Monomerization of TDP-43 is a key determinant for inducing TDP-43 pathology in amyotrophic lateral sclerosis.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization-deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization-deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell-derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Prostatic ischemia induces ventral prostatic hyperplasia in the SHR; possible mechanism of development of BPH.

In the light of increasing evidence that benign prostatic hyperplasia is associated with cardiovascular disease, we have investigated the relationship between prostatic blood flow and prostatic hyperplasia in the spontaneously-hypertensive-rat (SHR). Twelve-week-old male SHRs were treated with nicorandil for six weeks. Wistar-Kyoto rats were used as controls. Six weeks after nicorandil treatment, blood pressure and the prostatic blood flow were estimated, and tissue levels of malondialdehyde, HIF-1α, TGF-ß1, bFGF, dihydrotestosterone, and α-SMA were measured. SHRs showed significant increases in blood pressure, tissue levels of malondialdehyde, HIF-1α, TGF-ß1, bFGF, α-SMA and a significant decrease in the prostatic blood flow. Although treatment with nicorandil failed to alter the blood-pressure and α-SMA, it significantly ameliorated the increased levels of malondialdehyde, HIF-1α, TGF-ß1, and bFGF. There were no significant differences in tissue levels of dihydrotestosterone among any groups. These data indicate that development of prostatic hyperplasia may be associated with prostatic hypoxia, which nicorandil prevents via its effect to increase the blood flow.

Olmesartan ameliorates urinary dysfunction in the spontaneously hypertensive rat via recovering bladder blood flow and decreasing oxidative stress.

PURPOSE: As hypertension (HT) is one of the risk factors for lower urinary tract symptoms, we investigated the effect of an angiotensin II type I receptor blocker, olmesartan, on bladder dysfunction in the spontaneously hypertensive rat (SHR). MATERIALS AND METHODS: Twelve-week-old male SHRs were administered perorally with olmesartan (0, 1, or 3 mg/kg/day) or nifedipine (30 mg/kg/day) for 6 weeks. Wistar rats were used as normotensive controls. The effects of olmesartan or nifedipine on blood pressure (BP), bladder blood flow (BBF), urodynamic parameters, tissue levels of malondialdehyde (MDA), nuclear factor erythroid 2-related factor 2 (Nrf2), and nerve growth factor (NGF) were measured in the bladder. Localization of 4-hydroxy-2-nonenal (4-HNE), Nrf2, and NGF in the bladder was shown by immunohistochemistry. RESULTS: The SHRs showed significant increase in BP, micturition frequency, and expression of MDA, 4-HNE, Nrf2, and NGF when compared to the control Wistar rats. Conversely, there was a decrease in BBF and single voided volume in SHRs when compared to Wistar rats. Treatment with olmesartan and nifedipine significantly improved BP. However, only olmesartan significantly ameliorated urodynamic parameters and oxidative damage compared to the non-treated SHR. The immunoreactivities of 4-HNE, Nrf2, and NGF in SHR urothelium and blood vessels were increased compared to the control. Treatment with a high dose of olmesartan decreased the expressions of 4-HNE, Nrf2, and NGF in the bladder. CONCLUSION: Our data suggest that BP, BBF, and oxidative stress may be responsible for the functional changes in HT-related bladder dysfunction. Olmesartan significantly ameliorated this bladder dysfunction.

Characterization of silodosin and naftopidil in the treatment of bladder dysfunction in the spontaneously hypertensive rat.

