Adaptation to partial urethral obstruction in healthy aging LOU rats and the role of nerve growth factor signaling pathway in the bladder.

AIM: Aging is associated with poor ability to adapt to stress and abnormal nerve growth factor (NGF) profile. Lower urinary tract symptoms frequently disturb the quality of life of the aging population with no optimal treatment for both genders. The aim of the study was to compare the bladder response to bladder outflow obstruction in young and old LOU rats, a model of healthy aging that does not develop insulin resistance, and its relation to proNGF/NGF imbalance. METHODS: 6- and 36-month-old female LOU rats were subjected to partial bladder urethral obstruction (PUO) for 2 weeks. Morphometric parameters (body and bladder weight) and glycemia were evaluated. Cystometry was carried out to measure functional parameters followed by ex vivo assessment of muscle strip contractile characteristics. Tissue proteins were examined by immunoblotting and morphology was examined by microscopy. RESULTS: Body weight and glycaemia were not affected by surgery. PUO increases significantly bladder weight with increased thickness and fibrosis of the bladder wall as revealed by histological examination in both age groups. Cystometry showed that old PUO rats had a significant reduction in the intercontraction interval and the bladder capacity, a pattern opposite to young rats with PUO. Contractile properties of bladder strip were not affected by age or PUO. On the molecular level, the old rats had lower abundance of the mature NGF relative to proNGF, with signs of p75NTR activation suggested by the higher expression of TNF-α and JNK phosphorylation in the bladder tissue. CONCLUSION: Bladder adaptation to PUO occurs only in young LOU rats to maintain efficient bladder contractility. Old LOU rats display proNGF/NGF imbalance and the associated p75NTR activation. This can further induce tissue damage and degeneration through activation of JNK pathway and release of TNF-α which in turn interferes with the necessary bladder adaptation.

The role of renal biomarkers to predict the need of surgery in congenital urinary tract obstruction in infants.

INTRODUCTION: The diagnosis of renal function impairment and deterioration in congenital urinary tract obstruction (UTO) continues to be extremely challenging. The use of new renal biomarkers in this setting may favor early renal injury detection, allowing for a reliable choice of optimal therapeutic options and the prevention or minimization of definitive renal damage. OBJECTIVE: The aim of the study was to investigate a selection of promising biomarkers of renal injury with the intention of evaluating and comparing their profile with clinically based decisions for surgical intervention of infants with congenital obstructive uropathies. STUDY DESIGN: The first-year profile of renal biomarkers, serum creatinine (sCr), serum and urine cystatin C (CyC), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), transforming growth factor beta-1 (TGF-ß1), retinol-binding protein (RBP), and microalbuminuria (µALB), was analyzed in a cohort of 37 infants with congenital UTO, divided into three subgroups, 14 cases with grade III unilateral hydro(uretero)nephrosis, 13 cases with grade III bilateral hydro(uretero)nephrosis, and 10 cases with low urinary tract obstruction (LUTO), compared with 24 healthy infants matched by gestational age and birth weight. Serum and urine samples were stored at -70 °C and thereafter analyzed by quantitative enzymatic immunoassay. RESULTS: Compared with the control group (Figure), all renal biomarker values were significantly increased in patients (P ≤ 0.02). In the unilateral hydronephrosis and LUTO group, RBP (P ≤ 0.043), NGAL (P ≤ 0.043), KIM-1 (P ≤ 0.03), and TGF-ß1 (P ≤ 0.034) values dropped significantly after surgery. Neutrophil gelatinase-associated lipocalin alone and in combination with urine and serum CyC demonstrated the best performance in determining the need for surgery (area under the curve, 0.801 and 0.881, respectively). Biomarker profile analysis was suggestive of surgical intervention in 55.4% (7/13) of non-operated cases, and most of the biomarker values were above the cutoff levels within at least 3 months before the clinically based surgical decision in 58% (14/24) of all operated patients. DISCUSSION: To the best of the authors' knowledge, this is the first study to present the clinical use of selected group of serum and urinary biomarkers in the setting of UTO to distinguish between patients who would benefit from surgery intervention. The most promising results were obtained using NGAL, RBP, TGF-ß1, and KIM-1, especially in the unilateral hydro(uretero)nephrosis and LUTO subgroups when compared with the control group. CONCLUSIONS: Urine biomarkers, alone and in combination, demonstrated high potential as a non-invasive diagnostic tool for identifying infants who may benefit from earlier surgical intervention.

Effects of a combination of herbal extracts (modified Ojayeonjonghwan (Wuzi Yanzong wan)) on partial urethral obstruction-induced detrusor overactivity in rats: impact on the nitric oxide pathway and oxidative stress.

