Effects of herpes simplex virus vectors encoding poreless TRPV1 or protein phosphatase 1α in a rat cystitis model induced by hydrogen peroxide.

Enhanced afferent excitability is considered to be an important pathophysiological basis of interstitial cystitis/bladder pain syndrome (IC/BPS). In addition, transient receptor potential vanilloid-1 (TRPV1) receptors are known to be involved in afferent sensitization. Animals with hydrogen peroxide (HP)-induced cystitis have been used as a model exhibiting pathologic characteristics of chronic inflammatory condition of the bladder. This study investigated the effect of gene therapy with replication-defective herpes simplex virus (HSV) vectors encoding poreless TRPV1 (PL) or protein phosphatase 1 α (PP1α), a negative regulator of TRPV1, using a HP-induced rat model of cystitis. HSV vectors encoding green fluorescent protein, PL or PP1α were inoculated into the bladder wall of female rats. After 1 week, 1% HP or normal saline was administered into the bladder, and the evaluations were performed 2 weeks after viral inoculation. In HP-induced cystitis rats, gene delivery of PL or PP1α decreased pain behavior as well as a reduction in the intercontraction interval. Also, both treatments reduced nerve growth factor expression in the bladder mucosa, reduced bladder inflammation characterized by infiltration of inflammatory cells and increased bladder weight. Taken together, HSV-mediated gene therapy targeting TRPV1 receptors could be effective for the treatment of IC/BPS.

Intravesical hyaluronic acid treatment improves bacterial cystitis and reduces cystitis-induced hypercontractility in rats.

OBJECTIVE: To investigate the effect of intravesical hyaluronic acid on Escherichia coli-induced cystitis and cystitis-induced hypercontractility in rats. METHODS: Bacterial cystitis was induced in Wistar female rats by intravesical inoculation of E. coli. Isotonic saline was instilled in the control group (n = 6). The rats were either non-treated, treated with gentamycin (4 mg/kg, 5 days) or treated intravesically with hyaluronic acid (0.5 mL, 0.5%). On the eighth day, the bladder tissues were excised for histological examination, and the measurements of myeloperoxidase, superoxide dismutase and catalase activities. Contraction/relaxation responses to carbachol, isoprotrenol and papaverine were studied. RESULTS: Tissue myeloperoxidase activity was increased, but superoxide dismutase and catalase activities were decreased in bacterial cystitis, while hyaluronic acid treatment reversed these changes. In the hyaluronic acid-treated group, healing of the uroepithelium was observed, while decreased inflammatory cell infiltration was obvious in gentamycin-treated group. E. coli-induced cystitis in all rats resulted in increased contraction responses to carbachol compared with controls (P < 0.01). Treatment with hyaluronic acid, but not gentamycin, significantly (P < 0.05) depressed hypercontractility at maximum carbachol concentrations. In all rats with cystitis, papaverine-induced relaxation was increased, whereas isoproterenol-induced relaxation curves were not different between the studied groups. CONCLUSION: Gentamycin treatment, despite its ameliorative effect on inflammation, had no impact on the contractile dysfunction of the injured bladder. Intravesical hyaluronic acid, in addition to its supportive role in the healing of the epithelium, seems to lower the increased threshold for contraction and to reduce oxidative stress. These findings support a potential role for hyaluronic acid in the treatment of bacterial cystitis.

Telomerase reverse transcriptase promoter mutations in glandular lesions of the urinary bladder.