PURPOSE: As increasing evidence suggest that α(1)-blockers prevent benign prostatic hyperplasia related overactive bladder and nocturia in the human, we investigated the effects of silodosin and naftopidil on hypertension-related bladder dysfunction in the spontaneously hypertensive rat (SHR) model. MATERIALS AND METHODS: Twelve-week-old male SHRs received no treatment or treatment with silodosin (100 µg/kg, p.o.) or naftopidil (10 or 30 mg/kg, p.o.) once daily for 6 weeks. Wistar rats were used as normotensive controls. After 6-week treatment, voiding functions were estimated by metabolic cages (dark- and light-cycle separately) and cystometric studies. Furthermore, the bladder blood flow (BBF) was measured employing the hydrogen clearance method. RESULTS: SHRs showed significant increases in micturition frequency, and decreases in BBF and single voided volume in both metabolic cages and cystometrograms compared to the Wistar group. Treatment with silodosin normalized the decreased BBF, and treatment with naftopidil increased the BBF in a dose-dependent manner in the SHR group. Although treatment with silodosin and the high dose of naftopidil significantly inhibited micturition frequency in one day, only treatment with the high dose of naftopidil significantly inhibited micturition frequency and urine production in the light-cycle compared to the non-treated SHRs. Although treatment with silodosin and the high dose of naftopidil significantly increased single voided volume, only treatment with silodosin significantly inhibited non-voiding contractions in the cystometrgrams. CONCLUSION: Our data suggest that both silodosin and naftopidil improve hypertension-related bladder dysfunction in the SHR, and naftopidil but not silodosin improves urinary frequency in the light-cycle due to inhibition of urine production.

Fasudil improves the endothelial dysfunction in the aorta of spontaneously hypertensive rats.

We investigated the effects of fasudil, a Rho kinase inhibitor, in the endothelial dysfunction of aortas from spontaneously hypertensive rats (SHRs). SHRs were divided in three groups; intraperitoneally (i.p.) vehicle-treated SHRs (SHR), SHRs treated with fasudil 3 mg/kg i.p. (Fas3), and SHRs treated with fasudil 10 mg/kg i.p. (Fas10). Vehicle-treated Wistar rats were used as normo-tensive control group. After a six-week-treatment, blood pressure and heart rate were measured by the tail cuff method. Afterwards animals were sacrificed and aortas were examined in vitro by organ bath studies to evaluate the contraction and relaxation ability. Rho kinase activity, myosin light chain (MLC), phosphorylated MLC (phospho-MLC), eNOS, phospho-eNOS protein expression and eNOS mRNA levels were evaluated. SHR demonstrated a significant hypercontractility and impaired relaxation compared to the control. Fasudil 10mg/kg significantly corrected the hypercontractility, restored the relaxation, and significantly decreased the mean arterial blood pressure, while no change observed in the systolic blood pressure. Rho kinase activity was significantly higher in the SHR, and was significantly inhibited by the high dose of fasudil. There was a slight up-regulation in the MLC, and phospho-MLC protein levels in the SHR. eNOS and phospho-eNOS protein levels were significantly lower in the SHR, and this abnormality was significantly normalized by fasudil treatment. No significant difference was observed in the eNOS gene expression. This study suggests that fasudil by inhibiting the Rho kinase activity normalizes the eNOS expression and phosphorylation and ameliorates the endothelial dysfunction induced by hypertension in the SHR model.

Hydroxyfasudil ameliorates penile dysfunction in the male spontaneously hypertensive rat.

Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho-Rho kinase pathway is one of the key factors. To investigate whether administration of hydroxyfasudil, a Rho kinase inhibitor could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat), twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10 mg/kg, i.p.) once a day for 6 weeks. Wistar rats and SHRs treatment with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho-Rho kinase pathway and activation of NO-eNOS pathway in the SHR.

Nicorandil ameliorates hypertension-related bladder dysfunction in the rat.

AIMS: There is increasing evidence that ischemia is one of the main etiology in overactive bladder (OAB), and that nicorandil prevents OAB. We investigated the effect of nicorandil on hypertension-related bladder dysfunction in spontaneously hypertensive rats (SHRs). METHODS: Twelve-week-old SHRs received six-weeks treatment with nicorandil (0, 3, or 10 mg/kg, i.p. every day). Wistar rats were used for normotensive controls. Six weeks after nicorandil treatment, the bladder blood flow was estimated by hydrogen clearance method, and the bladder functions were estimated by voiding behavior studies and functional studies. Tissue levels of nerve growth factor (NGF) were measured by ELISA method. Furthermore, the participation levels of K(ATP) channel pores were investigated by real-time PCR. RESULTS: SHRs showed significant increases in blood pressure, micturition frequency, tissue levels of NGF and expressions of both K(IR) 6.1 and K(IR) 6.2 mRNAs, and a significant decrease in the bladder blood flow. The carbachol-induced contractile responses were similar in all groups. Although both doses of nicorandil failed to decrease the blood pressure, nicorandil significantly decreased the micturition frequency, tissue levels of NGF and increased the bladder blood flow in a dose dependent manner. The expressions of K(IR) 6.1 and K(IR) 6.2 mRNAs were slightly up-regulated by the low dose of nicorandil, whereas the high dose of nicorandil significantly up-regulated those expressions compared to non-treated SHRs. CONCLUSIONS: These data indicate that nicorandil prevents hypertension-related bladder dysfunction in the SHR, which may be related to its effect on the increased blood flow in the bladder.