BACKGROUND: We investigated the effects of a berry mixture formula (modified Ojayeonjonghwan (Wuzi Yanzong Wan, MO formula) on detrusor overactivity (DO). METHODS: The MO formula consisted of 5 seeds obtained from 5 types of berry plants. Twenty-four Sprague-Dawley rats were randomly assigned to four groups: sham-operated (control), partial urethral obstruction-induced DO (DO group), 0.03 mg/kg solifenacin-treated DO (solifenacin group) and 200 mg/kg MO formula -treated DO (berry mixture). The control and overactive groups were administered distilled water for 4 weeks, and the solifenacin and MO formula groups were treated with the respective medication for 4 weeks. After treatment, cystometrography was performed. At the endo of cystometrography, their bladder tissues were used for identifying the muscarinic receptors, endothelial nitric oxide synthase(eNOS), RhoA, Rock-I & II, 8-hydroxy-2' -deoxyguanosine(8-OHdG), superoxide dismutase(SOD), interleukin-6 &-8(IL-6, IL-8), and tumor necrosis factor-alpha(TNF-a). The tissues were stained and the muscle-to-collagen ratio was identified. RESULTS: The presence of the muscarinic receptors were not significantly different between the solifenacin and MO formula groups. However, significant differences were found between the solifenacin and MO formula groups in terms of eNOS, RhoA, Rock-I and -II levels. The muscle-to-collagen ratio was statistically lower in the DO and solifenacin groups; however, no significant difference was observed between the control and MO formula groups. Under oxidative stress, SOD showed a similar result as 8-OHgG. The MO formula group exhibited anti-inflammatory effects, showing that no significant difference was found between the control and MO formula groups regarding values of IL-6, IL-8, and TNF-a. However, the DO and solifenacin groups showed increased IL-6, IL-8, and TNF-a levels. Cystometrography showed that the OAB and solifenacin groups having a significantly lower value than the control and MO formula groups. The mean contraction interval was shorter in the DO, MO formula, and solifenacin groups and the highest in the control group. CONCLUSIONS: The MO formula exhibited a similar pharmacologic effect to that of solifenacin, with anti-inflammatory and antioxidant effects. Enhancement of the MO formula by the nitric oxide pathway affected DO including BPH-related DO. The MO formula may be one of the alternative choices of anticholinergics, a treatment for DO.

p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO.

The role of p53 in renal fibrosis has recently been suggested, however, its function remains controversial and the underlying mechanism is unclear. Here, we show that pharmacological and genetic blockade of p53 attenuated renal interstitial fibrosis, apoptosis, and inflammation in mice with unilateral urethral obstruction (UUO). Interestingly, p53 blockade was associated with the suppression of miR-215-5p, miR-199a-5p&3p, and STAT3. In cultured human kidney tubular epithelial cells (HK-2), TGF-ß1 treatment induced fibrotic changes, including collagen I and vimentin expression, being associated with p53 accumulation, p53 Ser15 phosphorylation, and miR-199a-3p expression. Inhibition of p53 by pifithrin-α blocked STAT3 activation and the expression of miR-199a-3p, collagen I, and vimentin during TGF-ß1 treatment. Over-expression of miR-199a-3p increased TGFß1-induced collagen I and vimentin expression and restored SOCS7 expression. Furthermore, SOCS7 was identified as a target gene of miR-199a-3p, and silencing of SOCS7 promoted STAT3 activation. ChIp analyses indicated the binding of p53 to the promoter region of miR-199a-3p. Consistently, kidney biopsies from patients with IgA nephropathy and diabetic nephropathy exhibited substantial activation of p53 and STAT3, decreased expression of SOCS7, and increase in profibrotic proteins and miR-199a-3p. Together, these results demonstrate the novel p53/miR-199a-3p/SOCS7/STAT3 pathway in renal interstitial fibrosis.

CD11c⁺ CD8⁺ T Cells Reduce Renal Fibrosis Following Ureteric Obstruction by Inducing Fibroblast Apoptosis.

