Changes in the function and expression of T-type and N-type calcium channels in the rat bladder after bladder outlet obstruction.

PURPOSE: We evaluated possible changes in the function and expression of T-type and N-type Ca(2+) channels in the bladder of rats with bladder outlet obstruction. MATERIALS AND METHODS: Female Sprague Dawley® rats were divided into a group with bladder outlet obstruction created by partial urethral ligation and a sham operated group. Six weeks postoperatively we determined the mRNA expression of T-type and N-type Ca(2+) channels in the bladder, dorsal root ganglion and spinal cord. We also cystometrically investigated expression by intravenous administration of the T-Ca blocker RQ-00311610 or the N-type Ca(2+) channel blocker ω-conotoxin GVIA. We then performed in vitro functional studies of detrusor strips using these blockers. RESULTS: mRNA expression of T-type Ca(2+) channels in the bladder detrusor and mucosa layers, and the spinal cord dorsal horn, and N-type Ca(2+) channels in the whole bladder and detrusor layer, and the spinal cord dorsal horn was greater in the obstructed group than the sham operated group. In obstructed rats bladder capacity and voided volume increased after RQ-00311610 administration but the number of nonvoiding contractions decreased after ω-conotoxin GVIA administration. Detrusor strips from obstructed rats showed weaker contractile responses to electrical field stimulation, particularly in regard to the purinergic component. ω-Conotoxin GVIA suppressed electrical field stimulation induced contractions only in the detrusor of obstructed rats, especially the cholinergic component. CONCLUSIONS: Blocking T-type Ca(2+) channels increased bladder capacity while N-type Ca(2+) channel blockade inhibited nonvoiding contractions in rats with bladder outlet obstruction. Decreased bladder efferent neurotransmission occurred after bladder outlet obstruction, predominantly in its purinergic component and detrusor contractions via cholinergic neurotransmission were activated in a compensatory manner, probably via N-type Ca(2+) channel up-regulation.

Strategies for the creation and maintenance of reconstructed arteriovenous fistulas using the forearm basilic vein.

Reconstruction of an arteriovenous fistula (AVF) after an initial failure to provide long-term patency has been desired in the era when hemodialysis patients' prognosis is improving. The forearm basilic vein AVF should be considered, before an artificial graft shunt or an AVF in the cubital region. The present study was designed to establish a strategy for the creation and maintenance of AVFs using the forearm basilic vein. This study reviewed 76 cases of reconstructed AVF including 18 cases using the basilic vein (23.7% of total cases). The following four points were considered: arm positioning of the cubital flexion position combined with the forearm supinated position; several small skin incisions with a subcutaneous tunnel; sufficient venous dilatation using Fogarty balloon catheter; and early percutaneous angioplasty introduction for immature AVF. The primary and secondary patency rates were examined. A radiobasilic AVF was created through a subcutaneous tunnel in two cases. The primary and secondary patency rates of AVF with the basilic vein were 54.7% and 76.7% respectively, whereas those of AVF with the cephalic vein were 49.3% and 71.3%. The basilic was not inferior to the cephalic vein (P-value of the log-rank test for primary and secondary patency rates were 0.927 and 0.811, respectively). Early stage percutaneous angioplasty was effective in five cases with immature AVF. The forearm basilic vein was useful in AVF reconstruction and equivalent to radiocephalic reconstruction. Careful observation and percutaneous angioplasty during the early period after the surgery were essential for long-term patency.

Reduction of prostate cancer incidence by naftopidil, an α1 -adrenoceptor antagonist and transforming growth factor-ß signaling inhibitor.

OBJECTIVES: Quinazoline-based α(1) -adrenoceptor antagonists are known to inhibit prostate tumor growth through induction of apoptosis. We investigated the effect of a naphthalene-based α(1) -adrenoceptor antagonist, naftopidil, on prostate cancer incidence, apoptosis of prostatic cell and transforming growth factor-ß signaling. METHODS: Prescription records were linked to pathological data for men who continued naftopidil (n = 766) or tamsulosin (n = 1015) for 3 months or longer between 2003 and 2010. Prostate cancer incidence was analyzed by log-rank test and the Cox proportional hazards model. Apoptosis and cell cycle arrest in human tissues were assessed by immunohistochemical detection of Bcl2 and p21, respectively. Growth inhibition and apoptosis treatment with naftopidil and tamsulosin were assessed in cancer cell lines. Interference with transforming growth factor-ß signaling was examined by western blot analysis. RESULTS: Prostate cancer incidence was significantly lower in men who received naftopidil for 3 months or longer compared with tamsulosin (P = 0.035). Multivariate analysis confirmed a decreased hazard ratio, 0.46, for naftopidil use (P = 0.013), which was more evident with longer treatment. Immunohistochemical positivity for Bcl2, a marker for resistance to apoptosis, was less frequently detected in prostate cancer cells of men who received naftopidil compared with tamsulosin (P < 0.05). Naftopidil inhibited cancer cell growth, induced apoptosis and blocked Smad2 phosphorylation activated by transforming growth factor-ß in cell lines, with a half maximal inhibitory concentration of 1.1 µmol/L. CONCLUSIONS: Naftopidil seems to reduce prostate cancer incidence, possibly by inducing apoptosis, preferentially in cancer cells, and blocking transforming growth factor-ß signaling.