The role of K ATP channels on ischemia-reperfusion injury in the rat testis.

AIMS: To investigate the participation of K(ATP) channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis. MAIN METHODS: Eight-week-old male Sprague-Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300 µg/kg intraperitoneally), 30 min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24h period of reperfusion followed. Then, expressions of K(IR)6.1, K(IR)6.2, caspase-3, PARP, Fas, FasL, and K(IR)6.1 and K(IR)6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining. KEY FINDINGS: Expressions of K(IR)6.1 protein and mRNA were more than 10-fold of those of K(IR)6.2 protein and mRNA in the testis. IR significantly increased the expressions of K(IR)6.1 protein and mRNA as well as K(IR)6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of K(IR)6.2 expressions between any of the groups. SIGNIFICANCE: Treatment with cromakalim has a protective effect against IR-induced testicular damage via activating K(ATP) channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR.

Pharmacological preconditioning of ATP-sensitive potassium channel openers on acute urinary retention-induced bladder dysfunction in the rat.

UNLABELLED: What's known on the subject? and What does the study add? Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both K(IR)6.1 and K(IR)6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder. OBJECTIVE: To investigate whether ATP-sensitive potassium (K(ATP)) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg). AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min. After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of K(ATP) channel pores were investigated using ELISA and real-time PCR methods, respectively. The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium. RESULTS: The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group. Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations. The expressions of K(IR)6.1 and K(IR)6.2 mRNAs were significantly up-regulated by the induction of AURC. Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of K(IR)6.1 and K(IR)6.2 mRNAs in the bladder compared with the AUR group. CONCLUSION: Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of K(ATP) channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

Effect of silodosin on detrusor overactivity in the male spontaneously hypertensive rat.

UNLABELLED: Recently, several studies have suggested that detrusor overactivity (DO) is the result of bladder ischaemia. Hypertension could affect pelvic arterial blood flow, resulting in loss of smooth muscle in the bladder with resultant loss of bladder compliance. Spontaneously hypertensive rats are considered a valuable tool for exploring the pathogenesis of DO. Some reports indicate that α(1) adrenoceptor antagonists improve chronic ischaemia of the lower urinary tract in patients with LUTS, with concomitant improvement of their symptoms as well as improvement of DO through an increased bladder blood flow (BBF) in the rat with bladder outlet obstruction. However, the mechanism of improvement of silodosin on storage or irritative symptoms is not well investigated and is still unclear. Silodosin prevents hypertension-related DO in the SHR via several possible mechanisms. One possible mechanism of the efficacy of silodosin to the DO includes the improvement of the BBF. OBJECTIVE: To investigate the effect of the α(1A) selective adrenoceptor antagonist, silodosin, on hypertension-related detrusor overactivity (DO) and its possible mechanism in spontaneously hypertensive rats (SHRs). MATERIALS AND METHODS: Twelve-week-old male SHRs received treatment with silodosin (100 µg/kg perorally) once daily for 6 weeks; vehicle-treated Wistar rats and vehicle-treated SHRs were used for our study. Six weeks after silodosin treatment, voiding functions were estimated by voiding behaviour and cystometric studies in all groups. The bladder blood flow was measured by the hydrogen clearance method, and tissue levels of nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the expressions of α1 adrenoceptor subtype mRNAs in the bladder were investigated by real-time PCR method. RESULTS: The SHRs showed significant increases in blood pressure, micturition frequency and tissue levels of NGF and CGRP in the bladder. Moreover, the SHRs showed significant decreases in bladder blood flow and single voided volume estimated by both voiding behaviour and cystometric studies compared with those in the Wistar rats. Six weeks of treatment with silodosin significantly ameliorated hypertension-related alterations of these variables with concomitant small changes of blood pressure. The expression levels of α1 adrenoceptor subtype mRNAs in the bladder were similar in all the groups and the rank order was α1A = α1D > α1B in all groups. However, there were no significant differences in the expressions of α(1A) adrenoceptor mRNAs between any groups. CONCLUSION: The data in the present study suggest that silodosin normalizes hypertension-related DO in SHRs, which could be related to its effect on the increased blood flow in the bladder.

