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Background and Purpose: Cyclooxygenase inhibitors function to reduce levels of prostaglandin E(2) (PGE(2)) and are broadly efficacious in models of bladder overactivity. We therefore investigated a regulation of urinary bladder function in conscious rats by modulation of the EP(3) receptor for PGE(2).Experimental Approach: The activity of the EP(3) receptor agonist GR63799X, and EP(3) receptor antagonists, CM9 and DG041, at recombinant EP(3) receptors was evaluated in vitro. In vivo, intraduodenal dosing during conscious, continuous-filling cystometry of spontaneously hypertensive rats was utilized to determine the urodynamic effect of EP(3) receptor modulation.Key Results: GR63799X dose-dependently (0.001-1 mg x kg(-1)) reduced bladder capacity, as indicated by a reduction in both the micturition interval and volume of urine per void. In contrast, CM9 (10 and 30 mg x kg(-1)) and DG041 (30 mg x kg(-1)) enhanced bladder capacity, as indicated by significantly longer micturition intervals and larger void volumes. CM9 and DG041 inhibited the responses to GR63799X supporting the in vivo activity of these pharmacological agents at the EP(3) receptor. In addition to its effect on bladder capacity, GR63799X increased endogenous urine production. Intra-arterial infusion of saline mimicked the enhancement of urine flow observed with GR63799X, and the response was inhibited by CM9.Conclusions and Implications: These data support the EP(3) receptor as a modulator of urinary bladder activity in the conscious rat, and in addition, indicate a role for EP(3) receptor activity in regulating urine flow. [ABSTRACT FROM AUTHOR]

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.

Neutropenia is a common consequence of radiation and chemotherapy in cancer patients. The resulting immunocompromised patients become highly susceptible to potentially life-threatening infections. Granulocyte colony-stimulating factor (G-CSF) is known to stimulate neutrophil production and is widely used as a treatment of chemotherapy-induced neutropenia. A small-molecule G-CSF secretagogue without a requirement for refrigerated supply chain would offer a more convenient and cost-effective treatment of chemotherapy-induced neutropenia. Bacterial lipopeptides activate innate immune responses through Toll-like receptor 2 (TLR2) and induce the release of cytokines, including G-CSF, from macrophages, monocytes, and endothelial. Pam 2 CSK 4 is a synthetic lipopeptide that effectively mimics bacterial lipoproteins known to activate TLR2 receptor signaling through the TLR2/6 heterodimer. Substrate-based drug design led to the discovery of GSK3277329, which stimulated the release of G-CSF in activated THP-1 cells, peripheral blood mononuclear cells, and human umbilical vein endothelial cells. When administered subcutaneously to cynomolgus monkeys ( Macaca fasciculari s), GSK3277329 caused systemic elevation of G-CSF and interleukin-6 (IL-6), but not IL-1β or tumor necrosis factor α, indicating a selective cytokine-stimulation profile. Repeat daily injections of GSK3277329 in healthy monkeys also raised circulating neutrophils above the normal range over a 1-week treatment period. More importantly, repeated daily injections of GSK3277329 over a 2-week period restored neutrophil loss in monkeys given chemotherapy treatment (cyclophosphamide, Cytoxan). These data demonstrate preclinical in vivo proof of concept that TLR2 agonism can drive both G-CSF induction and subsequent neutrophil elevation in the cynomolgus monkey and could be a therapeutic strategy for the treatment of chemotherapy-induced neutropenia., Competing Interests: Conflict-of-interest disclosure: The authors are employees of GlaxoSmithKline PLC.

Objectives: To study the relationship between myosin light chain phosphorylation of the detrusor muscle and spontaneous smooth muscle contractions in a rabbit model of partial outlet obstruction., Methods: New Zealand white rabbit urinary bladders were partially obstructed for 2 weeks. Rabbits were euthanized, detrusor muscle strips were hung on a force transducer and spontaneous activity was measured at varying concentrations (0-0.03 μM/L) of the Rho-kinase inhibitors GSK 576371 or 0.01 μM/L Y27632. Basal myosin light chain phosphorylation was measured by 2-D gel electrophoresis in control and GSK 576371-treated strips., Results: Both drugs suppressed the force of spontaneous contractions, whereas GSK 576371 had a more profound effect on the frequency of the contractions. The IC₅₀ values for the inhibition of frequency and force of spontaneous contractions were 0.17 μM/L and 0.023 μM/L for GSK 576371, respectively. The compound significantly decreased the basal myosin light chain phosphorylation from 28.0 ± 3.9% to 13.5 ± 1.9% (P < 0.05). At 0.01 μM/L, GSK 576371 inhibited spontaneous bladder overactivity by 50%, but inhibited carbachol-elicited contractions force by just 25%., Conclusions: These data suggest that Rho-kinase regulation of myosin light chain phosphorylation contributes to the spontaneous detrusor activity induced by obstruction. This finding could have therapeutic implications by providing another therapeutic option for myogenic, overactive bladder., (© 2013 The Japanese Urological Association.)