Tubulointerstitial fibrosis is a common consequence of various kidney diseases that lead to end-stage renal failure, and lymphocyte infiltration plays an important role in renal fibrosis. We previously found that depletion of cluster of differentiation 8⁺ (CD8⁺) T cells increases renal fibrosis following ureteric obstruction, and interferon-γ (IFN-γ)-expressing CD8⁺ T cells contribute to this process. CD8⁺ T cells are cytotoxic T cells; however, whether their cytotoxic effect reduces fibrosis remains unknown. This study showed that CD8⁺ T cells isolated from obstructed kidney showed mRNA expression of the cytotoxicity-related genes perforin 1, granzyme A, granzyme B, and FAS ligand; additionally, CD8 knockout significantly reduced the expression levels of these genes in obstructed kidney. Infiltrated CD8⁺ T cells were distributed around fibroblasts, and they are associated with fibroblast apoptosis in obstructed kidney. Moreover, CD11c⁺ CD8⁺ T cells expressed higher levels of the cytotoxicity-related genes than CD11c- CD8⁺ T cells, and infiltrated CD11c⁺ CD8⁺ T cells in obstructed kidney could induce fibroblast death in vitro. Results indicated that induction of fibroblast apoptosis partly contributed to the effect of CD8⁺ T cells on reduction of renal fibrosis. Given that inflammatory cells are involved in fibrosis, our results suggest that kidney fibrosis is a multifactorial process involving different arms of the immune system.

Reduction of obstruction related bladder overactivity by the guanylyl cyclase modulators BAY 41-2272 and BAY 60-2770 alone or in combination with a phosphodiesterase type 5 inhibitor.

AIMS: To assess the urodynamic effects of soluble guanylyl cyclase (sGC) stimulator, BAY 41-2272, and activator, BAY 60-2770, (which both are able to induce cGMP synthesis even in the absence of nitric oxide (NO)) alone or in combination with a phosphodiesterase type 5 (PDE5) inhibitor, vardenafil, in a model of partial urethral obstruction (PUO) induced bladder overactivity (BO). METHODS: Fifty-six male Sprague-Dawley rats were used, 31 of them underwent PUO. Fourteen rats were used for Western blots to assess PDE5 and sGC expression. For drug evaluation cystometry without anesthesia was performed three days following bladder catheterization. RESULTS: Obstructed rats showed higher micturition frequency and bladder pressures than non-obstructed animals (Intermicturition Interval, IMI, 2.28 ± 0.55 vs. 3.60 ± 0.60 min (± standard deviation, SD); maximum micturition pressure, MMP, 70.1 ± 8.0 vs. 48.8 ± 7.2 cmH2O; both P < 0.05). In obstructed rats vardenafil, BAY 41-2272, and BAY 60-2770 increased IMI (2.77 ± 1.12, 2.62 ± 0.52, and 3.22 ± 1.04 min; all P < 0.05) and decreased MMP (54.4 ± 2.8, 61.5 ± 11.3, and 51.2 ± 6.3 cmH2O; all P < 0.05). When vardenafil was given following BAY 41-2272 or BAY 60-2770 no further urodynamic effects were observed. PDE5 as well as sGC protein expression was reduced in obstructed bladder tissue. CONCLUSIONS: Targeting sGC via stimulators or activators, which increase the levels of cGMP independent of endogenous NO, is as effective as vardenafil to reduce urodynamic signs of BO. Targeting the NO/cGMP pathway via compounds acting on sGC might become a new approach to treat BO.

Changes of neuregulin-1 (NRG-1) expression in a rat model of overactive bladder induced by partial urethral obstruction: is NRG-1 a new biomarker of overactive bladder?

BACKGROUND: To determine whether neuregulin-1(NRG-1) is a potential new biomarker of overactive bladder (OAB) induced by partial urethral obstruction in a rat model of OAB and to evaluate the urothelium as a therapeutic target of OAB. METHODS: Female Sprague-Dawley rats were separated into three 20-animal groups: normal, OAB, and 5-hydroxymethyl tolterodine (5-HMT)-treated OAB. In the OAB and OAB + 5-HMT groups, the urethra of each animal was partially obstructed; the OAB + 5-HMT group received intravenous 5-HMT for 3 weeks. At the conclusion of the 5-HMT dosing, the rats in each group underwent cystometrography, and the bladders were histologically evaluated. The expression of brain derived-neurotrophic factor (BDNF) and NRG-1 were evaluated in the urothelium. RESULTS: Compared with the control group, the OAB group showed a markedly increased bladder weight and a significant decrease in the micturition interval and volume; rats in the OAB + 5-HMT group showed decreased bladder weights and an improved micturition interval and volume. BDNF and NRG-1 were expressed at significantly higher levels in the OAB group, and were significantly reduced in the OAB + 5-HMT group compared with the control group. CONCLUSIONS: The study suggests that NRG-1 is a potential new biomarker of OAB; the urothelium might be a therapeutic target for OAB treatment.

Impact of partial urethral obstruction on bladder function: time-dependent changes and functional correlates of altered expression of Ca²âº signaling regulators.