Glandular lesions of the urinary bladder include a broad spectrum of entities ranging from completely benign to primary and secondary malignancies. The accurate diagnosis of these lesions is both important and challenging. Recently, studies suggest that telomerase reverse transcriptase (TERT) promoter mutations could be a biomarker for urothelial carcinoma (UC). We hypothesized that these mutations can distinguish UC with glandular differentiation from nephrogenic adenoma, primary adenocarcinoma of the urinary bladder (PAUB), or secondary malignancies. Twenty-five cases of benign glandular lesions (including nephrogenic adenoma); 29 cases of UC with glandular differentiation; 10 cases of PAUB; and 10 cases each of metastatic colon cancer, prostatic carcinoma, and carcinoma from Mullerian origin were collected. Slides were reviewed and selected to make sure the lesion was at least 10% to 20% of all tissue. Macrodissection was performed in some of cases, and genomic DNA was extracted from the tissue. Telomerase reverse transcriptase promoter mutations were determined by standard polymerase chain reaction sequencing. Twenty-one cases (72%) of UC with glandular differentiation were positive for TERT promoter mutations. However, none of the remaining cases (total 65 cases of benign lesions, PAUB, and metastatic carcinomas) was positive for TERT promoter mutation. Telomerase reverse transcriptase promoter mutations were highly associated with UC including UC with glandular differentiation but not other glandular lesions of bladder. Therefore, in conjunction with morphologic features, Immunohistochemistry stain profile, and clinical information, TERT promoter mutations could distinguish UC with glandular differentiation from other bladder glandular lesions. In addition, lack of TERT promoter mutations in primary adenocarcinoma of bladder suggests that this entity may have different origin or carcinogenesis from those of UC.

Dual involvements of cyclooxygenase and nitric oxide synthase expressions in ketamine-induced ulcerative cystitis in rat bladder.

AIMS: The aims of the present study were to investigate voiding patterns, tissue constituents and the expressions of cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) involved in ketamine-induced ulcerative cystitis in rat urinary bladder. METHODS: Thirty Sprague-Dawley rats were distributed into three groups which received saline or ketamine (25 mg/kg/day) for a period of 14 and 28 days. In each group, cystometry was performed weekly and the concentration of ketamine and its metabolites (norketamine) was assayed. Paraffin-embedded sections were stained with Masson's trichrome stain, and ketamine-induced morphological changes were examined. Western blot analyses were carried out to examine the expressions of COX-2 and different NOS isoforms in bladder tissues. Immunofluorescence study was done to evaluate the expressions of COX-2 and macrophage infiltration (stained with ED-1 macrophage cell surface antigen) within the bladder. RESULTS: Ketamine treatment resulted in bladder hyperactivity and the non-voiding contractions were significantly increased. The urine concentrations of ketamine and norketamine were much higher in ketamine-treated group. Moreover, ulcerated urothelium and mononuclear cell infiltration were noted in ketamine-treated group. These alterations in urodynamic functions and tissue constituents were accompanied by increases in the expression of COX-2. Two NOS isoforms (iNOS and eNOS) were also overexpressed, but no significant change was observed for nNOS. COX-2 positive stained cells were significantly increased. Meanwhile, increased amounts of ED-1 positive stained macrophages were present and most of COX-2 expressed cells were co-stained with ED-1 in the early stage of ketamine treatment. CONCLUSIONS: Ketamine treatment affected bladder tissues by enhancing interstitial fibrosis and accelerating macrophages infiltration. Ketamine also initiated the up-regulations of COX-2 and iNOS and eNOS expressions. These up-regulated enzymes might play an important role in contributing to ketamine-induced alterations in micturition patterns and ulcerative cystitis.

Ca(2+)/calmodulin-dependent protein kinase II is associated with pelvic pain of neurogenic cystitis.

Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as a neurogenic cystitis. Interstitial cystitis is associated with urothelial lesions, voiding dysfunction, and pain in the pelvic/perineal area. In this study, we used a murine neurogenic cystitis model to identify genes participating in the development of pelvic pain. Neurogenic cystitis was induced by the injection of Bartha's strain of pseudorabies virus (PRV) into the abductor caudalis dorsalis (tail base) muscle of female C57BL/6J mice. Mice infected with PRV developed progressive pelvic pain. The sacral spinal cord was harvested on postinfection days (PID) 2 and 4, and gene expression was analyzed by microarrays and confirmed by quantitative RT-PCR. On PID 2, the overall expression profile was similar to that of uninfected sacral spinal cord; by PID 4, there were substantial differences in expression of multiple functional classes of genes, especially inflammation. Analysis of pain-signaling pathways at the dorsal horn suggested that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) contributes to neurogenic cystitis pelvic pain. Consistent with this, CaMKIIδ expression exhibited a mast cell-dependent increase in the sacral spinal cord at the mRNA level, and phospho-CaMKII immunoreactivity in the dorsal horn was increased on postinfection day (PID) 4 during PRV infection. Finally, intrathecal injection of the CaMKII inhibitor KN-93 attenuated the PRV pain response. These data suggest that CaMKII plays a functional role in pelvic pain due to neurogenic cystitis.