Three different approaches were employed to assess various markers associated with sex differences in responses to methamphetamine (MA). Bioassay measures reveal that MA treatment results in significantly greater reductions in body weight and increases in body temperature in male mice. Protein and mRNA determinations show significant increases in Bcl-2 and PAI-1 in male mice, while females show significant increases in GFAP and decreases in IGF-1R following treatment with MA. In mice with a heterozygous mutation of their dopamine transporter (+/- DAT), only female mice show significant differences in dopamine transporter binding and mRNA and associated reductions in striatal dopamine content along with increases in MA-evoked striatal dopamine output. The identification of these sex-dependent differences in markers provides a foundation for more exhaustive evaluation of their impact upon, and treatment of, disorders/neurotoxicity of the nigrostriatal dopaminergic system and the bases for the differences that exist between females and males.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Endometriosis, defined as the presence of endometrial glands and stroma at extra-uterine sites, is a gynecological condition that affects women of reproductive age. Consistent with its uterine origins, endometriotic lesions and resulting symptoms are hormonally responsive. To investigate Progesterone Receptor (PR)-based therapies, we measured physiological endpoints and gene expression in rat models of uterine cell estrogenic activity. Estrogen-induced ELT-3 rat leiomyoma cell proliferation was significantly inhibited by progesterone (P4), while the antiprogestin RU486 or the Selective PR Modulator (SPRM) asoprisnil, did not block proliferation. Stromal cell-derived factor-1 (SDF-1/Cxcl12) gene expression was induced by estrogen, and was repressed by the Selective Estrogen Receptor Modulators (SERMs), the antiestrogen ICI 182,780, and P4, but not by RU486 or the ERbeta-selective ligand ERB-041. In ELT-3 cells, asoprisnil demonstrated partial PR agonism on SDF-1 gene repression. Magnetic Resonance Imaging was used to monitor development of ectopic cysts in a rat surgical model of endometriosis. SERMs and P4 significantly decreased cyst volumes comparably by approximately 60%. However, ERB-041 and asoprisnil had no effect on cyst volume, and RU486 increased cyst volume by 20%. SDF-1 expression was modestly, but significantly, increased in the cyst compared to eutopic uterus, and P4 and raloxifene could repress the expression. We showed that SDF-1 was similarly regulated in human cells. These data suggest that transcriptional regulation of SDF-1 is a surrogate marker of estrogenic activities via ERalpha in rat uterine cells, and that SDF-1 repression by PR agonists can predict the ability to oppose the actions of estrogen in vivo.

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Objectives: To examine the effects of estrogen and/or progesterone on the cystometric profiles obtained using continuous-filling cystometry in the conscious Sprague-Dawley rat., Methods: Sprague-Dawley rats underwent ovariectomy (OVX) and were compared with controls by conscious continuous-filling cystometry. The effect of estrogen (10 microg/kg/d for 14 days) and/or progesterone (10 mg/kg/d for 14 days) replacement on OVX urodynamics was examined (n = 7-8/group)., Results: OVX rats demonstrated reduced micturition intervals and voided volumes compared with controls. These effects of OVX were reversed by estrogen replacement, but not by progesterone replacement. When combined with estrogen, progesterone functioned to partially antagonize the effects of estrogen in OVX rats., Conclusions: Estrogen enhances bladder capacity in the OVX rat and therefore is a likely contributor to the larger bladder capacity in the female compared with the male rat. Consistent with its established role in reproductive physiology, progesterone antagonizes the beneficial effects of estrogen on OVX rat urodynamics.

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

Two classes of amino acid-derived heterocyclic progesterone receptor ligands were developed to address the metabolic issues posed by the dimethyl amide functionality of the lead compound (1). The tetrazole-derived ligands behaved as potent partial agonists, while the 1,2,4-triazole ligands behaved as potent full agonists.

