Mouse Ex Vivo Kidney Culture Methods.

Kidney organogenesis has been a widely used classical model system to study inductive tissue interactions that guide differentiation of many organs. The basis for this is in the pioneering work done during the early 1950s when the conditions of how to support ex vivo growth and differentiation of developing kidneys were revealed. Importantly, culturing developing kidneys remains as an essential instrument to advance our understanding of molecular and cellular regulation of morphogenesis even today. Despite the fact that embryonic kidneys have been cultured for decades, it is not a trivial method and requires specific anatomical and developmental biology knowledge. This chapter outlines the general steps in organ culture and details the requirements for successful kidney explant differentiation.

Altered ureteral contractility with ageing: Role of the rho-kinase pathway.

This study investigated the role of calcium sensitisation in the regulation of ureteral contractility with ageing. Isolated ureteral strips from young (6-month old) and older (3-year old) pigs were mounted in Krebs bicarbonate solution and contractility induced by the α1-adrenoceptor agonist phenylephrine (30 and 300 µM) and 5-HT (10 and 100 µM), recorded in the absence and presence of the rho-kinase inhibitors Y-27632 (10 µM) and fasudil (30 µM). Ureteral strips developed bursts of contractile activity which was measured as area under the curve (AUC) and frequency. Maximum contraction to phenylephrine was significantly enhanced in tissues from older animals compared to younger animals (p < 0.001) while maximum contraction to 5-HT was greater in tissues from younger animals (p < 0.001). Both inhibitors significantly depressed AUC and frequency responses to both agonists in ureters from both age groups (p < 0.05). Inhibition by Y-27632 of phenylephrine (300 µM)- and 5-HT(100 µM)-induced contractions was greater in tissues from older animals than young (p < 0.05). Rho-kinase activity was also assayed in ureteral tissues, and basal activity was similar in ureters from both age groups. Neither phenylephrine nor 5-HT increased rho-kinase activity over basal levels. These data demonstrate the significant role rho-kinase plays in ureteral contractility and possible alterations with age.

Hydrogen sulfide plays a key role in the inhibitory neurotransmission to the pig intravesical ureter.

According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H2S) role in the neurogenic relaxation of the pig intravesical ureter. We have performed western blot and immunohistochemistry to study the expression of the H2S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS), measurement of enzymatic production of H2S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A2 analogue U46619, electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and NG-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H2S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K+ (KATP) channels, transient receptor potential A1 (TRPA1), transient receptor potential vanilloid 1 (TRPV1), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, HC030031, AMG9810, PACAP6-38 and CGRP8-37, respectively. These results suggest that H2S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via KATP channel activation. H2S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA1, TRPV1 and related ion channel activation.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

Impaired EphA4 signaling leads to congenital hydronephrosis, renal injury, and hypertension.

Experimental hydronephrosis induced by partial ureteral obstruction at 3 wk of age causes hypertension and renal impairment in adult rats and mice. Signaling by Ephrin receptors (Eph) and their ligands (ephrins) importantly regulates embryonic development. Genetically modified mice, where the cytoplasmic domain of the EphA4 receptor has been substituted by enhanced green fluorescent protein (EphA4gf/gf), develop spontaneous hydronephrosis and provide a model for further studies of the disorder. The present study aimed to determine if animals with congenital hydronephrosis develop hypertension and renal injuries, similar to that of experimental hydronephrosis. Ultrasound and Doppler techniques were used to visualize renal impairment in the adult mice. Telemetric blood pressure measurements were performed in EphA4gf/gf mice and littermate controls (EphA4+/+) during normal (0.7% NaCl)- and high (4% NaCl)-sodium conditions. Renal excretion, renal plasma flow, and glomerular filtration were studied, and histology and morphology of the kidneys and ureters were performed. EphA4gf/gf mice developed variable degrees of hydronephrosis that correlated with their blood pressure level. In contrast to EphA4+/+, the EphA4gf/gf mice displayed salt-sensitive hypertension, reduced urine concentrating ability, reduced renal plasma flow, and lower glomerular filtration rate. Kidneys from EphA4gf/gf mice showed increased renal injuries, as evidenced by fibrosis, inflammation, and glomerular and tubular changes. In conclusion, congenital hydronephrosis causes hypertension and renal damage, similar to that observed in experimentally induced hydronephrosis. This study further reinforces the supposed causal link between hydronephrosis and later development of hypertension in humans.