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons.

BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. RESULTS: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency. CONCLUSIONS: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.

Role for pAKT in rat urinary bladder with cyclophosphamide (CYP)-induced cystitis.

AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200-300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 µg/10 µl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function.

O-GlcNAc mediated glycosylation down-regulation in mice with cyclophosphamide induced cystitis.

PURPOSE: Cyclophosphamide induced cystitis is an established model for the study of bladder injury and wound healing. Glycosylation is an important modification mechanism that regulates the structure and function of secreted proteins and growth factors from inflammation sites. We determined the effect of cyclophosphamide induced cystitis on O-GlcNAc mediated glycosylation in the bladder. MATERIALS AND METHODS: Cystitis in WT C57BL6 mice was induced with intraperitoneal cyclophosphamide. Retrieved bladders were analyzed using histology, immunohistochemistry, reverse transcriptase-polymerase chain reaction and Western blot for glycosylation associated factors. RESULTS: Acute bladder injury was seen up to 168 hours (7 days) after injection. Reverse transcriptase-polymerase chain reaction revealed down-regulation of O-GlcNAc transferase, a key enzyme in O-GlcNAc mediated glycosylation, at the 8, 48 and 168-hour time points. Also, the glycosidase menangioma expressed antigen 5 was up-regulated at similar time points. Western blot analysis revealed decreased glycosylated protein during cyclophosphamide induced inflammation. CONCLUSIONS: To our knowledge we report the first study of alterations in O-GlcNAc mediated glycosylation activity in bladders with cyclophosphamide induced cystitis. Glycosylation may have a significant role in the bladder wound healing process. Future studies of the glycosylation signaling pathways in the bladder would assist in future potential therapy for bladder inflammatory disease and cancer by elucidating pathways that guide bladder development and wound healing.

Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice.

AIMS: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine found pre-formed in the urothelium. During inflammation, MIF is released into the bladder lumen and bladder MIF mRNA is upregulated. Since MIF also has tautomerase activity and blocking tautomerase activity also blocks MIF's biological activity, we hypothesized that blocking MIF's tautomerase activity would prevent bladder inflammation. Therefore, we examined the effects of a MIF tautomerase inhibitor (ISO-1; also blocks biological activity) on cyclophosphamide (CYP)-induced cystitis in mice. METHODS: Mice receiving CYP (300 mg/kg; i.p.) to induce cystitis or saline (control) were treated either with ISO-1 (20 mg/kg; i.p.; daily) or vehicle (20% DMSO; i.p.; daily) for 2 days. After 2 days, micturition volume and frequency in awake mice were recorded and also mechanical sensitivity to abdominal stimulation using von Frey monofilaments. Bladders were collected under anesthesia and examined histologically, nerve growth factor levels were assayed in bladder homogenates, and production of inflammatory cytokines in the bladder was determined using a targeted array. RESULTS: CYP treatment resulted in decreased micturition volume, increased frequency, decreased threshold, increased histological signs of cystitis, increased bladder NGF levels and production of inflammatory cytokines when compared to the control group. Treatment with ISO-1 prevented or greatly decreased all these changes. CONCLUSION: Antagonizing MIF's activity with a systemic MIF tautomerase inhibitor was able to prevent or greatly reduced chemical cystitis in mice, thus indicating the MIF mediates bladder inflammation in this model. MIF represents a novel and important modulator of cystitis.

Effects of estrogens and bladder inflammation on mitogen-activated protein kinases in lumbosacral dorsal root ganglia from adult female rats.