Purpose: Urodynamics have been traditionally recorded in anesthetized or conscious animals implanted with a bladder catheter that is used to artificially fill the bladder while measuring intravesicular bladder pressure. Anesthesia alters the urodynamics and in the conscious state this methodology requires that the dogs be tethered/restrained, which evokes stress and limits the period of continuous urodynamic assessment. A more physiological and chronic method of evaluating pharmacological responses on urodynamics is necessary., Materials and Methods: Adult female beagle dogs were surgically instrumented with radiotelemetry transmitters enabling urodynamic/hemodynamic recordings. Telemetered urodynamics were compared to those measured in anesthetized dogs receiving bladder infusion of saline. The response to diuresis with furosemide (Intervet, Millsboro, Delaware) and the M3 selective antimuscarinic darifenacin (Matrix Laboratories, Hyderabad, India) were evaluated., Results: Saline infused, anesthetized dogs demonstrated lower peak micturition pressure and higher threshold pressure than conscious, freely moving telemetered dogs. In telemetered dogs a single dose of furosemide increased voiding frequency and average urine volume per void. Darifenacin decreased peak voiding pressure without affecting voiding frequency., Conclusion: Telemetry provides the potential to significantly decrease animal use while enabling the continuous monitoring of urodynamics under more physiological conditions without tethering or artificial filling. In addition, this new model facilitates evaluation of the chronic efficacy of new urological therapies.

Aims: We tested cardiovascular and visceromotor reflex (VMR) responses to urinary bladder distension (UBD) in urethane anesthetized rats to see if it can replicate the response pattern and the inhibition of bladder nociceptive transmission by analgesics seen in isoflurane anesthetized animals., Methods: Female Sprague-Dawley rats under 3% isoflurane anesthesia were acutely instrumented with jugular venous, carotid arterial, and bladder cannulas for drug administration, blood pressure (BP) measurement, and bladder distension, respectively. Needle electrodes were placed directly into the abdominal musculature to measure myoelectrical activity subsequent to phasic UBD (30 sec in 3 min intervals). A cardiovascular response (pressor) and a VMR response (a contraction of abdominal and hind limb musculature) to UBD were evaluated in urethane (1.2 g/kg, i.v.) or isoflurane (1%) anesthetized rats., Results: Pressor and VMR responses to noxious UBD (60 mmHg) were generated under both anesthesics. The thresholds of stimulus response functions for both pressor and VMR responses were not affected by either anesthesics. However, the magnitude of the maximal pressor response was significantly reduced in urethane anesthesia. The analgesics, morphine, and mexiletine, significantly inhibited the VMR response to noxious UBD under both anesthetics, but the intensities of the inhibition from both analgesics under urethane anesthesia were much lower than under isoflurane anesthesia (ID50: 2.07 mg/kg vs. 0.88 mg/kg for morphine, >10 mg/kg vs. 0.47 mg/kg for mexiletine)., Conclusions: The rat urinary bladder distension model in urethane anesthetized rats demonstrates a blunted maximal pressor response and a reduced inhibition of visceral nociceptive transmission by analgesics. Neurourol. Urodynam. 28:442-446, 2009. (c) 2008 Wiley-Liss, Inc.

Prostaglandin EP3 receptors in the central nervous system (CNS) may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG041) and (2E)-N-{[5-bromo-2-(methyloxy)phenyl] sulfonyl}-3-[2-(2-naphthalenylmethyl)phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists with radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) were evaluated in female rats after intrathecal or intracerebroventricular administration. Both DG041 and CM9 showed a high affinity for EP3 receptors at subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the nonselective agonist PGE2. After intrathecal or intracerebroventricular administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude, of the bladder rhythmic contraction. With intrathecal administration DG041 and CM9 produced a long-lasting and robust inhibition on the VMR response to UBD, whereas with intracerebroventricular injection both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support the concept that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

The excitatory roles of EP3 receptors at the peripheral afferent nerve innervating the rat urinary bladder have been evaluated by using the selective EP3 antagonist (2E)-3-[1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl]-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG-041). The bladder rhythmic contraction model and a bladder pain model measuring the visceromotor reflex (VMR) to urinary bladder distension (UBD) have been used to evaluate DG-041 in female rats. In addition, male rats [spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD)] were anesthetized with pentobarbital sodium, and primary afferent fibers in the L6 dorsal root were isolated for recording the inhibitory response to UBD following intravenous injection of DG-041. Intravenous injection of DG-041 (10 mg/kg), a peripherally restricted EP3 receptor antagonist, significantly reduced the frequency of bladder rhythmic contraction and inhibited the VMR response to bladder distension. The magnitude of reduction of the VMR response was not different in the different strains of rats (SD, SHR, and WKY). Furthermore, quantitative characterization of the mechanosensitive properties of bladder afferent nerves in SHR, WKY, and SD rats did not show the SHR to be supersensitive to bladder distension. DG-041 selectively attenuated responses of mechanosensitive afferent nerves to UBD, with strong suppression on the slow-conducting, high-threshold afferent fibers, with equivalent activity in the three strains. We conclude that sensitization of afferent nerve activity was not one of the mechanisms of bladder hypersensitivity in SHR. EP3 receptors are involved in the regulation of bladder micturition and bladder nociception at the peripheral level.

