Integrin-Linked Kinase Improves Functional Recovery of Diabetic Cystopathy and Mesenchymal Stem Cell Survival and Engraftment in Rats.

OBJECTIVES: Diabetic cystopathy (DCP), characterized by peripheral neuropathy-associated bladder dysfunction, is a common urinary complication in patients with diabetes (~80%). Bone marrow mesenchymal stem cell (BMMSC) transplantation, a new and emerging regenerative therapy, provides a curative option for DCP. However, the application of this therapy is limited by the low survival rate and engraftment of transplanted stem cells. This study was undertaken to determine whether integrin-linked kinase (ILK) overexpression would improve stem cell survival and engraftment after BMMSC transplantation. METHODS: Diabetes was induced in rats by injection of streptozotocin. ILK expression was detected by qRT-PCR and Western blot. Bladder function was measured by urodynamic analyses. Smooth-muscle regeneration and vascularization were evaluated by immunohistochemistry staining. RESULTS: ILK overexpression by adenovirus promotes proliferation of BMMSCs in vitro. ILK overexpression enhanced the ability of BMMSCs to decrease the volume threshold for micturition and residual urine in the rats with diabetes. The contractile response of bladder strips, tissue structure of bladder and smooth-muscle regeneration/vascularization were also improved in the rats receiving ILK-modified BMMSCs. CONCLUSIONS: Our data highlight the clinical potential of transplantation of gene-modified BMMSCs in the treatment of DCP, thereby serving as a rapid and effective first-line strategy to cure the bladder dysfunction resulting from long-term diabetes.

[Effects of electroacupuncture treatment on nitrergic neurotransmitter in bladder neck and detrusor of rats with unstable bladder].

OBJECTIVE: To observe the effects of electroacupuncture treatment on nitrergic neurotransmitter in bladder neck and detrusor of rats with unstable bladder. METHODS: Rat models of unstable bladder were established by operation to induce urethral obstruction. Electroacupuncture treatment was given by acupuncturing Huiyang and Zhonglushu points for a week. Then the neuronal nitric oxide synthase (nNOS)-, endothelial nitric oxide synthase (eNOS)- and inducible nitric oxide synthase (iNOS)-positive cells in bladder neck and detrusor of the rats were observed. RESULTS: The nitrergic neurotransmitter in bladder neck and detrusor were obviously decreased in rats with unstable bladder. The electroacupuncture treatment could significantly increase the contains of NOS in bladder tissue. CONCLUSION: To promote the synthesis and secretion of nitrergic neurotransmitter in bladder tissue may be one of the mechanisms of acupuncture in adjusting bladder function.

Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract.

PURPOSE OF REVIEW: In recent years nitric oxide (NO) has gained increasing recognition as an important neurotransmitter and cell signaling molecule with a broad range of functions in the lower urinary tract. This review discusses recently published data related to the physiologic and pathophysiologic roles of NO and nitric oxide synthase (NOS) in the female lower urinary tract. RECENT FINDINGS: Expression of three isoforms of NOS, namely endothelial NOS, neuronal NOS and inducible NOS, has been identified in various tissues of the lower urinary tract in animals and humans. In addition to its relaxation effects on bladder and urethra, NO also serves as a neurotransmitter in the lower urinary tract. The physiologic roles of NO in overactive bladder, bladder outlet obstruction, diabetic cystopathy, interstitial cystitis, and bladder inflammation have been demonstrated. SUMMARY: NO plays an important role in the micturition process and in disorders of the lower urinary tract. Improved understanding of the pathophysiologic role of NO/NOS system and of the L-arginine-NO-cGMP pathway may allow us to identify suitable therapeutic targets for lower urinary tract disorders. However, there is a need for further investigation to determine the precise function of NO in human lower urinary tract because most work thus far has been done in animal models.

Mast cell chymase is a possible mediator of neurogenic bladder fibrosis.