Rhos and Rho kinases in the rat prostate: their possible functional roles and distributions.

As there is increasing evidence that Rho-Rho kinase (ROCK) pathway plays an important role in the proliferation and contraction in many tissues, we investigated the contractile role of a ROCK inhibitor, fasudil, and the distribution of RhoA, RhoB, RhoC, ROCK1, and ROCK2 in the rat prostate. Twelve-week-old Sprague-Dawley rat prostate was used in this study. Rat prostatic contractile responses induced by carbachol and norepinephrine were investigated in organ bath studies without or with 10(-7), 10(-6), and 10(-5) M of a non-selective ROCK inhibitor, fasudil. Immunoblot analysis and immunohistochemical staining were performed to investigate the participation levels of RhoA, RhoB, RhoC, ROCK1, and ROCK2. The E(max) values induced by carbachol and norepinephrine were similar in the rat prostate. Fasudil significantly inhibited carbachol- or norepinephrine-induced prostatic contractions in a dose-dependent manner. Fasudil 10(-5) M reduced the initial prostatic contraction (without fasudil) to 56.7 ± 5.9% for carbachol and to 45.7 ± 12.3% for norepinephrine. Amounts of RhoA, RhoB, RhoC, ROCK1, and ROCK2 were detected by immunoblot analysis in the prostate. Immunohistochemical study revealed that RhoA, RhoB, RhoC, ROCK1, and ROCK2 were all positive in the prostatic smooth muscle, while there were some differences of distributions of Immunoreactivities between these enzymes in the prostatic glandula. Our data indicated that rat prostate contains RhoA, RhoB, RhoC, ROCK1, and ROCK2, which play an important role in the autonomic nerve-mediated contractile responses in the prostate.

Protective effect of taurine on diabetic rat endothelial dysfunction.

As increasing evidence suggest that oxidative stress plays an important role in the developing angiopathy in diabetes, we studied the effects of taurine, a free radical scavenger, on diabetes induced angiopathy in the rat aorta. Six-week-old male Wistar rats were randomly divided into three groups; control group (Cont), diabetes group (DM) and diabetes group treated with taurine for four weeks, 500 mg/kg/day, intraperitoneally (i.p.) (DM+T). Diabetes was induced by streptozotocin (50 mg/kg i.p.). Four weeks after the induction of diabetes, serum glucose and malondialdehyde concentrations were measured. Additionally, organ bath studies and real-time PCR on muscarinic M(3) receptor and eNOS were performed. Although taurine treatment failed to decrease serum glucose levels, the increased serum malondialdehyde levels in diabetic rats were significantly decreased after taurine treatment. Norepinephrine-induced hyper-contractility as well as acetylcholine-induced, endothelium-dependent hypo-relaxation in diabetes were significantly prevented after taurine treatment. The differences in the expressions of muscarinic M(3) receptor mRNAs were statistically non-significant between groups. Moreover, diabetes-induced up-regulation of eNOS mRNAs was slightly prevented after taurine treatment. These data suggest that taurine acts beneficially against the diabetes-induced vascular dysfunction. Its potential action as a radical scavenger ameliorates the vascular disorders in diabetes.

Edaravone ameliorates diabetes-induced dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the rat.

