stimulated by the growth factors.The smooth muscle cell of bladder is very similar with the vascular smooth muscle cell in structure and function.Research have reported that the phenotype of bladder smooth muscle cell could be switched.Studies have shown that functional abnormalities of bladder outlet obstruction have the structural basis:phenotype transformation of the detrusor cells which means that phenotypic modulation of bladder smooth muscle cell plays important role in the physiopathology of bladder dysfunction in bladder outlet obstruction.However,whether dose diabetic causes the phenotypic modulation of bladder smooth muscle cell are still unclear at present.The smooth muscle cell phenotypic modulation means:the morphological,structural and functional changes of smooth muscle cell in body’s various stages of development or different disease.When the smooth muscle cell transforms from the contraction type to the synthetic type,proliferation and migration will be increased but inflammatory factor and expression of cell contract related genes like SMA,SMMHC, smoothelin,calponin and desmin will be decreased.myocardin belongs to the SAP (SAF-A/B,Acinus,PLAS) domain family of nuclear proteins.In the nucleus,myocardin associate with SRF,facilitating the binding of SRF to single or dual CArG boxes,activating transcription of genes encoding cytoskeletal and contractile proteins.myocardin is a remarkably potent transcriptional coactivator and the founding member of a class of muscle transcription factors expressed exclusively in cardiomyocytes and smooth muscle cell.Myocardin can regulate the phenotype of smooth muscle cell.The expression of myocardin is proportional to the contraction type of smooth muscle cell.It is well documented that the establishment of a differentiated phenotype in smooth muscle cell is dependent on the activation of myocardin.Meanwhile,our previous studies have shown that myocardin also plays a key role in the phenotypic modulation of corpus cavernosum smooth muscle cell,myocardin significantly decreased in corpus cavernosum of rats with diabetic erectile dysfunction,in turn over-expression of myocardin in corpus cavernosum can reverse corpus cavernosum smooth muscle cell phenotypic modulation and alleviate erectile dysfunction of rats with diabetes.In summary,we guess whether myocardin also decrease in bladder of rats with diabetic and then the phenotype of bladder smooth muscle cell transforms.However,up to now,few reports were found about the Phenotypic modulation of bladder smooth muscle in diabetic rats.Thus,in this study,to investigate the phenotypic modulation of bladder smooth muscle in diabetic rats, we detected the expression of contratile protein markers and Myocardin in bladder tissue of diabetic SD rats,which may provide a basic theory for the treatment of diabetic bladder dysfunction.METHODS1.Induction of Diabetes ratsFollow the way as we reported before,32 of 8-week-old male Sprague-Dawley rats weighing approximately 200g had been maintained in 12-hour light/dark facility with free access to food and water for a week,and then were randomly assigned to DM induction by STZ or to serve as a control.Rat were fasted for 12 h before being injection.Diabetes was induced after a 12-hour fast by a single intraperitoneal injection of 60mg/kg STZ diluted in 0.1 M sodium citrate buffer solution,PH 4.5.Controls were treated identically except a similar volume of buffer was injected instead of STZ.Three days after STZ treatment a blood sample was obtained by cutting the tail to estimate the blood glucose level.Animals with blood glucose 16.6 mmol/1 or greater, drinking more and polyuria were deemed diabetic and,therefore,suitable for study.All rats were housed in 12-hour light/dark cycle.The ambient temperature was kept at 25℃,and the rats had free access to food and water.9 week after STZ or vehicle treatment,body weights were measured.The bladder was removed through an abdominal incision,emptied and weighted before washed in PBS repeatedly.A part of bladder were fixed with 4% paraformaldehyde,the other were removed the mucosal and serosal layers then the bladder muscle layer from the mid body region was rapidly frozen before stored under at -80℃ for further analysis.2.H&E-staining and Masson’s Trichrome stainingAfter 9 week,bladder sections harvested from DM group and control group were stained with histological examination or Masson’s Trichrome that observe the morphology of bladder in different group.3.Measuring the expression of mycardin in DM group and control groupThe mRNA expressions of transcription factor myocardin in bladder were determined by real-time fluorescence quantitative PCR(qRT-PCR),the protein expression of myocardin in bladder were examined by western blotting.Then we compared the expression of myocardin in DM group with those in control group.4.Measuring the expression of a-SMA in DM group and control groupThe mRNA expressions of contractile phenotype markers a-SMA in bladder were determined by real-time fluorescence quantitative PCR(qRT-PCR),the protein expression of a-SMA in bladder were examined by western blotting.Then we compared the expression of a-SMA in DM group with those in control group.5.Measuring the expression of SMMHC in DM group and control groupThe mRNA expressions of contractile phenotype markers SMMHC in bladder were determined by real-time fluorescence quantitative PCR(qRT-PCR),the protein expression of SMMHC in bladder were examined by western blotting.Then we compared the expression of SMMHC in DM group with those in control group.RESULTS1.Induction of Diabetes ratsCompared with the starting weight,there are no significant difference between the DM group (215.9±25.0g) with the control group (218.7±28.8g) (P>0.05).After STZ treatment,the diabetes rats became drinking more and polyuria when compared with the control group.Compared with the control group (412.7±102.7g),the body weight of DM group (286.3±71.2g) were significantly decreased after 9 weeks(p=0.001),while the bladder wet weight were significantly increased (p=0.002).Two rats died in DM group when no death was found in the control group.2.H&E-staining and Masson’s Trichrome staining9 weeks after STZ treatment,medial disarray with SMC loss,deposition of extracellular matrix and loss of the characteristic spindle-like SMC morphology were observed in bladder of the DM group compared with controls by histological examination. Masson’s Trichrome staining revealed a marked reduction of SMC cytoplasm accompanied by the robust induction of fibrosis in bladder of the DM group compared with controls.3.The expression of myocardin,a-SMA,SMMHC in bladder between DM group and control groupWe examined the expression of myocardin and contractile phenotype markers at the mRNA level in the bladder tissue from DM group and control group by qRT-PCR.Compared with the control group, the levels of a-SMA,SMMHC and myocardin were significantly decreased in rats of the DM group(all P<0.05).In accordance with expressions at the mRNA level,the levels of a-SMA,SMMHC and myocardin at the protein level in the bladder were significantly decreased in rats of the DM group(all P<0.05).Obviously,the expression of myocardin and contractile phenotype markers a-SMA,SMMHC were reduced in the bladder of diabetic rat,which show that the phenotypic modulation of bladder smooth muscle cell could be induced by diabetic.CONCLUSION1.9 weeks after STZ treatment,the diabetic rats were more thin,drinking more and polyuria than the control rats,while the bladder wet weight of diabetic rats were significant increased compared with the control rats.It follows that the pathological of bladder in diabetic rats have changed which may be related to the bladder dysfunction.2.9 weeks after STZ treatment,the contractile phenotype markers a-SMA,SMMHC were significant decreased in the bladder of diabetic rat,which show that the phenotypic modulation of bladder smooth muscle cell have been occurred in diabetic rats and the phenotypic modulation of bladder smooth muscle cell in diabetic rats may related to the pathophysiology of bladder dysfunction in diabetic rats.3.The expression of myocardin were significant decreased in the bladder of diabetic which may involved in the phenotypic modulation of bladder smooth muscle cell in diabetic rats.