In animal models of partial urethral obstruction (PUO), altered smooth muscle function/contractility may be linked to changes in molecules that regulate calcium signaling/sensitization. PUO was created in male rats, and urodynamic studies were conducted 2 and 6 wk post-PUO. Cystometric recordings were analyzed for the presence or absence of nonvoiding contractions [i.e., detrusor overactivity (DO)]. RT-PCR and Western blots were performed on a subpopulation of rats to study the relationship between the expression of RhoA, L-type Ca(2+) channels, Rho kinase-1, Rho kinase-2, inositol 1,4,5-trisphosphate, ryanodine receptor, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 kDa, and urodynamic findings in the same animal. Animals displayed DO at 2 (38%) and 6 wk (43%) post-PUO, increases were seen in in vivo pressures at 2 wk, and residual volume at 6 wk. Statistical analysis of RT-PCR and Western blot data at 2 wk, during the compensatory phase of detrusor hypertrophy, documented that expression of molecules that regulate calcium signaling and sensitization was consistently lower in obstructed rats without DO than those with DO or control rats. Among rats with DO at 2 wk, linear regression analysis revealed positive correlations between in vivo pressures and protein and mRNA expression of several regulatory molecules. At 6 wk, in the presence of overt signs of bladder decompensation, no clear or consistent alterations in expression of these same targets were observed at the protein level. These data extend prior work to suggest that molecular profiling of key regulatory molecules during the progression of PUO-mediated bladder dysfunction may shed new light on potential biomarkers and/or therapeutic targets.

Recurrent Johanson-Blizzard syndrome in a triplet pregnancy complicated by urethral obstruction sequence: a clinical, molecular, and immunohistochemical approach.

We report on a triplet pregnancy of consanguineous parents with one fetus being affected by recurrent Johanson-Blizzard syndrome (JBS). At autopsy in the 35th gestational week, the affected triplet presented with an especially severe and lethal manifestation of the disorder as compared to his elder affected brother and to cases in the literature, thus exemplifying great interfamilial and intrafamilial phenotypic variability. Arhinencephaly and cystic renal dysplasia associated with urethral obstruction sequence were features not described previously in the literature. In addition to the lack of exocrine acini as the characteristic feature of JBS, the pancreas revealed a resorptive inflammatory reaction with infiltration by eosinophilic granulocytes that focally dispersed onto islets of Langerhans, thus favoring a progressive destructive rather than primary dysplastic process and possibly explaining the occurrence of diabetes mellitus in later life. JBS maps to chromosome 15q15-q21.1 and is associated with mutations in the UBR1 gene. Testing the fetus and the affected sibling revealed a homozygous truncating mutation in UBR1. The resulting absence of the UBR1 protein was confirmed by Western blot. Immunohistochemical staining using a commercial anti-UBR1 antibody demonstrated staining, presumably artifactual. This finding suggests that, until an appropriately validated antibody has been identified, this modality should not be utilized for diagnosis or confirmation of this disorder.

Beta3-adrenoceptors in the rat sacral spinal cord and their functional relevance in micturition under normal conditions and in a model of partial urethral obstruction.

AIMS: Beta3-adrenoceptor selective agonists are evaluated as a new treatment for patients with lower urinary tract symptoms . It is believed that ß3-AR selective agonists exert their effects via a peripheral site of action. However, ß3-ARs have been found in brain tissue. This study examined whether ß3-ARs are present in rat sacral spinal cord, and whether there are differences in ß3-AR expression between normal and partial urethral obstruction (PUO) animals, and furthermore assessed the functional relevance of spinal ß3-ARs for micturition. METHODS: Thirty-eight male Sprague-Dawley rats underwent either PUO or sham-operation. Two weeks after operation, half of the animals were used for histomorphological analysis. Remaining animals were used for functional experiments, where a ß3-AR selective agonist, BRL 37344, was given intrathecally. Bladder function was assessed by continuous cystometry in non-anesthetized animals before and after drug administration. RESULTS: Beta3-ARs were found in sacral spinal cord segments with an accumulation in the ventral horn. There was a significant increase of ß3-AR expression in obstructed rats. In functional experiments obstructed rats showed increased bladder weight, micturition frequency, spontaneous activity, and bladder pressures (all P < 0.05) compared to controls. Intrathecally administered BRL 37344 showed no effect in non-obstructed rats. In obstructed rats intrathecal BRL 37344 significantly reduced bladder pressures, spontaneous activity, and micturition frequency (all P < 0.05). CONCLUSIONS: Beta3-ARs are present in rat sacral spinal cord, and are significantly up-regulated after PUO. Besides their well-established peripheral site of action in the treatment of voiding dysfunction, ß3-AR selective agonists might exert relevant effects at a central nervous site of action.

Partial urethral obstruction: ATF3 and p-c-Jun are involved in the growth of the detrusor muscle and its motor innervation.