INTRODUCTION: Diabetes mellitus (DM) represents a major risk factor for erectile dysfunction (ED). Although the etiology of diabetes-induced ED is multifactorial and still unknown, reactive oxygen species are thought to be one of the key factors. AIM: The aim of this article is to investigate whether administration of edaravone, a free radical scavenger, could prevent type 1 diabetes-induced dysfunction of nitric oxide (NO)-induced relaxation in corpus cavernosum smooth muscle in the rat. METHODS: Six-week-old male Wistar rats were randomly divided into three groups. One group was treated with citrate-phosphate buffer plus normal saline (group Cont), whereas in the other two groups, diabetes was induced by streptozotocin (50 mg/kg intraperitoneally [i.p.]). Subsequently, the diabetic rats were treated for 4 weeks either with edaravone (10 mg/kg/day, i.p.; group DM + E) or with normal saline (group DM). MAIN OUTCOME MEASURES: Serum glucose and malondialdehyde levels as well as penile cyclic guanosine monophosphate (cGMP) concentrations were determined, and penile function was estimated by organ bath studies with norepinephrine-mediated contractions and acetylcholine-mediated relaxations. The participation mRNA levels of muscarinic M(3) receptors, neuronal nitrous oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), and participation protein levels of nNOS, eNOS, phosphorylated nNOS, and phosphorylated eNOS were investigated by quantitative real-time polymerase chain reaction (PCR) and immunoblot analysis, respectively. RESULTS: Treatment with edaravone prevented partially but significantly the decreased body and penile weight induced by diabetes. Treatment with edaravone significantly improved the increased diabetes-induced malondialdehyde levels, the decreased penile cGMP concentrations, the increased diabetes-induced norepinephrine-mediated contractions, and the decreased acetylcholine-mediated relaxation. Although there were no significant differences in expression levels of mRNAs in nNOS, diabetes-induced upregulation of muscarinic M(3) receptor and iNOS mRNAs as well as diabetes-induced downregulations of eNOS, phosphorylated nNOS, and phosphorylated eNOS were significantly prevented by edaravone. CONCLUSIONS: Edaravone decreases the oxidative insult in the penile corpus cavernosum by ameliorating the NO-NOS system and thus preventing partially the developing ED in DM in the rat.

Nicorandil ameliorates ischaemia-reperfusion injury in the rat kidney.

BACKGROUND AND PURPOSE: Nicorandil, an ATP-sensitive potassium (K(ATP) ) channel opener and nitric oxide donor, is used in the treatment of angina and acute heart failure. Here we investigated the effects of two K(ATP) channel openers, nicorandil and cromakalim on ischaemia reperfusion (I-R) injury in the kidney. EXPERIMENTAL APPROACH: Right nephrectomy was performed in 8-week-old male Sprague-Dawley rats and they were then divided into six groups: control group; I-R, including 30 min of left renal ischaemia followed by 24 h of reperfusion; I-R groups plus nicorandil 3 or 10 mg·kg⁻¹ i.p.; and I-R groups plus cromakalim 100 or 300 µg·kg⁻¹ i.p. After reperfusion, renal function was estimated by serum creatinine (SCr), urinary albumin:creatinine ratio (ACR) and urinary ß2-microglobulin (ß2-MG). Levels of K(ATP) channel subtypes were investigated by Western blot. Kidney sections were stained for 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine. KEY RESULTS: Renal I-R induced significant increases in SCr, ACR and ß2-MG levels compared with the control animals. Treatment with K(ATP) channel openers reduced urinary ß2-MG levels, raised by I-R. Both K(IR) 6.1 and K(IR) 6.2 channels were expressed. Expression of K(IR) 6.2 channels in the I-R group was lower than in the control group, which was restored to normal by treatment with K(ATP) channel openers. Histologically, severe acute tubular damage was observed in the I-R kidney and this damage was ameliorated by K(ATP) channel openers, dose-dependently. CONCLUSIONS AND IMPLICATIONS: ATP-sensitive potassium channel openers protected against proximal tubule damage after I-R injury. Nicorandil could represent a powerful additional component in the treatment of patients undergoing partial nephrectomy or renal transplantation.

Protective effect of sivelestat, a neutrophil elastase inhibitor, on ipsilateral and contralateral testes after unilateral testicular ischaemia-reperfusion injury in rats.