Nonsteroidal anti-inflammatory cyclooxygenase inhibitors that function to reduce prostaglandin E2 (PGE2) production have been widely reported as effective agents in models of urinary bladder overactivity. We therefore investigated a potential role for the PGE2 receptor, EP3, in urinary bladder function by performing conscious, freely moving cystometry on EP3 receptor knockout (KO) mice. EP3 KO mice demonstrated an enhanced bladder capacity compared with wild-type (WT) mice ( approximately 185% of WT) under control conditions, based on larger voided and infused bladder volumes. Infusion of the EP3 receptor agonist GR63799X into the bladder of WT mice reduced the bladder capacity. This was ineffective in EP3 KO mice that demonstrated a time-dependent increase in bladder capacity with GR63799X, an effect similar to that observed with vehicle in both genotypes. In addition, infusion of PGE2 into WT mice induced bladder overactivity, an effect that was significantly blunted in the EP3 KO mice. The data reported here provide the first evidence supporting a functional role for EP3 receptors in normal urinary bladder function and implicate EP3 as a contributor to bladder overactivity during pathological conditions of enhanced PGE2 production, as reported previously in overactive bladder patients.

The present study investigated whether beta3-adrenoceptor activation acts on the bladder afferent pathway by examination of the visceromotor reflex (VMR) and pressor responses to urinary bladder distension (UBD) and whether beta3-adrenoceptor activation produces urinary bladder relaxation in hyperactive spontaneously hypertensive rats (SHRs) in comparison with their normotensive control rats [Wistar-Kyoto (WKY)]. Using the VMR responses to noxious UBD as a measure of bladder afferent signal transmission, SHRs did not present a sensitized bladder phenotype. However, reduced bladder compliance accompanied by a reduced void threshold was detected in the SHR detrusor. Furthermore, the selective beta3-adrenoceptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) (i.v.) failed to attenuate VMR or pressor responses to UBD in either SHRs or WKY rats, but it dose-dependently inhibited rhythmic contraction (RC) in SHRs. The minimal effective dose was 0.001 mg/kg. Using the same model in WKY rats, CL-316243 did not elicit significant inhibition of contractions in the bladder RC assay. These results suggest that SHRs represent abnormal efferent/detrusor function (detrusor overactivity) without mechanosensory afferent hypersensitivity. The beta3-adrenoceptor agonist CL-316243 acts on the detrusor muscle to increase urine storage in SHRs.

Purpose: High voltage activated calcium channels have been implicated in nociceptive transmission in several animal pain models. To our knowledge this is the first study to evaluate the ability of various high voltage activated calcium channel blockers to inhibit the transmission of noxious stimuli from the bladder at the level of the spinal cord., Materials and Methods: The nociceptive response was measured by analyzing the visceromotor reflex and cardiovascular (pressor) responses to bladder distention. The role of Cav2.2 (N-type), Cav2.1 (P/Q-type) and Cav1 (L-type) calcium channels in bladder nociceptive reflex responses was examined using omega-conotoxin-GVIA, omega-agatoxin IVA/omega-conotoxin MVIIC and verapamil (Sigma-Aldrich), respectively. Female Sprague-Dawley rats were acutely instrumented with intrathecal catheters, carotid arterial and bladder cannulas. Needle electrodes were placed directly into the abdominal musculature to measure myoelectric activity subsequent to repeat phasic bladder distention at 60 mm Hg for 30 seconds at 3-minute intervals with the rats under 1% isoflurane. Drugs were administered by intrathecal injection 2 minutes before distention and responses were recorded for 15 minutes per dose., Results: When administered intrathecally, omega-conotoxin-GVIA and omega-conotoxin MVIIC (10 microg/kg each) significantly attenuated reflex responses to noxious bladder distention to 12% and 65% of the maximal visceromotor reflex response, and to 45% and 59% of the control pressor response, respectively. However, agatoxin and verapamil were less effective., Conclusions: The study suggests that spinal Cav2.2 and Q-type Cav2.1 calcium channels contribute to acute bladder nociception, while Cav1 channels have a limited role.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