BACKGROUND: Interstitial cystitis is a chronic condition associated with bladder inflammation and, like a number of other chronic pain states, symptoms associated with interstitial cystitis are more common in females and fluctuate during the menstrual cycle. The aim of this study was to determine if estrogens could directly modulate signalling pathways within bladder sensory neurons, such as extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. These signalling pathways have been implicated in neuronal plasticity underlying development of inflammatory somatic pain but have not been as extensively investigated in visceral nociceptors. We have focused on lumbosacral dorsal root ganglion (DRG) neurons projecting to pelvic viscera (L1, L2, L6, S1) of adult female Sprague-Dawley rats and performed both in vitro and in vivo manipulations to compare the effects of short- and long-term changes in estrogen levels on MAPK expression and activation. We have also investigated if prolonged estrogen deprivation influences the effects of lower urinary tract inflammation on MAPK signalling. RESULTS: In studies of isolated DRG neurons in short-term (overnight) culture, we found that estradiol and estrogen receptor (ER) agonists rapidly stimulated ER-dependent p38 phosphorylation relative to total p38. Examination of DRGs following chronic estrogen deprivation in vivo (ovariectomy) showed a parallel increase in total and phosphorylated p38 (relative to beta-tubulin). We also observed an increase in ERK1 phosphorylation (relative to total ERK1), but no change in ERK1 expression (relative to beta-tubulin). We observed no change in ERK2 expression or phosphorylation. Although ovariectomy increased the level of phosphorylated ERK1 (vs. total ERK1), cyclophosphamide-induced lower urinary tract inflammation did not cause a net increase of either ERK1 or ERK2, or their phosphorylation. Inflammation did, however, cause an increase in p38 protein levels, relative to beta-tubulin. Prior ovariectomy did not alter the response to inflammation. CONCLUSIONS: These results provide new insights into the complex effects of estrogens on bladder nociceptor signalling. The diversity of estrogen actions in these ganglia raises the possibility of developing new ways to modulate their function in pelvic hyperactivity or pain states.

Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells.

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 microM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 microM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.

Induction of COX-2 expression by acrolein in the rat model of hemorrhagic cystitis.

AIM: Acrolein (ACR) is a urinary metabolite of cyclophosphamide (CPS) and ifosfamide (IFS), which has been demonstrated to be the causative agent of hemorrhagic cystitis (HC), induced by these compounds. In this study, we investigate the participation of cyclooxygenase-2 (COX-2) on ACR-induced HC. METHODS: Male Wistar rats (150-200g; six rats per group) were treated with distilled water or intravesical ACR and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters and COX-2 expression. RESULTS: COX-2 immunohistochemical expression was significant 12h after ACR administration mainly in subepithelial cells. ACR injection also alters some macroscopic and microscopic parameters in bladder of rats analyzed by Gray's criteria. CONCLUSIONS: COX-2 participates in the pathogenesis of ACR-induced HC first seen 12h after initial contact between ACR and urothelium.

Increased expression of neuronal nitric oxide synthase in bladder afferent pathways following chronic bladder irritation.

Immunocytochemical techniques were used to examine alterations in the expression of neuronal nitric oxide synthase (NOS) in bladder pathways following acute and chronic irritation of the urinary tract of the rat. Chemical cystitis was induced by cyclophosphamide (CYP) which is metabolized to acrolein, an irritant eliminated in the urine. Injection of CYP (n = 10, 75 mg/kg, i.p.) 2 hours prior to perfusion (acute treatment) of the animals increased Fos-immunoreactivity (IR) in neurons in the dorsal commissure, dorsal horn, and autonomic regions of spinal segments (L1-L2 and L6-S1) which receive afferent inputs from the bladder, urethra, and ureter. Fos-IR in the spinal cord was not changed in rats receiving chronic CYP treatment (n = 15, 75 mg/kg, i.p., every 3rd day for 2 weeks). In control animals and in animals treated acutely with CYP, only small numbers of NOS-IR cells (0.5-0.7 cell profiles/sections) were detected in the L6-S1 dorsal root ganglia (DRG). Chronic CYP administration significantly (P < or = .002) increased bladder weight by 60% and increased (7- to 11-fold) the numbers of NOS-immunoreactive (IR) afferent neurons in the L6-S1 DRG. A small increase (1.5-fold) also occurred in the L1 DRG, but no change was detected in the L2 and L5 DRG. Bladder afferent cells in the L6-S1 DRG labeled by Fluorogold (40 microliters) injected into the bladder wall did not exhibit NOS-IR in control animals; however, following chronic CYP administration, a significant percentage of bladder afferent neurons were NOS-IR: L6 (19.8 +/- 4.6%) and S1 (25.3 +/- 2.9%). These results indicate that neuronal gene expression in visceral sensory pathways can be upregulated by chemical irritation of afferent receptors in the urinary tract and/or that pathological changes in the urinary tract can initiate chemical signals that alter the chemical properties of visceral afferent neurons.

Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma.

Accumulating experimental evidence has linked the overproduction of extracellular matrix-degrading metalloproteinases with tumor cell invasion. In the present study one member of the metalloproteinase family, type IV collagenase (M(r) 72,000 gelatinase), is shown to be elevated in the urine of patients with transitional cell carcinoma of the bladder. The form of the enzyme in the urine was studied by three independent methods: enzyme-linked immunosorbent assay, Western immunoblotting; and gelatin zymography. Immunoblotting revealed that the enzyme was present as a series of fragments, each retaining the amino terminus of the mature proenzyme. A prominent M(r) 43,000 fragment was associated with the transitional cell carcinoma cases. Zymography demonstrated that multiple enzyme species with gelatinase activity were present in urine and that high-molecular-weight bands of substrate lysis corresponded to complexes between type IV collagenase and tissue inhibitor of metalloproteinases 2. The total amount of type IV collagenase antigen was significantly elevated in the urine of 37 transitional cell carcinoma patients (range, 0-1081 ng/ml; mean, 318.4 +/- 147.3) compared to 19 normal controls (P < or = 0.004) and 17 inflammatory disease controls (P < or = 0.011). Immunohistochemical staining of bladder tumor biopsies verified that the transitional cell carcinoma cells were producing the M(r) 72,000 enzyme. Thus, M(r) 72,000 type IV collagenase, which is present in the urine in many forms including fragments and complexes with inhibitors, may be a useful marker for bladder cancer diagnosis or prognosis.

Urinary N-acetyl-beta-glucosaminidase and the selection of children for radiologic evaluation after urinary tract infection.

Urinary levels of N-acetyl-beta-glucosaminidase (NAG) were measured in 147 consecutively enrolled children younger than 13 years of age with urinary tract infection to determine whether elevated levels were a predictor of urologic abnormalities. The children were classified as having cystitis if results of 0 or 1 of the following tests were positive and as having pyelonephritis if results of greater than or equal to 2 tests were positive: (1) temperature greater than 38 degrees C, (2) serum C-reactive protein greater than 1 mg/dL, (3) erythrocyte sedimentation rate greater than 25 mm/h, and (4) 1-deamino-8-D-arginine vasopressin-renal concentrating protein less than 810 mOsm/kg. Urinary NAG to creatinine ratios did not distinguish cases of cystitis from those of pyelonephritis. Urinary NAG was useful in identifying children with cystitis who had vesicoureteral reflux of grades II through V. Of 6 children with cystitis and vesicoureteral reflux, 5 had levels of NAG more than 1 SD above the mean, whereas of 75 children without vesicoureteral reflux, only 15 had such an elevation (P = .003). Of those children with a normal NAG level, 60 (98.4%) had normal radiologic evaluation results, and only 1 child (1.6%) had vesicoureteral reflux. Levels of NAG did not identify children with pyelonephritis who had vesicoureteral reflux. It is concluded that (1) urinary NAG is of no value in localizing the site of urinary tract infection, and (2) an NAG level within 1 SD of the mean in a child with cystitis indicates a low risk of urologic abnormalities, and radiologic evaluation may be omitted unless infection recurs.

Pharmacological evaluation of the role of cyclooxygenase isoenzymes on the micturition reflex following experimental cystitis in rats.