Rho-kinase, a common final path of various contractile bladder and ureter stimuli.

Normal urinary bladder function is based on the proper contraction and relaxation of smooth muscle (SM), which constitutes the majority of the bladder wall. The contraction and relaxation of all SM involves a phosphorylation-dephosphorylation pathway involving the enzymes smooth muscle myosin light chain kinase (SMMLCK) and smooth muscle myosin light chain phosphatase (SMMLCP), respectively. Although originally thought to function just as a passive opposition to SMMLCK-driven SM contraction, it is now clear that SMMLCP activity is under an extremely complex molecular regulation via which SMMLCP inhibition can induce "calcium sensitization." This review provides a thorough summary of the literature regarding the molecular regulation of the SMMLCP with a focus on one of its major inhibitory pathways that is RhoA/Rho-kinase (ROK) including its activation pathways, effector molecules, and its roles in various pathological conditions associated with bladder dysfunction. Newly emerging roles of ROK outside of SM contractility are also discussed. It is concluded that the RhoA/ROK pathway is critical for the maintenance of basal SM tone of the urinary bladder and serves as a common final pathway of various contractile stimuli in rabbits, rats, mice, and pigs as well as humans. In addition, this pathway is upregulated in response to a number of pathological conditions associated with bladder SM dysfunction. Similarly, RhoA/Rho-kinase signaling is essential for normal ureteral function and development and is upregulated in response to ureteral outlet obstruction. In addition to its critical role in bladder SM function, a role of ROK in the urothelium is also beginning to emerge as well as roles for ROK in bladder infection and invasion and metastasis of bladder cancer.

RhoA/Rho-kinase as a therapeutic target for the male urogenital tract.

INTRODUCTION: Rho-kinase (ROCK) is a serine/threonine kinase and is one of the major downstream effectors of the small guanosine triphosphatase Rho. In the past few years, evidence has been accumulating to suggest that the RhoA/ROCK system may play an important role in the pathogenesis of a number of cardiovascular and urogenital disorders. AIM: The aim of this study is to review the literature pertaining to the role of the RhoA/ROCK system in male urogenital function. METHODS: Comprehensive literature review was performed using PubMed. MAIN OUTCOME MEASURES: Inhibitors of ROCK may have potential therapeutic applications, as derived from preclinical and a few clinical studies. RESULTS: Published reports suggest that elevated RhoA/Rho-kinase signaling plays a role in the development of benign prostatic hyperplasia, erectile dysfunction, kidney failure, ejaculation disorders, prostate and bladder cancer initiation, and eventual metastasis. CONCLUSIONS: This review focuses on our current understanding of the role of the RhoA/Rho-kinase pathway in the regulation of the male urogenital system. Rho-kinase inhibitors may evolve into an important pharmacologic option in the future treatment of urogenital system disorders.

Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.

The integrin-linked kinase (ILK), pinch and parvin ternary complex connects the cytoplasmic tails of beta1 integrins to the actin cytoskeleton. We recently showed that constitutive expression of ILK and alpha parvin in both the ureteric bud and the metanephric mesenchyme of the kidney is required for kidney development. In this study, we define the selective role of ILK in the ureteric bud of the mouse kidney in renal development by deleting it in the ureteric cell lineage before the onset of branching morphogenesis (E10.5). Although deleting ILK resulted in only a moderate decrease in branching, the mice died at 8 weeks of age from obstruction due to the unprecedented finding of intraluminal collecting duct cellular proliferation. ILK deletion in the ureteric bud resulted in the inability of collecting duct cells to undergo contact inhibition and to activate p38 mitogen-activated protein kinase (MAPK) in vivo and in vitro. p38 MAPK activation was not dependent on the kinase activity of ILK. Thus, we conclude that ILK plays a crucial role in activating p38 MAPK, which regulates cell cycle arrest of epithelial cells in renal tubulogenesis.

Histone deacetylases are critical regulators of the renin-angiotensin system during ureteric bud branching morphogenesis.