OBJECTIVE: Infravesical obstruction leads to growth of urinary bladder smooth-muscle cells. The ganglion cells innervating the bladder muscle also increase in size. Stretch of detrusor muscle cells rapidly activates c-Jun NH2-terminal kinase (JNK), which phosphorylates the transcription factor c-Jun, and stimulates the synthesis of the cotranscription factor ATF3. The aim of the study was to determine whether ATF3 and p-c-Jun were involved in growth of bladder smooth-muscle and ganglion cells. MATERIAL AND METHODS: The urethra was partially obstructed in female rats. After 3 days or 10 weeks bladders were weighed, fixated and cut for immunohistochemistry to demonstrate ATF3 and p-c-Jun. Ganglia were processed separately. Unoperated and sham-operated rats were used as controls. RESULTS: There was no ATF3 or p-c-Jun in control detrusor muscle. After 3 days of obstruction bladder weight had nearly doubled. Almost all nuclei in the detrusor showed immunofluorescence for ATF3 and p-c-Jun. After 10 weeks bladder weight had increased 10-fold. Almost all detrusor nuclei still showed p-c-Jun, but few had ATF3 activity. In control ganglia there was no ATF3 and only faint nuclear p-c-Jun activity. After 3 days of obstruction the ganglion cells had increased in size and many nuclei showed intense immunofluorescence for ATF3 and p-c-Jun. After 10 weeks the ganglion cell size had increased further. There was no ATF3 activity and no more p-c-Jun than in control ganglia. CONCLUSION: ATF3 and p-c-Jun seem to be involved in the growth of the detrusor muscle and its motor innervation following infravesical outlet obstruction.

Afferent nerve regulation of bladder function in health and disease.

The afferent innervation of the urinary bladder consists primarily of small myelinated (Adelta) and unmyelinated (C-fiber) axons that respond to chemical and mechanical stimuli. Immunochemical studies indicate that bladder afferent neurons synthesize several putative neurotransmitters, including neuropeptides, glutamic acid, aspartic acid, and nitric oxide. The afferent neurons also express various types of receptors and ion channels, including transient receptor potential channels, purinergic, muscarinic, endothelin, neurotrophic factor, and estrogen receptors. Patch-clamp recordings in dissociated bladder afferent neurons and recordings of bladder afferent nerve activity have revealed that activation of many of these receptors enhances neuronal excitability. Afferent nerves can respond to chemicals present in urine as well as chemicals released in the bladder wall from nerves, smooth muscle, inflammatory cells, and epithelial cells lining the bladder lumen. Pathological conditions alter the chemical and electrical properties of bladder afferent pathways, leading to urinary urgency, increased voiding frequency, nocturia, urinary incontinence, and pain. Neurotrophic factors have been implicated in the pathophysiological mechanisms underlying the sensitization of bladder afferent nerves. Neurotoxins such as capsaicin, resiniferatoxin, and botulinum neurotoxin that target sensory nerves are useful in treating disorders of the lower urinary tract.

Vardenafil-induced relaxation and cyclic nucleotide levels in normal and obstructed rat urinary bladder.

OBJECTIVE: To study the effects of the phosphodiesterase-5 inhibitor, vardenafil, on contraction and cyclic nucleotide levels in isolated detrusor preparations with and without mucosa, from control rats and rats with partial urethral obstruction (PUO) and intact mucosa. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into groups subjected to PUO for 14 days (six), and sham-operated control rats (12). Detrusor preparations were mounted in organ baths and effects of increasing concentrations of vardenafil (1 nm to 100 microm) assessed on carbachol-activated (1 microm) preparations, and on contractions induced by transmural activation of nerves (electrical field stimulation, EFS). Levels of cGMP and cAMP were determined using radioimmunoassays. RESULTS: Vardenafil caused concentration-dependent relaxations of carbachol-contracted detrusor, the mean (sd) of which at 100 microm was 91 (4)% in control and 100% in PUO rats. The -log 50% inhibitory concentration (IC(50)) was 4.41 (0.08) and 4.73 (0.05) (P < 0.01), respectively. Removing the mucosa increased the relaxant effect of vardenafil at 1-10 microm (P < 0.05) although -log IC(50) values were unaffected compared to the control. The cGMP levels ( pmol/mg protein) in control preparations increased from 2.5 (0.6) to 5.0 (0.8), and from 1.4 (0.2) to 7.2 (1.3) in obstructed bladders. In mucosa-denuded preparations the cGMP content increased from 0.6 (0.1) to 1.6 (0.4) in response to vardenafil. In control rats, the levels of cAMP increased from 12.8 (2.5) to 18.9 (0.9) (P < 0.05) after vardenafil. In mucosa-denuded preparations the cAMP levels after vardenafil increased from 16.5 (2.11) to 37.8 (3.4) (P < 0.01). In PUO bladders, the tissue content of cAMP increased from 12.6 (2.4) to 20.6 (3.4) (P < 0.01). Vardenafil concentration-dependently inhibited nerve-induced contractions in all groups studied. At 100 microm 19 (3)% of the control contraction remained, vs 8 (1)% for preparations from obstructed rats, and 11 (4)% in mucosa-denuded preparations. CONCLUSION: In normal rats, vardenafil relaxed carbachol- and inhibited EFS-induced contractions of detrusor preparations with and without urothelium, and in PUO rats with urothelium. Relaxations were accompanied by increases in both cAMP and cGMP content. It is proposed that vardenafil-induced relaxation of rat detrusor, also in obstructed and mucosa-denuded preparations, is mediated via cAMP.

