Role Of Endothelial Nitric Oxide Synthase (eNOS) In The Pathogenesis And Development Of Diabetic Cystopathy In A STZ-induced Diabetic Rat Model

BackgroundDiabetic cystopathy(DC)is one of the.most common complications in diabetes mellitus(DM)and becoming increasingly a concern in DM patients.Up to date,the mechanism mediating the pathogenesis and development of DC remains unclear.It has been proven to be difficult to restore bladder function in diabetic patients clinically. Therefore,it's in great need to develop new therapeutic approaches for DC. Physiologically,endothelial nitric oxide synthase(eNOS)-derived NO plays an essential role in the control of blood vessel dilation and blood pressure regulation.NO can inhibit platelet aggregation,leukocyte adhesion as well as smooth muscle proliferation in the blood wall,which are common phenomenon in the pathogenesis of vascular diseases.Abnormal expression levels of eNOS mRNA and protein leading to endothelial dysfunction and vascular injury occurred in most diabetic vascular complications,such as nephropathy,neuropathy and retinopathy.Diabetic cystopathy may partly due to the abnormal expression levels of eNOS mRNA and protein in the bladder.PurposeTo evaluate the urodynamical changes of diabetic cystopathy in a STZ- induced diabetic rat model and to detect the expression levels of eNOS mRNA and protein. Using selective eNOS antagonist L-NIO and eNOS coenzyme BH4,we assess the function state of eNOS.To examine the state of oxidative stress and to investigate the ultra-structure change of micro vascular endothelium;to testify the efficiency of ant oxidative treatment on diabetic cystopathy and to explore the possible mechanism.Methods1.Diabetic rat model was induced by a single intraperitoneal injection of STZ (60mg/kg body weight)in male Sprague-Dawley rats.Rats were divided into 3 groups: normal control,diuresis control and diabetes groups.Examinations were performed at 6W and 12W after injection of STZ.Cystometry was taken to evaluate the bladder function;Expression levels of eNOS mRNA and protein were assessed with RT-PCR and Western blot assays respectively and their correlation with bladder function index were analyzed.We evaluated the expression profile of eNOS in rat bladder by immunohistochemistry(IHC)assay.We utilized electro-microscope technique to analyze the ultra-structure change of bladder micro vessels.2.6W after induction of diabetes,L-NIO and BH4 were injected intraperitoneally to testify the function state of eNOS through evaluating expression levels of eNOS mRNA and protein Expression level of 3-NT protein was evaluated with western blot assay.3.After treated withα-LA for 6W,cystometry was performed to evaluate the bladder function;expression levels of eNOS mRNA,eNOS protein and 3-NT protein were examined by RT-PCR and western blot assays.Activities of SOD and CAT and level of MDA were quantified by biochemical assays.Results1.Compared with normal control group,blood glucose level and 24h urine volume of diabetic group increased(P＜0.01),while body weight decreased(P＜0.01).The result of cystometry revealed that after 6W of injection of STZ,bladder capacities of normal,diuresis and diabetic group were 0.75±0.04,1.62±0.15,and 2.65±0.26ml, respectively,while at 12W,those values were 0.86±0.06,1.93±0.10 and 2.65±0.26ml with significant difference.Post voiding volumes of each group at 6W were 0.064± 0.003,0.112±0.013,and 0.232±0.010ml,while at 12W,the volumes were 0.071±0.004,0.129±0.024,and 0.561±0.061ml;single voiding volumes of each group at 6W were 0.70±0.02,1.52±0.30和2.06±0.25ml,while at 12W,the volumes are 0.80±0.04,1.77±0.11和2.09±0.21ml;voiding efficiencies of each group at 6W were 93.3±2.1%,93.2±2.7%和90.0±2.5%,while at 12W they were 93.1±2.2%,93.8±3.2%, and 78.1±2.6%.No significant difference was observed among bladder pressures of each group.As revealed by RT-PCR assay,expression levels of eNOS mRNA from each group at 6W were 0.81±0.12,0.90±0.12,and 1.98±0.16,while at 12W they were 0.87±0.17,1.05±0.11,and 2.89±0.23 respectively.Expression levels of eNOS protein measured by western blot were 0.98±0.12,0.93±0.14,and 2.28±0.16,while at 12W they were 1.03±0.13,1.14±0.11,and 3.12±0.20,respectively.The expression of eNOS was found only in bladder vessels by IHC.Compared with normal rats,the diabetic rats showed significantly stronger stain of eNOS at both 6W and 12w.We observed remarkable alteration of bladder micro-vessel under electro-microscope characterized by swollen endothelial cells with disappeared mitochondrial cristae,ruffled nucleus, and condensed chromatin.There was increased number of lysosome while decreased ribosome and endoplasmic reticulum in the endothelial cells.Collagen accumulation was prominent in the perivascular area.2.Treatment with L-NIO and BH4 showed no influence on the expression levels of both eNOS mRNA and protein in diabetic bladder.As shown by the result of western blot assay,3-NT production in diabetic bladder was increased(0.39±0.05 vs 0.05±0.01,P＜0.01),and both L-NIO and BH4 administration reduced the 3-NT expression level(0.25±0.05 and 0.21±0.02,P＜0.01 compared with diabetic rats).3.Treatment withα-LA influenced neither blood glucose level nor body weight of diabetic rats.Compared with DM+NS group,α-LA treatment significantly reduced bladder capacity(2.65±0.15 vs 1.93±0.09 ml,P＜0.05),single voiding volume (2.09±0.10 vs 1.73±0.06,P＜0.05),and post voiding volume(0.561±0.060 vs 0.202±0.013ml,P＜0.01),meanwhile increased the voiding efficiency(78.1±2.6%vs 89.6±2.6%,P＜0.05).Oxidative stress occurred in diabetic bladder demonstrated by increased level of MDA(0.64±0.05 vs 0.31±0.03 nmol/mg protein,P＜0.01),and decreased activity of SOD(35.70±2.05 vs 15.23±1.17 ug protein,P＜0.01)and CAT (83.45±8.48 vs 29.86±4.97 ug protein,P＜0.01).Treatment withα-LA significantly relieved oxidative stress by decreasing MDA level(0.474±0.05 nmol/mg protein,P＜0.01)and increasing activity of SOD(27.91±2.09 ug protein,P＜0.01)and CAT(48.50±3.75,P＜0.01).α-LA treatment reduced expression level of eNOS mRNA(3.01±0.27 vs 1.85±0.28,P＜0.05)and protein(3.89±0.33 vs 2.75±0.24,P＜0.05),as well as 3-NT protein(0.61±0.07 vs 0.31±0.05,P＜0.01).Conclusion1.Diabetes mellitus in rats,with similar urodynamical changes to human,is a reliable and useful model for research on diabetic cystopathy.2.Expression of eNOS is upregulated at both mRNA and protein levels in a time-dependent manner in the diabetic bladder.3.Diabetic cystopathy develops in a time-dependent manner and is correlated negatively with expression levels of eNOS mRNA and protein.4.Injury of bladder micro vessel occurs in line with severity of bladder dysfunction and may serve as a morphological index for bladder function.5.eNOS in diabetic bladder is inappropriately activated and in the state of uncoupling,leading to increased formation of peroxynitrite as an oxidative source.6.Oxidative stress occurs in diabetic bladder due to imbalanced oxidant and anti-oxidant system.7.Antioxidant treatment can reverse impaired function of bladder in diabetic rats through down-regulating expression levels of eNOS mRNA and protein and may serve as a new therapeutic way for diabetic cystopathy.