Mutations in the genes encoding components of the renin-angiotensin system (RAS) in mice or humans cause congenital abnormalities of the kidney and urinary tract. We hypothesized that absence of angiotensin (Ang) II in angiotensinogen (AGT)-deficient mice leads to defects in ureteric bud (UB) branching and that RAS genes are critically dependent on histone deacetylase (HDAC) activity. The number of UB tips was lower in AGT-/- compared with AGT+/+ embryonic (E) day E13.5 metanephroi (24+/-1.5 versus 36+/-3.7, p<0.05). Real-time RT-PCR demonstrated that pharmacological inhibition of HDAC activity with Scriptaid increases AGT, renin, angiotensin-converting enzyme (ACE), and AT1 receptor (AT1R) mRNA levels in E12.5 mouse metanephroi and early mesenchymal (MK3) cells. Furthermore, Scriptaid enhanced Ang II induced decrease in Sprouty (Spry) 1 gene expression in cultured UB cells. Treatment of intact E12.5 mouse metanephroi grown ex vivo with Ang II (10(-5) M, 24 h) increased HDAC-1 and decreased total acetylated histone H3 protein levels. These findings indicate that lack of endogenous Ang II in AGT-deficient mice inhibits UB branching. We conclude that intact RAS is critical in structural integrity of the renal collecting system and that UB morphogenetic program genes, such as AGT, renin, ACE, AT1R, or Spry1, are epigenetically controlled via HDACs.

Maturation of ureter-bladder connection in mice is controlled by LAR family receptor protein tyrosine phosphatases.

Congenital anomalies affecting the ureter-bladder junction are frequent in newborns and are often associated with other developmental defects. However, the molecular and morphological processes underlying these malformations are still poorly defined. In this study, we identified the leukocyte antigen-related (LAR) family protein tyrosine phosphatase, receptor type, S and F (Ptprs and Ptprf [also known as Lar], respectively), as crucially important for distal ureter maturation and craniofacial morphogenesis in the mouse. Embryos lacking both Ptprs and Ptprf displayed severe urogenital malformations, characterized by hydroureter and ureterocele, and craniofacial defects such as cleft palate, micrognathia, and exencephaly. The detailed analysis of distal ureter maturation, the process by which the ureter is displaced toward its final position in the bladder wall, leads us to propose a revised model of ureter maturation in normal embryos. This process was deficient in embryos lacking Ptprs and Ptprf as a result of a marked reduction in intrinsic programmed cell death, thereby causing urogenital system malformations. In cell culture, Ptprs bound and negatively regulated the phosphorylation and signaling of the Ret receptor tyrosine kinase, whereas Ptprs-induced apoptosis was inhibited by Ret expression. Together, these results suggest that ureter positioning is controlled by the opposing actions of Ret and LAR family phosphatases regulating apoptosis-mediated tissue morphogenesis.

NRAGE: a potential rheostat during branching morphogenesis.

Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38(MAPK)) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38(MAPK) activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38(MAPK) activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38(MAPK) activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.

Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Inflammatory myofibroblastic tumors of the urinary tract: a clinicopathologic study of 46 cases, including a malignant example inflammatory fibrosarcoma and a subset associated with high-grade urothelial carcinoma.