Prostanoids, generated from cyclooxygenase (COX) isoenzymes, play a role in the physiological function of the lower urinary tract and are important mediators of inflammatory hyperalgesia. The present work evaluates the effects of the COX-1/COX-2 inhibitor dexketoprofen as well as of a selective COX-2 inhibitor, NS-398, on urodynamic function following endotoxin (LPS) or cyclophosphamide (CYP)-induced inflammation of the urinary bladder. The application of arachidonic acid (330 microgram rat(-1)) onto the serosal surface of the urinary bladder in control rats elicited bladder contractions which could be blocked in a dose-dependent manner by dexketoprofen (0.1 - 3 mg kg(-1), i.v.) but not by NS-398 (0.2 - 6 mg kg(-1), i.v. ). Dexketoprofen (3 mg kg(-1), i.v.) decreased the micturition frequency and increased the pressure threshold for triggering the micturition either when administered within 15 min or 3 h following surgery in control animals. NS-398 (6 mg kg(-1), i.v.) decreased the micturition frequency and increased the pressure threshold when administered 3 h but not 15 min following surgery. Administration of LPS (2 mg kg(-1), i.v., 90 - 120 min) increased both the micturition frequency and the pressure threshold for triggering the micturition reflex. Changes in urodynamic parameters induced by LPS were prevented by doses of either dexketoprofen (1 mg kg(-1), i.v.) or NS-398 (2 mg kg(-1), i.v.) which were ineffective in control animals. Pretreatment with CYP (150 mg kg(-1), i.p., 48 h) increased the micturition frequency, pressure threshold, and the minimal intravesical pressure but decreased the mean amplitude of micturition contractions. In CYP-treated rats, dexketoprofen (1 mg kg(-1), i.v.) or NS-398 (2 mg kg(-1), i.v.) blocked the CYP-induced urodynamic changes with exception of the micturition contraction amplitude. These results indicate that COX-1 may be involved in modulating the threshold for activating the micturition reflex in the normal rats and also demonstrates that inhibition of COX-2 prevents or reverses the urodynamic changes associated with bladder inflammation induced either by surgery, LPS or CYP treatments.

Nitric oxide synthases and cyclophosphamide-induced cystitis in rats.

The role of inducible (iNOS) and neuronal nitric oxide (nNOS) synthases and of tachykinin NK1 receptors on the pathogenesis of cyclophosphamide (CYP)-induced cystitis was investigated, in rats. CYP-induced cystitis was characterized by large increases in bladder-protein plasma extravasation (PPE), increases in the urinary excretion of nitric oxide (NO) metabolites and histological evidences of urothelial damage, edema, extensive white blood cell infiltrates and vascular congestion of the bladder. The specific iNOS inhibitor, S-methylthiourea (MITU), produced marked inhibition (>90%) of CYP-induced increases in PPE associated with amelioration of tissue inflammatory changes. Treatment with 7-nitroindazole (7-NI; 20, 40 and 80 mg/kg), a selective nNOS inhibitor, did not significantly reduce CYP-induced increases in PPE and failed to produce histological improvement. In addition, treatment with MITU, but not with 7-NI, inhibited the increases in the urinary excretion of NO metabolites induced by CYP treatment. WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-androstano[3,2-b]pyrimido[1,2-a]benzimidazole; WIN), a selective NK1-receptor antagonist, reduced the increases in EPP and ameliorated the inflammatory changes in the bladder induced by CYP. However, the maximal degree of protection achieved with WIN was significantly less than that produced by MITU. Combined treatment with the iNOS inhibitor and the NK1 antagonist produced no greater effect than that produced by the iNOS inhibitor alone. Our results suggest that NO plays a fundamental role in the production of the cystitis associated with CYP treatment. The iNOS, and not nNOS, seems responsible for the inflammatory changes. Part of the increases in NO may due to activation of NK1 receptors by neuropeptides such as substance P possibly released from primary afferent fibers.

Arylsulfatase A activity of urine in patients with various genitourinary tract disorders.