OBJECTIVE: To investigate the effect of a neutrophil elastase inhibitor, sivelestat sodium hydrate, on testicular ischaemia-reperfusion (IR)-injury. MATERIAL AND METHODS: Eight-week-old male Sprague-Dawley rats were divided into four groups: sham-operated control rats; IR rats (group IR); and IR rats that received intra-abdominal administration of 15 mg/kg or 60 mg/kg sivelestat (group IR15 and group IR60, respectively). Right testicular vessels were clamped for 90 min in groups IR, IR15 and IR60. Sivelestat had been administered 45 min after the induction of the ischaemia in groups IR15 and IR60. In subpopulations of IR, IR15 and IR60 rats, reperfusion was performed after ischaemia for 2 h (groups IR-A, IR15-A and IR60-A, respectively) or 48 h (groups IR-B, IR15-B and IR60-B, respectively). At the end of the reperfusion period, blood samples were aspirated from both spermatic veins of each rat and testosterone was evaluated. Then both testes from all rats were collected and tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), and heat-shock protein-70(HSP-70) were evaluated. Testicular tissue samples were also processed for histological evaluation and TUNEL staining. RESULTS: MDA, MPO and HSP-70 levels in the ischemic testis were significantly higher in the IR group compared with the control group. MDA and HSP-70 in the contralateral testis were significantly higher in the IR group compared with the control group. Bilateral testosterone levels were lower in all rat groups in comparison with the control group. Bilateral testicular samples in group IR showed extensive histopathologic degenerative alterations and increased percentage of apoptotic cells. Sivelestat treatment lowered the MDA concentration and the percentage of apoptotic cells bilaterally and ameliorated the testicular histological pattern bilaterally. CONCLUSIONS: Unilateral testicular ischaemia causes significant contralateral testicular damage. Sivelestat may be a novel adjunct tool for reducing oxidative stress and partially preventing bilateral testicular damage.

Real-Time Monitoring of Blood Flow and Intravesical Pressure in the Rat Bladder.

OBJECTIVES: Because there is little information available about blood flow in the voiding cycle of the bladder, we performed a study in which we simultaneously monitored blood flow and intravesical pressure during the micturition cycle in a rat model. METHODS: Approximately 300 g male Wistar rats were used in this study. Cystometric studies were performed according to our previous report, and simultaneously blood flow was monitored. RESULTS: Before the micturition reflex occurred, a significant increase in bladder blood flow was observed, and this increased blood flow continued during the micturition reflex. Under the maximum contraction pressure, blood flow rapidly decreased (within 10% compared to the max level). This low level of blood flow continued for more than half a minute. CONCLUSION: Our data indicated that the blood flow in the bladder was dynamically changed during voiding. This technique may represent a strong tool to investigate bladder function under drug administrations and/or pathophysiological conditions.

Effects of cyclohexenonic long-chain fatty alcohol in type 2 diabetic rat nephropathy.

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (CHLFA) on the alterations of type 2 diabetes-induced nephropathy. Forty-week-old male Goto-Kakizaki (GK) and Wistar rats were divided into four groups of 6 to 8 animals. Group A consisted of eight Wistar rats and served as an age-matched control group. Group B (7 GK rats) received no treatment and served as a diabetic group. Group C (6 GK rats) was treated daily with low-dose CHLFA (2 mg/ kg/body weight, subcutaneously) for 30 weeks, and Group D (6 GK rats) with high-dose CHLFA (8 mg/kg/body weight) for 30 weeks. At the end of the treatment period, urinary protein excretion, blood chemistry, renal histological, and immunohistological analyses were conducted. Although CHLFA administration did not influence serum glucose or insulin levels, it reversed diabetes-induced increases in urinary protein excretion and serum creatinine. Light microscopically, CHLFA treatment ameliorated the otherwise elevated glomerular sclerotic scores in the diabetic group.Immunohistochemically, increased expression of desmin and decreased expression of rat endothelial cell antigen-1 in the group with untreated diabetes both showed a reversal to control levels in the high-dose CHLFA treatment group. In conclusion, CHLFA may ameliorate type 2 diabetes-induced nephropathy.

Muscarinic receptors and their mRNAs in type 2 Goto-Kakizaki diabetic rat prostate.