Inflammatory myofibroblastic tumor (IMT) of the urinary tract, also termed postoperative spindle cell nodule, inflammatory pseudotumor, and pseudosarcomatous fibromyxoid tumor, is rare and in the past was believed to reflect diverse entities. We reviewed a series of 46 IMTs arising in the ureter, bladder, and prostate, derived primarily from a large consultation practice. There were 30 male and 16 females aged 3 to 89 years (mean 53.6). Lesions were 1.2 to 12 cm (mean 4.2). There was a history of recent prior instrumentation in 8 cases. Morphology was similar to that previously described for IMT occurring in this region, with the exception of 1 case that focally appeared sarcomatous. Polypoid cystitis coexisted in 5 patients (11%). Mitoses were typically scant (0 to 20/10 hpf, mean 1). Necrosis was seen in 14 (30%) cases. Invasion of the muscularis propria was documented in 19 (41%). By immunohistochemistry (IHC), lesions at least focally expressed anaplastic lymphoma kinase (ALK) (20/35, 57%), AE1/3 (25/34, 73%), CAM5.2 (10/15, 67%), CK18 (6/6, 100%), actin (23/25, 92%), desmin (15/19, 79%), calponin (6/7, 86%), caldesmon (4/7, 57%, rare cells), p53 (10/13, 77%), and most lacked S100 (0/14), CD34 (0/13), CD117 (2/13, 15%), CD21 (0/5), and CD23 (0/3). ALK gene alterations were detected by fluorescence in situ hybridization (FISH) in 13/18 (72%) tested cases, including 2 with prior instrumentation; 13/18 (72%) showed agreement between FISH ALK results and ALK protein results by IHC. Most bladder IMTs were managed locally, but partial cystectomy was performed as the initial management in 7 cases and cystectomy in 1 (1 IMT was initially misinterpreted as carcinoma, 1 IMT was found incidentally as a separate lesion in a cystectomy specimen performed for urothelial carcinoma). Follow-up was available in 32 cases (range 3 to 120 mo; mean 33; median 24). There were 10 patients with recurrences (2 with 2 recurrences). Recurrences were unassociated with muscle invasion or with ALK alterations. In 2 cases, tumors of the urinary tract (TURs) showing IMT preceded (1 and 2 mo, respectively) TURs showing sarcomatoid carcinoma with high-grade invasive urothelial carcinoma accompanied with separate fragments of IMT. Even on re-review the IMT in these 2 cases were morphologically indistinguishable from other cases of IMT, with FISH demonstrating ALK alterations in the IMT areas in one of them. Both these patients died of their carcinomas. Lastly, there was 1 tumor with many morphological features of IMT and an ALK rearrangement, yet overtly sarcomatous. This case arose postirradiation for prostate cancer 4 years before the development of the lesion, with tumor recurrence at 4 months and death from intra-abdominal metastatic disease at 9 months. In summary, urinary tract IMTs are rare and share many features with counterparts in other sites, displaying similar morphology and immunogenotypic features whether de novo or postinstrumentation. Typical IMTs can be locally aggressive, sometimes requiring radical surgical resection, but none of our typical cases metastasized, although they can rarely arise contemporaneously with sarcomatoid urothelial carcinomas. For these reasons, close follow-up is warranted.

Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to pelvic pressure increase after obstruction.

Prostanoids exert physiological effects on ureteral contractility that may lead to pressure changes and pain during obstruction. In the present study, we examined whether (1) obstruction changes the expression of the two cyclooxygenase (COX) isoforms, COX-1 and COX-2 in human and rat ureters and (2) administration of a selective COX-2 inhibitor influences the pelvic pressure change after experimental ureteral obstruction. Rats were subjected to bilateral ureter obstruction. Ureters were removed and dissected into a proximal dilated and distal non-dilated segment. RNA and protein were extracted and analyzed for cyclooxygenase expression by quantitative polymerase chain reaction and Western blotting. Human ureter samples were obtained from patients undergoing radical nephrectomy. Rat and human ureteral samples were processed for immunohistochemistry. COX-1, but not COX-2 mRNA, was readily detected in the normal rat ureter. COX-2 mRNA and protein expression was increased in the proximal dilated ureter compared to distal non-dilated ureter. This increased COX-2 expression was associated with increased urinary prostaglandin E2 (PGE2) excretion after release of obstruction. Immunohistochemistry showed increased COX-2 labeling in surface epithelium and smooth muscle layers in both rat and human obstructed ureters compared to control ureters. Furthermore, contractile PGE2-EP1 and thromboxane TP receptors were expressed in ureteral smooth muscle. Systemic treatment with the COX-2 selective inhibitor parecoxib (5 mg/kg/day) attenuated the pelvic pressure increase during obstruction. In summary, COX-2 expression is significantly increased in the ureteral wall in response to obstruction in the rat and human ureter and COX-2 activity contributes to increased pelvic pressure after obstruction.

Neuronal apoptosis inhibitory protein is expressed in developing kidney and is regulated by PAX2.