Aims: Several mechanisms that are involved in acute rat bladder nociception were examined. The nociceptive response was measured by analyzing both cardiovascular and visceromotor reflex responses to urinary bladder distension. The contributions of micro-opioid receptor, kappa-opioid receptor, sodium channels, muscarinic receptors, and cyclooxygenase, were explored with morphine, U50,488, mexiletine, oxybutynin, and naproxen, respectively., Methods: Female Sprague-Dawley rats were acutely instrumented with jugular venous, carotid arterial, and bladder cannulas. Needle electrodes were placed directly into the abdominal musculature to measure myoelectrical activity subsequent to repeated phasic urinary bladder distension (60 mmHg for 20 sec in 3 min intervals) under 1% isoflurane. Drugs were administered by i.v. bolus injection 2 min prior to distension., Results: The analgesics morphine (ID50 0.69 mg/kg), U50,488 (1.34 mg/kg), and mexiletine (2.60 mg/kg) significantly inhibited the visceromotor reflex response to noxious urinary bladder distension. Oxybutynin also attenuated reflex responses to noxious urinary bladder distension to 41% of the maximal pressor response and 32% of the control visceromotor reflex response (3.01 and 5.05 mg/kg), respectively, indicating a role of muscarinic receptors in bladder nociception. Naproxen did not attenuate the pressor response, but moderately inhibited visceromotor reflex to 45% of control at 30 mg/kg (P < 0.05)., Conclusions: Current results using the rat urinary bladder distension model are consistent with previous research demonstrating a role of the analgesics (morphine, U50,488, and mexiletine) in the inhibition of visceral nociceptive transmission. The utility of the reflex responses to urinary bladder distension may provide a method useful to examine mechanisms which target the bladder sensory pathway., ((c) 2007 Wiley-Liss, Inc.)

Transforming growth factor (TGF)-beta plays a critical role in diabetic nephropathy. To isolate the contribution of one of the signaling pathways of TGF-beta, the Smad3 gene in the mouse was knocked out at exons 2 and 3, and the effect was studied in streptozotocin (STZ)-induced diabetes over a period of 6 wk. TGF-beta activity was increased in the diabetic mice but was not able to signal via Smad3 in the knockout (KO) mice. As expected in the wild type, the kidneys of the STZ-diabetic mice showed both structural and functional defects that are characteristic of diabetic renal involvement. In the Smad3-KO mice, however, the defects that were improved were renal hypertrophy, mesangial matrix expansion, fibronectin overproduction, glomerular basement membrane thickening, plasma creatinine, and the blood urea nitrogen. The parameters not significantly altered by the Smad3-KO were albuminuria, reduction in podocyte slit pore density, and the increase in vascular endothelial growth factor abundance and activity. It seems that the absence of Smad3 modifies the natural course of murine diabetic nephropathy, providing renal functional protection and preventing structural lesions relating to kidney hypertrophy and matrix accumulation, even though albuminuria and changes in podocyte morphology persist. In conclusion, the effects of the Smad3-KO mirror the effects of anti-TGF-beta therapy in diabetes, suggesting that the chief component of TGF-beta signaling that is relevant to kidney disease is the Smad3 pathway.

Purpose: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions., Experimental Design: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency., Results: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals., Conclusion: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Inhibitors of transforming growth factor beta (TGF-beta) type I receptor (ALK5) offer a novel approach for the treatment of fibrotic diseases such as renal, hepatic, and pulmonary fibrosis. The optimization of a novel phenylpyridine pyrazole series (1a) led to the identification of potent, selective, and orally active ALK5 inhibitors. The cellular potency and pharmacokinetics profiles of these derivatives were improved and several compounds presented antifibrotic activity when orally administered to rats in an acute liver model of dimethylnitrosamine- (DMN-) induced expression of collagen IA1 mRNA, a major gene contributing to excessive extra cellular matrix deposit. One of the most potent ALK5 inhibitors identified in this chemical series, compound 13d (GW788388), reduced the expression of collagen IA1 by 80% at a dose of 1 mg/kg twice a day (b.i.d.). This compound significantly reduced the expression of collagen IA1 mRNA when administered orally at 10 mg/kg once a day (u.i.d.) in a model of puromycin aminonucleoside-induced renal fibrosis.

SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC(50) of 14.3 nM and was approximately 4-fold less potent as an inhibitor of ALK4 (IC(50) = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC(50) > 10,000 nM). In cell-based assays, SB-525334 (1 microM) blocked TGF-beta1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen alpha1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha1(I) and procollagen alpha1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-beta1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that promotes malignant glioma invasion, angiogenesis, and immunosuppression. Antisense oligonucleotide suppression of TGF-beta(2) ligand expression has shown promise in preclinical and clinical studies but at least two ligands mediate the effects of TGF-beta in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGF-beta receptor, on a panel of human malignant glioma cell lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGF-beta signaling, with decreased TGF-beta-mediated transcription. Furthermore, SB-431542 inhibited the expression of two critical effectors of TGF-beta-vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGF-beta-mediated morphologic changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGF-beta receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

Clinically, there is a great need for small molecule inhibitors that could control pathogenic effects of transforming growth factor (TGF-beta) and/or modulate effects of TGF-beta in normal responses. Inhibition of TGF-beta signaling would be predicted to enhance re-epithelialization of cutaneous wounds and reduce scarring fibrosis. Selective small molecule inhibitors of the TGF-beta signaling pathway developed for therapeutics will also be powerful tools in experimentally dissecting this complex pathway, especially its cross-talk with other signaling pathways. In this study, we characterized 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124), a member of a new class of small molecule inhibitors related to imidazole inhibitors of p38, which inhibit the TGF-beta type I receptor serine/threonine kinase known as activin receptor-like kinase (ALK) 5. We demonstrate that this compound selectively and concentration-dependently inhibits ALK4-, ALK5-, and ALK 7-dependent activation of downstream cytoplasmic signal transducers, Smad2 and Smad3, and of TGF-beta-induced mitogen-activated protein kinase pathway components but does not alter ALK1, ALK2, ALK3 or ALK6-induced Smad signaling. SB-505124 also blocks more complex endpoints of TGF-beta action, as evidenced by its ability to abrogate cell death caused by TGF-beta1 treatment. SB-505124 is three to five times more potent than a related ALK5 inhibitor described previously, SB-431542.

Cleft palate is among the most common birth defects in humans, caused by a failure in the complex multistep developmental process of palatogenesis. It has been recently shown that transforming growth factor beta3 (Tgf-beta3) is an absolute requirement for successful palatal fusion, both in mice and humans. However, very little is known about the mechanisms of Tgf-beta3 signaling during this process. Here we show that putative Tgf-beta type I receptors, Alk-1, Alk-2, and Alk-5, are all endogenously expressed in the palatal epithelium. Activation of Alk-5 in the Tgf-beta3 (-/-) palatal epithelium is able to rescue palatal fusion, whereas inactivation of Alk-5 in the wild-type palatal epithelium prevents palatal fusion. The effect of Alk-2 is similar, but less pronounced. The induction of fusion by activation of Alk-5 or Alk-2 is stronger in the posterior parts of the palates at the embryonic day 14 (E14), while their activation at E13.5 also restores anterior fusion, reflecting the natural anterior-posterior direction of palate maturation in vivo. We also show that Smad2 is endogenously activated in the palatal midline epithelial seam (MES) during the fusion process. By using a mutant Alk-5 receptor that is an active kinase but is unable to activate Smads, we show that activation of Smad-independent Tgf-beta responses is not sufficient to induce fusion of shelves deficient in Tgf-beta3. Based on these observations, we conclude that the Smad2-dependent Alk-5 signaling pathway is dominant in palatal fusion driven by Tgf-beta3.

Recent advances have identified novel small molecule inhibitors of the transforming growth factor-beta (TGF-beta) type I receptor kinase as a potential therapy in organ remodeling diseases, such as chronic renal disease. Because TGF-beta is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment in many fibrotic diseases. The rationale for inhibition of TGF-beta signaling in renal disease includes prevention of fibrosis, tubular dedifferentiation and vascular effects.