Differential expression profile of cold (TRPA1) and cool (TRPM8) receptors in human urogenital organs.

OBJECTIVES: To examine the expressions of transient receptor potential (TRP) channel A1 and TRPM8 in the human urogenital organs (urinary bladder and prostate) and how these expressions change in association with bladder outlet obstruction (BOO) or benign prostatic hyperplasia (BPH). In addition to TRPM8, a cool receptor, TRPA1 is recently recognized as a cold receptor. TRPA1 is also a candidate for mechanosensor and/or nociceptor. METHODS: Urinary bladder mucosa and muscular layer were taken from 9 controls and 9 patients with BOO. Prostatic specimens were obtained from 5 controls and 6 patients with BPH. Expressions of TRPA1 and TRPM8 messenger RNAs (mRNAs) were quantified by real-time revere transcriptase-polymerase chain reaction. Localization of TRPA1 protein was explored with immunohistochemistry. RESULTS: The expression levels of TRPA1 mRNA in the bladder mucosa, bladder muscular layer, and prostate were in the ratio of 639:1:16. TRPA1 mRNA in the bladder mucosa with BOO was significantly upregulated to 2.32 times of control. TRPA1 protein was localized in the epithelial cells of both urinary bladder and prostate gland. The expression of TRPM8 mRNA in the prostate was much higher than that in the bladder mucosa (3024:1), but was not found in the bladder muscle layer. BPH or BOO did not significantly affect the expression of TRPM8. CONCLUSIONS: TRPA1 and TRPM8 were differentially expressed in the human urinary bladder and prostate. TRPA1 in the bladder epithelium might be involved in the bladder sensory transduction and the induction process of overactive bladder by BOO.

Captopril reduces deposition of collagen in lamina propria and muscular layers of the bladder and ureter in neonatal dogs with partial urethral obstruction.

OBJECTIVE: It has been recognized that most boys with posterior urethral valve ablation have varying degrees of ureteral dilation and abnormal bladder function. It has been shown that deposition of collagen in the bladder layers increases the intravesical pressure that is transmitted to the pelvicalyceal system. The purpose of this study was to evaluate, using stereological methods, the effect of captopril on prevention of collagen formation in different layers of the ureter and bladder under conditions of partial urethral obstruction (PUO). MATERIAL AND METHODS: PUO was induced in 10 neonatal dogs. These animals were then divided into two equal groups: the experimental group received captopril (35 mg/kg/day) and the positive control group received no treatment. After 6 weeks, the neonatal dogs underwent left nephroureterectomy and cystectomy. The ureter and bladder volumes, the volume fraction and absolute volume of the histological parameters of the ureter and bladder, the collagen content of the lamina propria, muscular and adventitial layers, and the muscle content of the muscular layer were estimated using stereological methods. RESULTS: The ureter and bladder volumes, the volumes of the layers including mucosa, lamina propria and muscular layer adventitia, the muscle content of the muscular layer and the collagen content of the adventitial layer did not show any significant differences between the groups. The collagen content (absolute volume) of lamina propria and muscular layer in the experimental group was lower than that in the positive control group for both the ureter and bladder. CONCLUSION: Administration of captopril decreases the collagen content of the lamina propria and muscular layer in the ureter and bladder of neonatal dogs with PUO.

Downregulation of Smad transcriptional corepressors SnoN and Ski in the fibrotic kidney: an amplification mechanism for TGF-beta1 signaling.