AIMS: Urinary bladders of patients with myelomeningocele, owing to spina bifida, are often functionally impaired, fibrotic organs. Common to this condition are repeated occurrences of bladder infection and inflammation. Since mast cells have been associated with a fibrogenic response in inflammatory conditions, we investigated the role of mast cell granule product, chymase, as a mediator of myleodysplastic bladder fibrosis. METHODS: Human control and myelodysplastic bladder tissues were stained with Unna's stain and chymase antibody to determine mast cell number and localization. Cell specific localization of collagen mRNAs was determined by in situ hybridization (ISH). In vitro, normal human bladder fibroblasts were treated with recombinant chymase, heparin and inhibitors, and collagen subtype concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: Myelodysplastic bladders were characterized by increased mast cells in the detrusor muscle layer compared to control bladders, as well as mast cell degranulation and increased connective tissue deposition. Both types I and III collagen mRNA localized to fibroblasts surrounding detrusor muscle fascicles, whereas only collagen III mRNA localized to cells within connective tissue infiltrated muscle bundles in myelomeningocele bladder tissue. Chymase treatment of bladder fibroblasts, in vitro, was dose-dependent and resulted in significant increases in both types I and III collagen. Heparin did not alter collagen protein expression, whereas heparin-chymase combination modulated type III collagen expression. Serine protease inhibitor, phenylmethylsulfonlyfluoride, did not inhibit collagen synthesis, whereas denatured chymase resulted in decreased collagenous protein levels. CONCLUSIONS: Bladder fibrosis may be mediated by mast cell chymase stimulation of collagen synthesis.

Endothelial nitric oxide synthase expression in neurogenic urinary bladders treated with intravesical resiniferatoxin.

OBJECTIVE: To investigate endothelial nitric oxide synthase (eNOS) immunoreactivity in bladder biopsies from patients with neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, and compare this with control material; the distribution of two other vascular markers, von Willebrand Factor (vWF) and the vascular endothelial growth factor (VEGF), was also studied. PATIENTS AND METHODS: Flexible cystoscopic bladder biopsies from eight controls investigated for asymptomatic microhaematuria and 19 patients with refractory spinal NDO enrolled in a clinical trial of intravesical treatment with escalating doses of resiniferatoxin were immunostained with polyclonal rabbit antibodies for eNOS, vWF and VEGF. Fewer baseline NDO specimens (eight) were available for vWF and VEGF staining. Computerized image analysis was used to quantify immunoreactivity, and the Mann-Whitney test for statistical analysis. RESULTS: eNOS immunoreactivity was found in the suburothelium and less often in the urothelium, with a distribution indicating a location in small blood vessels at the urothelium-suburothelium junction. Immunostaining for vWF showed a similar location. There was a trend to higher eNOS values before treatment in those responding than in those not responding to resiniferatoxin (P = 0.059), and a significant reduction in eNOS immunoreactivity after successful treatment (P = 0.016). VEGF staining was weaker but there was a significant increase in pretreatment biopsies of responders to resiniferatoxin (P = 0.048). Clinical and histopathology features were similar in both groups. CONCLUSIONS: The trend for higher eNOS expression in patients with NDO who responded to resiniferatoxin suggests that increased vasculature or vasodilatation in the suburothelium may be necessary for successful intravesical treatment. Further studies with more patients are required to confirm this relationship and to examine the mechanisms underlying changes in vasculature with levels of bladder overactivity.

The contractile potency of adenosine triphosphate and ecto-adenosine triphosphatase activity in guinea pig detrusor and detrusor from patients with a stable, unstable or obstructed bladder.