Arylsulfatase A activity was measured in urine from patients with various genitourinary tract disorders such as bladder tumor and inflammation. No significant difference in enzyme activity was found between normal and affected urine on the basis of either the volume or protein content of urine, a finding which differed from previous results. However, it was demonstrated that urine from affected patients was more labile to heat treatment in comparison with the control. Upon pretreatment of urine arylsulfatase A at 62.5 degrees C for 10 min, an average of 57% of the original activity was lost in samples from patients with bladder tumor, 58% in those with testicular tumor and 62% in cystitis and urethritis, respectively, while the enzyme activity in the control urine lost only 27% with similar heat treatment. These results were statistically significant with p < 0.001. The arylsulfatase A from patients with advanced bladder tumors demonstrated the presence of a variant form (pI 5.3) which was not detected in normal urine. This variant of arylsulfatase A was also demonstrated in nephrostomy urine from patients with congenital obstruction of the pelvi-ureteric junction.

Urinary lactate dehydrogenase isoenzyme analysis in adult population.

This investigation was a systemic study on an adult population of urinary lactate dehydrogenase (LDH) isoenzyme analysis for the distinction between upper and lower urinary tract infections. The study included 160 urine samples from patients and healthy individuals. On the basis of clinical symptoms, urinary bacterial colony counts, renal function tests and radiologic findings, the adults were divided into pyelonephritis group, cystitis group, pelvic lesion group, and control group. This technique correctly identified 23 of 26 patients with pyelonephritis by the presence of elevated LDH-V (over 10 percent) and all of 12 patients with cystitis by the presence of elevated LDH-I (over 60 relative units) but low LDH-V (below 10 percent or lower than LDH-I). In the pelvic group, the results of eight patients were consistent with cystitis and four with pyelonephritis. Our study confirms the sensitivity and specificity of the LDH isoenzyme technique for the differential diagnosis of urinary tract infection on adult patients and is consistent with previous studies on pediatric patients. However, one should be cautious to interpret the results of LDH isoenzymogram before extra-urinary tract lesions are excluded.

Involvement of interleukin-6-regulated nitric oxide synthase in hemorrhagic cystitis and impaired bladder contractions in young rats induced by acrolein, a urinary metabolite of cyclophosphamide.

Hemorrhagic cystitis is a common complication in children receiving cyclophosphamide, a chemotherapeutic alkylating agent. Acrolein is a urinary metabolite from cyclophosphamide and can induce hemorrhagic cystitis. Here, we investigated the effects and mechanisms of acrolein by intravesical instillation on urinary bladder muscle contractions and pathological alterations in rats. Acrolein instillation significantly increased the muscle contractions of rat bladder detrusor after 1 and 6 h but markedly decreased detrusor contractions after 24 h. Acrolein increased phosphorylated protein kinase C (pan-PKC) expressions in bladders after 1 and 6 h but inhibited it after 24 h. Inducible nitric oxide (NO) synthase (iNOS) protein expressions were markedly induced in bladders 24 h after acrolein treatment. Twenty-four-hour acrolein instillation increased the levels of nitrite/nitrate and interleukin-6 (IL-6) in the urinary bladder. The iNOS inhibitors significantly inhibited the acrolein-increased nitrite/nitrate levels, but not IL-6 levels. IL-6-neutralizing antibodies effectively inhibited the acrolein-increased NOx levels. The increased detrusor contractions by 1-h acrolein treatment were significantly reversed by the PKC inhibitor RO32-0432, and the decreased detrusor contractions by 24-h acrolein treatment were significantly reversed by the iNOS inhibitor and IL-6-neutralizing antibody. Both the iNOS inhibitor and IL-6-neutralizing antibody effectively reversed the increased iNOS expression, decreased PKC phosphorylation, increased bladder weight, and hemorrhagic cystitis in rats 24 h after acrolein treatment. Taken together, these results suggest that an IL-6-regulated iNOS/NO signaling pathway participates in the acrolein-triggered detrusor contraction inhibition and hemorrhagic cystitis. These findings may help us to find a new strategy to treat cyclophosphamide-induced hemorrhagic cystitis.