TGF-beta1 is a profibrotic cytokine that plays a central role in the onset and progression of chronic renal diseases. The activity of TGF-beta1 is tightly controlled by multiple mechanisms, in which antagonizing Smad-mediated gene transcription by co-repressors is an important regulatory component. This study examined the expression of Smad transcriptional co-repressors in the fibrotic kidney and investigated their potential functions in controlling TGF-beta1 response. Western blot analysis demonstrated that the protein levels of Smad transcriptional co-repressors SnoN and Ski were progressively reduced in a time-dependent manner in the fibrotic kidney induced by unilateral ureteral obstruction in mice, whereas renal Smad abundance was relatively unaltered. Consistently, SnoN and Ski staining was diminished in the nuclei of renal tubular epithelium and interstitium after obstructive injury. In vitro, knockdown of SnoN expression by RNA interference in tubular epithelial cells dramatically sensitized their responsiveness to TGF-beta1 stimulation. Conversely, ectopic expression of exogenous SnoN or Ski after transfection conferred tubular epithelial cell resistance to TGF-beta1-induced epithelial to myofibroblast transition. Both SnoN and Ski could block Smad-mediated activation of TGF-beta1-responsive promoter and exhibited additive effect in abrogating the profibrotic actions of TGF-beta1. These results indicate that as a result of loss of Smad transcriptional co-repressors, the profibrotic TGF-beta1 signaling in diseased kidney is markedly amplified in a magnitude much greater than previously thought. Therefore, new strategy aimed to increase Smad transcriptional co-repressors expression may be effective in antagonizing TGF-beta1 signaling and thereby blocking the progression of chronic renal fibrosis.

Fetal surgery for posterior urethral valves: long-term postnatal outcomes.

OBJECTIVE: Fetal intervention for obstructive uropathy was first performed at the University of California, San Francisco in 1981. Indications for treatment were bilateral hydronephrosis with oligohydramnios. Preintervention criteria included fetal urinary electrolytes with beta-microglobulin levels, karyotyping, and detailed sonography specifically looking for renal cortical cysts. We reviewed the outcomes of children who underwent fetal intervention with specific long-term follow-up in patients who were found postnatally to have posterior urethral valves. METHODS: A retrospective review of the University of California, San Francisco fetal surgery database was performed for patients with a prenatal diagnosis of obstructive uropathy. Medical records from 1981 to 1999 were reviewed. Long-term follow-up was documented if the cause of the urinary tract obstruction was posterior urethral valves. We collected data points, focusing on time and type of intervention, fetal urinary electrolytes, appearance of fetal kidneys, present renal function, length of follow-up, and present status of the urinary tract. RESULTS: Forty patients were evaluated for fetal intervention; 36 fetuses underwent surgery during this time period. Postnatal confirmation of posterior urethral valves was demonstrated in 14 patients. All patients had favorable fetal urinary electrolytes. Mean gestational age at intervention was 22.5 weeks. The procedures performed included creation of cutaneous ureterostomies in 1, fetal bladder marsupialization in 2, in utero ablation of valves in 2, and placement of vesicoamniotic catheter in 9. Six deaths occurred before term delivery with premature labor and the newborns succumbing to respiratory failure. One pregnancy was terminated electively because of shunt failure and declining appearance of fetal lungs and kidney. The remaining 8 living patients had a mean follow-up of 11.6 years. Chronic renal disease with abnormal serum creatinine was present in 5 patients. Two patients have undergone renal transplantation, and 1 is awaiting organ donation. Five of the 8 living patients have had urinary diversion with vesicostomy, cutaneous ureterostomy, or augmentation cystoplasty with later reconstruction. CONCLUSIONS: Fetal intervention for posterior urethral valves carries a considerable risk to the fetus with fetal mortality rate of 43%. The long-term outcomes indicate that intervention may not change the prognosis of renal function or be a predictor for possible urinary diversion. Despite all of these patients' having favorable urinary electrolytes, this did not seem to have any implication postnatally. When counseling families about fetal intervention, efforts should be focused on that intervention may assist in delivering the fetus to term and that the sequelae of posterior urethral valves may not be preventable. Fetal surgery for obstructive uropathy should be performed only for the carefully selected patient who has severe oligohydramnios and "normal"-appearing kidneys.