In this report female and male CD-1 mice were treated with a neurotoxic regimen of methamphetamine (MA) to compare gender differences in striatal dopamine depletion and concordant changes in mRNA markers of the transforming growth factor-beta injury response associated with neurodegeneration. Striatal dopamine concentrations of MA-treated female mice were less depleted and significantly greater than that of identically treated males. Associated with this gender difference in striatal dopamine depletion were significantly decreased mRNA levels of plasminogen activator inhibitor-1 and a trend for increased (p = 0.06) mRNA levels of glial fibrillary acidic protein within females. No statistically significant differences between MA-treated female and male mice were obtained in mRNA levels for transforming growth factor-beta, transforming growth factor-beta type 2 receptor, activin-like kinase-5 or fibronectin. These data demonstrate the presence of changes in two specific molecular markers of the transforming growth factor-beta injury response which are in accordance with gender differences in MA-induced striatal dopamine depletion. The results suggest that the neuroprotective advantage displayed by females may in part be related to reductions in the transforming growth factor-beta injury response as indicated by decreased mRNA plasminogen activator inhibitor-1 and an increased response of reactive astrocytes which promote neuronal survival as indicated by augmented glial fibrillary acidic protein mRNA levels., (Copyright 2003 S. Karger AG, Basel)

The role of angiotensin II (AII) and angiotensin IV (AIV) as inducers of PAI-1 expression during hypertension was studied in vivo. A 2-week infusion of AII (300 ng/kg/min) via an osmotic pump increased systolic blood pressure (171 +/- 2 vs. 138 +/- 6 mm Hg), urinary protein excretion (32 +/- 6 vs. 14 +/- 2 mg/day), and renal (2.2 +/- 0.5 vs. 1.0 +/- 0.1) and cardiac (1.8 +/- 0.3 vs. 1.0 +/- 0.1) gene expression of plasminogen activator inhibitor 1 (PAI-1). AIV infusion did not affect any of the above with the exception of PAI-1 gene expression which was increased in the left ventricles (1.7 +/- 0.3 vs. 1.0 +/- 0.1). AII-infused rats displayed a decreased creatinine clearance (538 +/- 75 vs. 898 +/- 96 ml/min) and hypertrophic left ventricles (0.275 +/- 0.006 vs. 0.220 +/- 0.011 g/100 g). Our results demonstrate that AII but not AIV infusion is associated with increased renal PAI-1 gene expression., (Copyright 2002 S. Karger AG, Basel)

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.

The effects of the angiotensin type 1 (AT(1)) receptor antagonist, eprosartan, were studied in a model of severe, chronic hypertension. Treatment of male spontaneously hypertensive stroke prone rats (SHR-SP) fed a high-fat, high-salt diet with eprosartan (60 mg/kg/day i.p.) for 12 weeks resulted in a lowering of blood pressure (250 +/- 9 versus 284 +/- 8 mm Hg), renal expression of transforming growth factor-beta mRNA (1.5 +/- 0.2 versus 5.4 +/- 1.4) and the matrix components: plasminogen activator inhibitor-1 (5.2 +/- 1.4 versus 31.4 +/- 10.7), fibronectin (2.2 +/- 0.6 versus 8.2 +/- 2.2), collagen I-alpha 1 (5.6 +/- 2.0 versus 23.8 +/- 7.3), and collagen III (2.7 +/- 0.9 versus 7.6 +/- 2.1). Data were corrected for rpL32 mRNA expression and expressed relative to Wistar Kyoto (WKY) rats [=1.0]. Expression of fibronectin protein was also lowered by eprosartan (0.8 +/- 0.1 versus 1.9 +/- 0.5), relative to WKY rats. Eprosartan provided significant renoprotection to SHR-SP rats as measured by decreased proteinuria (22 +/- 2 versus 127 +/- 13 mg/day) and histological evidence of active renal damage (5 +/- 2 versus 195 +/- 6) and renal fibrosis (5.9 +/- 0.7 versus 16.4 +/- 1.9) in vehicle- versus eprosartan-treated rats, respectively. Our results demonstrated that AT(1) receptor blockade with eprosartan can reduce blood pressure and preserve renal structure and function in this model of severe, chronic hypertension. These effects were accompanied by a decreased renal expression of transforming growth factor-beta1, plasminogen activator inhibitor-1, and several other extracellular matrix proteins compared with vehicle-treated SHR-SP.

Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

1. The effect of carvedilol on renal function, structure and expression of TGF beta and the matrix proteins fibronectin, collagen I and collagen III, was evaluated in spontaneously hypertensive stroke-prone (SHR-SP) rats fed a high fat, high salt diet. 2. Carvedilol treatment for 11 to 18 weeks did not alter systolic blood pressure in SHR-SP rats, however, it resulted in a significant reduction in heart rate. 3. Carvedilol treatment reduced renal fibrosis and total, active and chronic renal damage to levels approaching those of WKY rats on a normal diet. 4. Urinary protein excretion was higher in SHR-SP rats (51+/-10 mg day(-1)) than WKY rats (18+/-2 mg day(-1)) and this was further increased when SHR-SP rats were fed a high fat, high salt diet (251+/-120 mg day(-1)). Treatment with carvedilol resulted in significantly lower urinary protein excretion (37+/-15 mg day(-1)). 5. The expression of TGF beta mRNA was significantly higher in SHR-SP rats compared to WKY rats and a further increase was observed when rats were fed a high fat, high salt diet. Renal TGF beta expression was significantly reduced by treatment with carvedilol. The expression of fibronectin and collagen I and collagen III mRNA showed a pattern similar to that observed with TGF beta mRNA expression. Collagen I mRNA expression followed a pattern similar to renal fibrosis. 6. These data indicate that carvedilol can provide significant renal protection in the absence of any antihypertensive activity and that the mechanisms involved in this action may include reduced expression of profibrotic factors such as TGF beta.

In the present study, the role of transforming growth factor-beta (TGFbeta) on the production of the extracellular matrix component, fibronectin, in the prostate has been studied. The mRNA levels of fibronectin, TGFbeta and the two TGFbeta receptors, ALK5 (activin like kinase) and type II, were measured using reverse-transcription polymerase chain reaction (RT-PCR). TGFbeta increased fibronectin mRNA and protein (7-fold) in a concentration-dependent fashion. An interesting relationship between the two TGFbeta receptors was found in that TGFbeta caused an upregulation of its type I receptor mRNA (5-6-fold) and a downregulation of the type II receptor mRNA (5-fold). Time-course experiments revealed that the change in expression of the TGFbeta receptors reached maximum at 24 h with an early increase at 4-5 h, whereas the fibronectin gene expression was not significantly stimulated until about 24 h. These data provide evidence that TGFbeta stimulates extracellular matrix production in prostate cells.

A novel guanosine triphosphate-binding protein, chronic renal failure gene (CRFG), was discovered by differential display PCR to be regulated differentially in renal disease. Within the rat kidney, CRFG mRNA was localized to the outer medulla and was highly expressed in epithelial cells. The specific renal expression of CRFG mRNA in the outer medulla was reduced dramatically in several rat models of renal disease, including diabetic nephropathy, partial nephrectomy, ischemia, and anti-Thy1.1-induced nephritis. CRFG was localized selectively in the nucleus of human and rodent cells, as determined by immunocytochemistry and green fluorescence fusion protein. Cellular mRNA levels of CRFG were also increased after serum administration, when cells proliferate. These data suggest that CRFG may be involved in regulating guanosine triphosphate-dependent nuclear events that are associated with cell proliferation and that are important in normal renal function and essential for growth and development.

The role of ANG II on renal and cardiac gene expression of matrix proteins was studied in rats with progressive renal disease. Induction of renal failure by five-sixths nephrectomy of Sprague-Dawley rats resulted in hypertension (163 +/- 19 vs. control pressures of 108 +/- 6 mmHg), proteinuria (83 +/- 47 vs. 14 +/- 2 mg/day), and increased renal expression of fibronectin, thrombospondin, collagen I and III, transforming growth factor-beta (TGF-beta), and plasminogen activator inhibitor-1 (PAI-1) mRNA. Treatment with the ANG II receptor antagonist, eprosartan (60 mg. kg(-1).day(-1)), lowered blood pressure (95 +/- 5 mmHg) and proteinuria (19 +/- 8 mg/d) and abrogated the increased TGF-beta, fibronectin, thrombospondin, collagens I and III, and PAI-1 mRNA expression. An increase in left ventricular weight was observed in five-sixths nephrectomized rats (0.13 +/- 0.01 vs. 0.08 +/- 0.01 g/100 g body wt), a response that was inhibited by eprosartan treatment (0.10 +/- 0.01 g/100 g). Left ventricular expression of TGF-beta and fibronectin was also increased in rats with renal disease; however, the small decreases in expression observed in eprosartan-treated rats did not reach statistical significance. These data suggest that eprosartan may be beneficial in progressive renal disease and that the mechanism of action includes inhibition of cytokine production in addition to antihypertensive activity.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth factor-beta1 in the rodent brain support a major role of microglia in neural activities of transforming growth factor-beta1 and give a new link between transforming growth factor-beta1 and the complement system. The auto-induction of the transforming growth factor-beta1 system has implications for transgenic mice that overexpress transforming growth factor-beta1 in brain cells and for its potential role in amyloidogenesis.