During fetal kidney development, the extent of ureteric bud (UB) branching will determine final nephron endowment for life. Nephron number varies widely among normal humans and those who are born at the low end of the nephron number spectrum may be at risk for essential hypertension in adulthood. Little is known about how nephron number is set. However, we previously showed that the transcription factor, Pax2, suppresses apoptosis in UB cells during kidney development and optimizes branching morphogenesis. Here, we report that PAX2 directly binds to a specific recognition motif in the human neuronal apoptosis inhibitory protein (NAIP) gene promoter. NAIP is an endogenous inhibitor of apoptosis, inactivating caspase-3 and caspase-7 in neuronal tissues. PAX2 activates NAIP gene transcription (7-fold) in vitro and NAIP transcript level is increased fourfold in HEK293 cells stably transfected with PAX2. We show that Naip is expressed in embryonic day 15 (E15) fetal kidney tissue (RT-PCR) and NAIP protein is demonstrated by immunohistochemistry in E15 mouse kidney collecting ducts and P1 proximal tubules. Naip mRNA is significantly reduced (50%) in heterozygous Pax2 mutant mice. Finally, we show that an antisense Naip1 cDNA transfected into murine collecting duct cells doubles caspase-3/7 activity induced by Baxalpha. These observations suggest that the powerful effects of PAX2 on renal branching morphogenesis and final nephron number may be mediated by activation of Naip which then suppresses apoptosis in UB cells.

Evaluation of urothelial stretch-induced cyclooxygenase-2 expression in novel human cell culture and porcine in vivo ureteral obstruction models.

Obstruction and stretch induce cyclooxygenase (COX)-2 expression and prostanoid synthesis in urinary tissues, causing pain, inflammation, hypercontractility, and cell proliferation. Our objective was to characterize acute COX-2 induction during in vivo ureteral obstruction, establish a cell culture model of urothelial stretch-induced COX-2 expression, and evaluate whether mechanotransduction could alter transcriptional and post-transcriptional regulation of COX-2. We performed laparoscopic unilateral ureteral ligation in pigs and allowed progression for 1, 2, 6, 24, or 48 h. We evaluated COX-2 expression with reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunoblotting. We cultured primary human urothelial cells on stretch plates, applied stretch for up to 48 h, and measured COX-2 expression by RT-PCR and immunoblotting, transcription with run-on assays, and mRNA stability with actinomycin mRNA decay assays. In vivo ureteral obstruction induced COX-2 expression 4-fold within 6 h, maintaining induction for 24 h. In cell culture, stretch induced COX-2 steady-state mRNA and protein within the first 3 h of stretch, maintaining this induction for over 6 h. Three hours of stretch doubled COX-2 transcription relative to unstretched controls and increased COX-2 mRNA half-life 3-fold. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in the urothelium and establish a primary urothelial cell culture model for the study of stretch-induced COX-2 mechanisms. This is also the first report to identify alterations in steady-state COX-2 mRNA having components of both transcriptional and post-transcriptional regulation of stretch-regulated COX-2. Future elucidation of COX-2 signaling may identify novel therapeutic targets for treating stretch and distension of urinary tissues.

H-Ras, R-Ras, and TC21 differentially regulate ureteric bud cell branching morphogenesis.

The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras-expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras-expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.

Expression of cytochrome P450 enzymes CYP1A1, CYP1B1, CYP2E1 and CYP4B1 in cultured transitional cells from specimens of the human urinary tract and from urinary sediments.

Expression of cytochromes P450 CYP1A1, CYP1B1, CYP2E1 and CYP4B1 was analysed on the transcript level in human urothelial cells obtained by various methods. As a source of urothelial cells, exfoliated cells in urine samples were used. Their expression profiles were determined either immediately after centrifugal enrichment (n=4) or after their cultivation and propagation (n=8). Another source of urothelial cells were ureter specimens from surgical subjects (n=4). Generally, expression was most prominent for CYP1B1 and CYP4B1 among the CYP transcripts analysed. CYP1B1 mRNA was detected in all samples investigated except for one ureter specimen. CYP4B1 mRNA was present in cell cultures from three out of eight healthy subjects, in three out of four directly investigated urinary sediments and in the cells of all five ureter specimens of four donors investigated after resection and subsequent cell culture. In most cases, CYP2E1 transcript levels were lower than those of CYP1B1 and CYP4B1. CYP2E1 mRNA was detected in cell cultures of six out of eight healthy subjects, in one out of four urinary sediments and in three out of five ureter specimens. CYP1A1 mRNA was clearly observed only in cells from resected ureters. In cell cultures the relative mRNA expression levels varied with subjects interindividually, intraindividually and also during the time of cell culture. The study demonstrates constitutive mRNA expressions of xenobiotic metabolising CYP enzymes in human urothelial cells obtained by different methods. In particular, transcripts of CYP1B1 and CYP4B1 are present, coding for enzymes which are active in the metabolism of polycyclic aromatic hydrocarbons and arylamines, respectively.