Heparin-binding EGF-like growth factor expression increases selectively in bladder smooth muscle in response to lower urinary tract obstruction.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an activating ligand for the epidermal growth factor receptor (ErbB1) tyrosine kinase and at least one isoform of the ErbB4 receptor tyrosine kinase, is synthesized by the smooth muscle of the human bladder wall. In this study we tested the hypothesis that HB-EGF plays a role in the bladder-wall thickening that occurs in response to obstructive syndromes affecting the lower urinary tract, possibly by acting as an autocrine smooth muscle cell (SMC) growth factor. HB-EGF was mitogenic for primary culture human bladder SMC, and cell growth in serum-containing medium was inhibited more than 70% by [Glu52]-diphtheria toxin/CRM197, a specific HB-EGF inhibitor, consistent with a physiologic role for HB-EGF as an autocrine bladder SMC mitogen. Human and mouse bladder SMC in vivo and cultured human bladder SMC expressed the primary HB-EGF receptor, ErbB1, but not mRNA for the secondary HB-EGF receptor, ErbB4, thereby identifying ErbB1 as the cognate HB-EGF receptor in the bladder wall. Reverse transcription-polymerase chain reaction analysis also demonstrated ErbB2 and ErbB3 expression in human bladder muscle tissue, suggesting the possibility of receptor cross-talk after ErbB1 activation. Urethral ligation in mice resulted in an increase in steady-state HB-EGF mRNA expression up to 24 hours in whole bladder tissue in comparison with ErbB1 and glyceraldehyde 3-phosphate dehydrogenase mRNA levels, which did not change in a demonstrable pattern. HB-EGF protein increased coordinately with HB-EGF mRNA levels. Dissection of bladder tissue into muscle and mucosal layers demonstrated that the increase in HB-EGF mRNA occurred predominantly in the muscle layer, with peak levels (13-fold higher than sham controls) occurring 12 hours after obstruction. These data support a physiologic role for HB-EGF as a mediator of hypertrophic bladder tissue growth.

Nitric oxide synthase--its distribution and alteration in the intramural ganglia of the urinary bladder in normal and urethra-obstructed guinea pigs.

Nitric oxide (NO) has been proposed to function as an inhibitory neurotransmitter in the lower urinary tract. This study investigates the distribution of NO-containing neurons and its changes following urethral obstruction in the guinea-pig. By using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and NO synthase (NOS) immunohistochemistry, the highest frequency of NO-containing neurons was observed in the bladder base. Double labelling studies showed that 70.9% of NADPH-d reactive neurons co-expressed NOS immunoreactivity. Acetylcholinesterase reactivity was present in the majority of the intramural neurons with 54% of them expressed NOS immunoreactivity. NADPH-d reactivity was colocalized with vasoactive intestinal polypeptide, calcitonin gene-related peptide and substance P immunoreactivities in both neurons and fibres. Colocalization study also revealed that NADPH-d reactive neurons formed a distinct cell population from tyrosine hydroxylase positive neurons. At 12 hours after urethral obstruction, NADPH-d reactivity in the intramural ganglion cells was noticeably enhanced and this was sustained till 24 hours whence some intensely stained neurons appeared to undergo degenerative changes. Neuronal degeneration was more drastic at 48 hours so that the number of NADPH-d positive neurons was significantly reduced. The present study suggests that NO is an important neurotransmitter in the urinary bladder and that it may be involved in the relaxation activity in the bladder base during micturition. It is speculated that the increased NADPH-d reactivity in intramural ganglion cells elicited by urethral obstruction may be responsible for the cell death. It is suggested that the resulting cell loss or bladder denervation may account for the urinary dysfunction such as frequency and urgency of micturition in patients with urethral obstruction.

Cytokine gene expression during experimental Escherichia coli pyelonephritis in mice.

PURPOSE: We studied nine inflammatory and immunoregulatory cytokines in acute pyelonephritis and urethral obstruction in mice to better understand the processes underlying kidney inflammation and scarring. MATERIALS AND METHODS: Experimental acute pyelonephritis was established in Bki NMRI outbred mice by bladder inoculation of Escherichia coli, followed by 6 h urethral obstruction. The numbers of cytokine mRNA expressing cells for interleukin-1 (IL-1), IL-4, IL-6, IL-10, IL-12, tumor necrosis factor alpha (TNF-alpha), TNF-beta, transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) were determined in the kidneys and spleens from the infected, non-infected but obstructed and untouched mice using in situ hybridization with radio-labelled oligonucleotide probes at 12 h, 48 h and 6 d after release of the urethral obstruction. RESULTS: Kidney cell expression of IL-1, IL-6 and TNF-alpha mRNA was observed already at 12 h and persisted on day 6 in the infected animals. A significant proinflammatory cytokine response occurred also in the non-infected obstructed animals, albeit later and at lower levels. A marked increase of IL-4, IL-10, TGF-beta and IFN-gamma mRNA producing cells was also found in the kidneys of these two groups again with higher levels in the infected animals. Very high numbers of splenocytes expressing mRNA for IL-1 were observed especially in the infected animals. A high proportion of splenocytes further expressed mRNA for IL-6, TNF-alpha, IL-4, IL-10, IFN-gamma and TGF-beta, again with highest numbers in the infected group of animals. CONCLUSIONS: The present study extends previous knowledge about the local and systemic cytokine expression profiles during acute pyelonephritis and after urethral obstruction. Of particular interest was the marked kidney cell expression of mRNA for TGF-beta, presumed to be important both for obstructive and post-infectious renal scarring.