Angiopoietin-1 mediates adipose tissue-derived stem cell-induced inhibition of neointimal formation in rat femoral artery.

BACKGROUND: Adipose tissue-derived stem cells (ASC) produce a variety of cytokines that potentially mediate the proangiogenic and antiapoptotic effects of the ASC. We examined whether ASC produced angiopoietin-1 (Ang1) and whether Ang1 functionally mediated ASC-induced suppression of neointimal formation. METHODS AND RESULTS: Ang1 production was measured by enzyme-linked immunosorbent assay. Production of endogenous Ang1 by ASC was inhibited with small interfering RNA (siRNA) for Ang1. Overproduction of Ang1 was achieved with an adenovirus that expresses Ang1 (AdAng1). ASC expressing Ang1 siRNA, or AdAng1 were administered around the femoral artery after wire injury, and immunohistochemical analysis was performed to examine their effects on neointimal formation. ASC produced Ang1 in a time-dependent manner, especially when cultured in medium containing growth factors for vascular endothelial cells. When ASC were treated with Ang1 siRNA, the inhibitory effect of ASC on neointimal formation was significantly reduced. Knockdown of Ang1 significantly increased macrophage infiltration in the neointima, and significantly decreased endothelial regeneration. In contrast, forced expression of Ang1 using AdAng1 significantly suppressed neointimal formation and macrophage infiltration, and stimulated reendothelialization. CONCLUSIONS: Ang1 was implicated in ASC-induced suppression of neointimal formation. The results also suggested that Ang1 inhibited neointimal formation via stimulation of reendothelialization and suppression of macrophage infiltration in the neointima.

Adrenomedullin mediates adipose tissue-derived stem cell-induced restoration of erectile function in diabetic rats.

INTRODUCTION: Erectile dysfunction (ED) is a major health problem. It is known that diabetic patients are more refractory to common treatments for ED. AIM: To explore the better treatment for ED, we examined the effects of adipose-derived stem cells (ASC) on ED using a diabetic rat model. We also analyzed the cytokines produced by ASC and implicated in ASC-induced restoration of erectile function. METHODS: Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. ASC or adenoviruses were injected into the penis 6 weeks after STZ administration. Erectile function, penile histology and protein expression were analyzed 4 weeks after the injection of ASC or adenoviruses. MAIN OUTCOME MEASURES: Intracavernous pressure and mean arterial pressure were measured to evaluate erectile function. The morphology of the penis was analyzed by Elastica van Gieson stain and immunohistochemistry. The expression of proteins specific for vascular endothelial cells (VEC) was assessed by Western blot analysis. RESULTS: ASC restored erectile function especially when they were cultured in medium containing growth factors for VEC. This restoration was associated with improvement in the histology of the cavernous body, and increased expression of VEC markers such as VE-cadherin and endothelial nitric oxide synthase (eNOS). When the expression of adrenomedullin (AM), a vasoactive peptide originally isolated from human pheochromocytoma tissue, was knocked down, the effect of ASC on ED was significantly diminished. Knockdown of AM was associated with decreased expressions of VE-cadherin and eNOS. Furthermore, overexpression of AM induced by adenovirus infection significantly improved erectile function in these diabetic rats. Overexpression of AM was associated with increased expressions of VE-cadherin and eNOS. CONCLUSIONS: These results suggested that ASC have the potentials to restore erectile function and that AM produced by ASC plays a major role in the restoration of erectile function.

Renoprotective effect of erythropoietin in ischemia/reperfusion injury: possible roles of the Akt/endothelial nitric oxide synthase-dependent pathway.

OBJECTIVES: It has been reported that erythropoietin protects the kidneys from ischemia/reperfusion injury. In the present study, we examined the role of Akt and endothelial nitric oxide synthase in the protective effect of erythropoietin on ischemia/reperfusion injury of the kidney. METHODS: Erythropoietin was injected in the peritoneal space of ICR mice after ischemia/reperfusion injury and its effect was assessed by measuring blood urea nitrogen and creatinine, and by histological analysis. Phosphorylation of Akt and endothelial nitric oxide synthase was examined by western blot analysis. Endothelial nitric oxide synthase gene null mice were also used to examine the role of endothelial nitric oxide synthase in the renoprotective effect of erythropoietin. RESULTS: Erythropoietin administration significantly inhibited the increase in blood urea nitrogen and creatinine after ischemia/reperfusion injury compared with control mice. Accordingly, erythropoietin administration significantly ameliorated the histological damages, including apoptotic cell death. Erythropoietin significantly stimulated phosphorylation of Akt and endothelial nitric oxide synthase in the kidneys. When endothelial nitric oxide synthase gene null mice were subjected to ischemia/reperfusion injury, erythropoietin did not significantly suppress the increase in blood urea nitrogen or creatinine. CONCLUSIONS: Erythropoietin seems to activate the Akt/endothelial nitric oxide synthase-dependent pathway in the kidneys. This pathway might be implicated in the renoprotective effect of erythropoietin in the ischemia/reperfusion injury model.

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.