Distribution and functional significance of phosphodiesterase isoenzymes in the human lower urinary tract.

To date, it is widely accepted that several disorders of the male and female urogenital tract, such as erectile dysfunction, bladder overactivity, urinary stone disease and the benign prostatic syndrome, can be therapeutically approached by influencing the function of the smooth musculature of the respective organs. In order to achieve a pronounced drug effect without significant adverse events, especially on the cardiovascular system, a certain degree of tissue selectivity is mandatory. Selective intervention in intracellular pathways regulating smooth muscle tone has become a promising strategy to modulate tissue function. Since the concept of taking a pill as a cure for an illness or the relief of symptoms has become widely accepted by the consumers, the pharmacological treatment of urological diseases has focused on selective, orally available drugs, acting via influencing intracellular regulatory mechanisms, thus combining a high response rate and the advantage of an on-demand intake. PDEs play a central role in controlling the levels of cyclic nucleotides (i.e. cAMP and/or cGMP), which are important second messengers in many transmitter pathways involved in the regulation of biological processes of urogenital tissues. Specifically, the use of isoenzyme selective phosphodiesterase (PDE) inhibitors offers great hope in the medical treatment of various genitourinary diseases. These agents are regarded efficacious, having a fast onset of drug action in the target tissue and an improved effect-to-side-effect ratio. The growing experience with the use of this class of compounds in urology is mainly based on basic research efforts and this field will remain the most exciting and innovative subject in genitourinary physiology and pharmacology for the next few years. These tremendous research efforts may lead to a vast pharmacological armamentarium of possible new treatment options. The purpose of this review is to summarize the current knowledge on the distribution and potential functional significance of PDE isoenzymes in the human lower urinary tract.

Expression of Rho-kinase (ROCK-1 and ROCK-2) and its substantial role in the contractile activity of the sheep ureter.

Expression of two isoforms of Rho-kinase (ROCK) and its functional role in the physiological control of smooth muscle contraction in the sheep ureter were investigated. Helical strips of the ureteric smooth muscle were stimulated by electrical field stimulation (EFS, 60 V, 1 mS, 2, 4, 8, 16 and 32 Hz, for 20 S), KCl (80 mm), carbachol (CCh, 10(-8)-10(-4) m) or phenylephrine (Phe, 10(-8)-10(-4) m). EFS produced a reproducible contractile activity, which was abolished by tetrodotoxin (3 x 10(-6) m), a Na(+) channel blocker. A muscarinic receptor antagonist, atropine (2 x 10(-6) m), and an adrenergic neuron blocker, guanethidine (10(-5) m), significantly suppressed the contraction induced by EFS. However, this contraction was augmented in the presence of N(G)-nitro-l-arginine (l-NA, 10(-4) m), a nitric oxide synthase inhibitor. Two Rho-kinase inhibitors, Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m), markedly attenuated the EFS-elicited contraction. CCh and Phe produced concentration-dependent contraction in the sheep ureter. pD(2) values for Phe and CCh were 5.04+/-0.11 and 5.00+/-0.22, respectively. Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m) also significantly inhibited CCh- and Phe-induced contractions. Moreover, these ROCK inhibitors produced relaxations in the KCl-elicited contraction in a concentration-dependent manner. pD(2) values for Y-27632 and fasudil were, respectively, 5.17+/-0.07 and 4.58+/-0.08 (P<0.001). Furthermore, the influences of these agents were also tested on spontaneous phasic contractions of the tissue. Among Y-27632, fasudil, TTX, l-NA, guanethidine and atropine, only the ROCK inhibitors (10(-6)-10(-5) m) were able to suppress the spontaneous contractile activity. Western blot analysis has revealed that both isoforms of Rho-kinase (ROCK-1 and ROCK-2) are expressed in the sheep ureter. Densitometric analysis has indicated that these enzymes are less expressed in the sheep ureter than are in the sheep aorta in a significant manner. These results show that a contractile enzyme, Rho-kinase, is expressed, and it mediates agonist- and EFS-induced contractions as well as spontaneous contractile activity of the isolated sheep ureter. Since Y-27632 and fasudil depressed the contractions, it seems plausible to postulate that Rho-kinase inhibitors may be beneficial in the treatment of renal colic.