PURPOSE: We compared the potency of adenosine triphosphate (ATP) and its nonhydrolyzable analogue alpha,beta-methylene ATP for generating contractions in human detrusor smooth muscle from patients with a stable, unstable and obstructed bladders. The different ATP potencies were compared with the ecto-adenosine triphosphatase (ATPase) of these samples. MATERIALS AND METHODS: Contractile experiments were done in vitro by superfusing samples with purines and dose-response curves were generated. Ecto-ATPase activity was measured from the rate of ATP hydrolysis sensitive to the ecto-ATPase inhibitor ARL 67156 with a luciferin-luciferase assay. RESULTS: ATP generated contractions with a mean EC50 of 933 microM. in tissue from stable bladders and was significantly more potent in tissue from unstable and obstructed bladders (EC50 141 and 172 microM., respectively). alpha,beta-methylene ATP was more potent in tissue from stable and unstable bladders (mean combined EC50 3 microM.). In guinea pig detrusor the mean EC50 for ATP and alpha,beta-methylene ATP was 138 and 5.5 microM., respectively. Mean total ATPase activity in unstable bladder biopsies plus or minus standard deviation was about 50% of that in stable bladder biopsies (2.54 +/- 1.50 versus 1.37 +/- 0.46 nmol. per second per mg. protein ). The ARL 67156 sensitive fraction was also significantly less in samples from unstable compared with stable bladders (mean 0.94 +/- 0.41 versus 0.36 +/- 0.26 nmol. per second mg. protein ). CONCLUSIONS: The greater potency of ATP for generating contractions in detrusor from unstable bladders may be due to reduced extracellular hydrolysis, allowing purine greater access to detrusor smooth muscle. This finding may explain atropine resistant purine based contractions in detrusor from unstable bladders.

Familial mitochondrial intestinal pseudo-obstruction and neurogenic bladder.

Intestinal dysmotility and neurogenic bladder have been described as part of two autosomal-recessive mitochondrial disorders assumed to be due to a defect in communication between the nuclear and mitochondrial genomes: myoneurogastrointestinal encephalopathy (MNGIE) and diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome). Partial cytochrome c oxidase deficiency has been described in both. We describe three Ashkenazi Jewish siblings with progressive intestinal dysmotility, neurogenic bladder, and autonomic manifestations but no central nervous system involvement. Cytochrome c oxidase deficiency was demonstrated in peripheral and multiple intestinal muscle biopsies. Mitochondrial DNA analysis of an intestinal biopsy of patient 1 showed heteroplasmy consisting of a normal 16.5-kb band and an approximately 28-kb band, suggestive of a duplication. Mitochondrial DNA analysis of a muscle biopsy of patient 2 showed multiple deletions, mainly 10- and 11-kb bands. We suggest that this unique combination of intestinal pseudo-obstruction and neurogenic bladder could comprise a new autosomal-recessive mitochondrial disorder.

Nitric oxide synthase expression in neurogenic bladder disease: a pilot study.

Neurogenic bladders are susceptible to bladder cancer development, especially in the case of chronic indwelling catheters. The classic carcinogenesis theory involves the formation of carcinogenic nitrosamines by bacteria due to chronic infection. We designed a pilot study to evaluate the expression of the of Nitric Oxide Synthase (NOS) isoforms in bladder tissue to study the role of the endogenous formation of NO (Nitric Oxide) in neurogenic bladders. Immunohistochemistry was performed on bladder biopsies from neurogenic, normal, and obstructed bladders. The neurogenic bladder had a higher expression of endothelial NOS (eNOS), but especially of neuronal NOS (nNOS). Besides, this extra-neuronal expression of nNOS by urothelium and interstitial cells was observed. This study proves the important role of endogenous formation of NO by suburothelial nerves but also by urothelium and interstitial cells. This overexpression could possibly be a factor in the higher incidence of bladder cancer in neurogenic bladders. On the other hand, it shows the plasticity of the NO pathways in these cases and raises important research questions concerning the physiological role of these changes.

Antioxidant deficiency following clam enterocystoplasty.

OBJECTIVE: To assess whether there is a reduction in serum antioxidant activity in patients who have undergone a clam enterocystoplasty procedure. PATIENTS AND METHODS: Serum glutathione peroxidase (GPase) activity was measured in 20 patients who had undergone clam enterocystoplasty. Serum selenium concentration was also measured in 62 similar patients and compared with 56 healthy controls and 44 patients with a neuropathic bladder, mainly patients with spinal cord injuries who had not undergone surgery. RESULTS: GPase activity correlated well with serum selenium measurement. There was a significant reduction (P < 0.001) in serum selenium level in young (< 50 years old) non-neuropathic bladder patients following clam enterocystoplasty. This reduction in serum selenium was similar to that found in both unoperated patients with a neuropathic bladder (who are known to have an increased risk of developing bladder cancer) and those patients with a neuropathic bladder who had undergone augmentation. This reduction was not related to urinary tract infection nor the time since surgery. CONCLUSION: A reduction in serum selenium has been shown to increase susceptibility to bladder cancer following carcinogenic exposure to compounds such as nitrosamines. This study suggests that patients with idiopathic and congenital instability may be at an equally high risk as a result of undergoing this procedure.

Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury.

Changes in the distribution of neuronal nitric oxide synthase immunoreactivity (NOS-IR) after chronic (5-6 weeks) spinal cord injury (SCI) were examined in bladder afferent and spinal neurons in the region of the sacral parasympathetic nucleus (SPN) in the L6-S1 spinal segments. Bladder afferent neurons in the L1, L2, L6 and S1 dorsal root ganglia (DRG) were identified by retrograde axonal transport following injection of fluorogold (FG) into the urinary bladder. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of spinal cord intact animals with significantly greater numbers of NOS-IR cells present in thoracic (T8, T12; 30-40 NOS-IR cell profiles/section) and rostral lumbar (L1) DRGs (18 NOS-IR cell profiles/ section) compared to caudal lumbosacral (L5-S1) DRGs (0.2-0.4 NOS-IR cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 12-20 NOS-IR cell profiles/section) and in the L1-L2 DRG (15-40 NOS-IR cell profiles/section) but not in the L5 DRG following SCI. In these ganglia, an average of 41.2 +/- 7.8% (L6) and 36.3 +/- 0.9% (S1) of FG-labeled bladder afferent neurons were NOS-IR. In contrast, in spinal cord intact animals, no FG-labeled bladder afferent neurons were NOS-IR. Following SCI, NOS-IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN (lateral collateral pathway of Lissauer) of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L5, S2). SCI also significantly (p < or = 0.001) increased the number of spinal neurons in the region of the SPN (presumptive preganglionic neurons) in the L6-S1 spinal segments exhibiting NOS-IR. These results indicate that NOS-IR in bladder afferent and spinal neurons is plastic and can be up-regulated by chronic SCI. Changes in the neurochemical properties of these neurons after SCI may be mediated by pathological changes in the target organ (i.e., urinary bladder) and/or spinal cord.

Experimental neurogenic bladder in rats and effect of Robaveron, a biological prepared from swine prostate, on it.

An experimental neurogenic bladder was induced in rats by an intraspinal injection of 10% phenol-glycerin solution. The functional and biochemical changes in the bladder were studied in vivo and ex vivo, and the effect of Robaveron, a biological prepared from swine prostate, on these changes were examined. The forced voiding pressure in the control rats which had been caused by the neurogenic bladder was reduced to 1/4-1/5 that of the intact animals. Robaveron recovered a decrease in the pressure, and the effect of 150-250 mg/kg, p.o., corresponded nearly to that of 40 mg/kg, i.m. In the ex vivo study with the strips of isolated neurogenic bladder muscle, the time required to cause 50% of the maximum contraction by transmural electric stimulation prolonged to about four times that of the intact one. Such a prolongation was recovered by Robaveron in a dose-dependent fashion, and the effect of Robaveron given p.o. corresponded to about 1/6 that of the drug given i.m. No change in Ca2+-influx, was found, although Ca2+-efflux was inhibited in the strip of neurogenic bladder muscle. Such an inhibition was significantly relieved in that of animals treated with Robaveron. In the neurogenic bladder rat, the ratio of bladder weight to body weight increased, and the activities of total ChE and AChE in the bladder muscle decreased, on which Robaveron did not show any influence. Phasic and tonic contractions in the strips of intact rat bladder and guinea pig ileum were inhibited by La3+, particularly phasic response in both the strips. Robaveron recovered the inhibition in a dose-dependent fashion. The present study suggests that the urinary dysfunction in neurogenic bladder may be caused not only by nervous disorders but also by changes in the bladder muscle itself. Robaveron was found to be effective for most of such changes. These findings may support the clinical efficacy of Robaveron for improving the attenuated bladder function.