BACKGROUND: As increasing evidence is pointing towards the relationship between diabetes and benign prostatic hyperplasia/lower urinary tract symptoms, we investigated the pharmacological properties and gene expressions of the muscarinic receptors in type 2 diabetes rat prostate. METHODS: Twelve- and 70-week-old male Goto-Kakizaki (GK) rats and age-matched male Wistar rats were used in this study. The densities of muscarinic receptors (B(max) values) were determined by saturation studies with [(3)H]NMS ([N-methyl-(3)H] scopolamine methyl chloride) in the prostatic membrane particulates. The participation levels of M(1), M(2), and M(3) receptor protein and mRNA levels in the prostate were investigated by immunoblot analysis and real-time polymerase chain reaction (PCR), respectively. RESULTS: The B(max) values in 12-week-old Wistar and GK, and in 70-week-old Wistar and GK rat prostates were 36.0 +/- 2.8, 49.4 +/- 11.4, 22.0 +/- 2.2, and 47.0 +/- 4.1 fmol/mg protein, respectively. However, there were no significant differences in the affinity constants between any groups. Immunoblot analysis showed the existence of significant amounts of M(1), M(2), and M(3) receptor subtypes in each rat prostate. According to real-time PCR studies the rank order of expression levels of muscarinic receptors mRNA subtypes in the prostate were M(3) > M(2) > M(1). In each receptor subtype in each group, diabetes induced up-regulation of mRNAs while the advanced age of the rats was related with down-regulation of mRNAs. CONCLUSIONS: Our data indicated that type 2 diabetes induced up-regulation and age-related down-regulation of the expressions of muscarinic receptors and their mRNAs in the rat prostate.

The role of ATP-sensitive potassium channel on acute urinary retention and subsequent catheterization in the rat.

We investigated the role of K(ATP) channel on acute urinary retention (AUR) induced bladder dysfunction. Eight-week-old female Sprague-Dawley rats were divided into seven groups: a sham-operated control group, an AUR group, and five AUR groups treated with: two different K(ATP) channel openers namely nicorandil (3 or 10mg/kg), or cromakalim (100 or 300microg/kg), or one K(ATP) channel inhibitor namely glibenclamide (5mg/kg). The drugs were administered 30min before induction of AUR. After the urethra was obstructed with a clip, AUR was induced by intravesical infusion of 2.5ml of saline via cystostomy. Following a 30min obstruction the bladder was allowed to drain with a catheter in place for 60min with real-time monitoring of intravesical pressure and blood flow. After the experimental period, the bladder function was assessed, using organ bath techniques (carbachol and 100mM KCl). AUR increased the intravesical pressure and decreased the blood flow. The subsequent catheterization decreased the intravesical pressure and increased the blood flow. AUR group reduced significantly the contractile responses to both carbachol and KCl compared with the control group. Nicorandil and cromakalim but not glibenclamide prevented the bladder dysfunction after AUR suggesting that K(ATP) channel openers may prevent the bladder dysfunction caused by AUR and subsequent catheterization.

Protective effect of edaravone, a free-radical scavenger, on ischaemia-reperfusion injury in the rat testis.

OBJECTIVE: To investigate the effect of edaravone, a radical scavenger, on ischaemia-reperfusion (I-R) injury in the testes. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were allocated to one of four groups: a no-drug group subjected to induction of 30-min of ischaemia and 60-min reperfusion; two drug groups administered edaravone at 1 or 10 mg/kg intraperitoneal and then subjected to 30-min ischaemia and 60-min reperfusion; and a sham-operated control group administered edaravone at 10 mg/kg intraperitoneal. To induce testicular I-R, the right testis was exposed outside of the body and the testicular artery was clamped with a small clip for 30 min. Blood flow and nitric oxide (NO) release were monitored in real time simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. After death the tissue levels of NO(2)-NO(3) (a marker of NO production), malondialdehyde (a marker of lipid peroxidation), 8-hydroxydeoxyguanosine (a marker of oxidative DNA damage), myeloperoxidase (a marker of neutrophil infiltration), and heat-shock protein 70 (HSP 70) and its mRNA were measured. The testicular tissue was also analysed histologically. RESULTS: Clamping the testicular artery resulted in a decrease of blood flow to 0-5% of the basal level measured before clamping. NO release was increased during clamping and gradually recovered to the basal level on removing the clip. Interestingly, the peak of NO release in rats of the no-drug group occurred at the start of reperfusion, while that in the high-dose drug group occurred several minutes later. The levels of NO(2)-NO(3), malondialdehyde, 8-hydroxydeoxyguanosine, myeloperoxidase and HSP 70 and its mRNA, and histological variables, were significantly greater in the no-drug I-R group than in the control, and these variables were ameliorated by treatment with edaravone. CONCLUSION: These results indicate that edaravone reduces the oxidative stress and prevents the testicular damage induced by I-R.