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Aims: The nuclear hormone receptor estrogen-related receptor α (ERRα) regulates the activation of mitochondrial genes in various human tissues, but its role in the adrenal gland and its disorders has not been defined. Therefore, we examined ERRα expression in both normal adrenal cortex (NAC) and adrenocortical tumor (ACT) in order to study the possible correlation of ERRα with adrenal development and tumor development., Methods: Human adrenal specimens (non-pathological fetal n=7; non-pathological post-birth n=40; aldosterone producing adenoma (APA) n=11; cortisol producing adenoma (CPA) n=11; adrenocortical carcinoma (ACC) n=8) were immunohistochemically examined in this study. NAC (n=13) and ACT (n=28) frozen tissue specimens were also available for studying ERRα mRNA levels., Key Findings: In fetal NAC tissues, ERRα labeling index (LI) in fetal zone (FZ) was significantly higher that that in neocortex (NC), and the differences among age groups for overall mean LI was statistically significant when analyzed according to individual cortical layers. ERRα LI was also significantly higher in ACC than in other types of ACT. ERRα mRNA was detected in NAC and all types of ACT., Significance: Results of our present study suggest a possible role of ERRα in adrenal development and ACC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Context: Although steroid hormones produced by the adrenal gland play critical roles in human physiology, a detailed quantitative analysis of the steroid products has not been reported. The current study uses a single methodology (liquid chromatography-tandem mass spectrometry, LC-MS/MS) to quantify ten corticosteroids in adrenal vein (AV) samples pre- and post-adrenocorticotropic hormone (ACTH) stimulation., Design/methods: Three men and six women with a diagnosis of an adrenal aldosterone-producing adenoma (APA) were included in the study. Serum was collected from the iliac vein (IV) and the AV contralateral to the diseased adrenal. Samples were collected, before and after administration of ACTH. LC-MS/MS was then used to quantify serum concentrations of unconjugated corticosteroids and their precursors., Results: Prior to ACTH stimulation, the four most abundant steroids in AV were cortisol (90%), cortisone (4%), corticosterone (3%) and 11-deoxycortisol (0.8%). Post-ACTH administration, cortisol remained the major adrenal product (79%); however, corticosterone became the second most abundantly produced adrenal steroid (11%) followed by pregnenolone (2.5%) and 17α-hydroxypregnenolone (2%). ACTH significantly increased the absolute adrenal output of all ten corticosteroids measured (P < 0.05). The four largest post-ACTH increases were pregnenolone (300-fold), progesterone (199-fold), 17α-hydroxypregnenolone (187-fold) and deoxycorticosterone (82-fold)., Conclusion: Using LC-MS/MS, we successfully measured 10 corticosteroids in peripheral and AV serum samples under pre- and post-ACTH stimulation. This study demonstrates the primary adrenal steroid products and their response to ACTH., (© 2012 Blackwell Publishing Ltd.)

Context: In patients who seek surgical cure of primary aldosteronism (PA), The Endocrine Society Guidelines recommend the use of adrenal vein sampling (AVS), which is invasive, technically challenging, difficult to interpret, and commonly held to be risky., Objective: The aim of this study was to determine the complication rate of AVS and the ways in which it is performed and interpreted at major referral centers., Design and Settings: The Adrenal Vein Sampling International Study is an observational, retrospective, multicenter study conducted at major referral centers for endocrine hypertension worldwide., Participants: Eligible centers were identified from those that had published on PA and/or AVS in the last decade., Main Outcome Measure: The protocols, interpretation, and costs of AVS were measured, as well as the rate of adrenal vein rupture and the rate of use of AVS., Results: Twenty of 24 eligible centers from Asia, Australia, North America, and Europe participated and provided information on 2604 AVS studies over a 6-yr period. The percentage of PA patients systematically submitted to AVS was 77% (median; 19-100%, range). Thirteen of the 20 centers used sequential catheterization, and seven used bilaterally simultaneous catheterization; cosyntropin stimulation was used in 11 centers. The overall rate of adrenal vein rupture was 0.61%. It correlated directly with the number of AVS performed at a particular center (P = 0.002) and inversely with the number of AVS performed by each radiologist (P = 0.007)., Conclusions: Despite carrying a minimal risk of adrenal vein rupture and at variance with the guidelines, AVS is not used systematically at major referral centers worldwide. These findings represent an argument for defining guidelines for this clinically important but technically demanding procedure.

Adrenal vein sampling (AVS) is fundamental for subtype diagnosis in patients with primary aldosteronism. AVS protocols vary between centers, especially for diagnostic indices and for use of adrenocorticotropic hormone (ACTH) stimulation. We investigated the role of both continuous ACTH infusion and bolus on the performance and interpretation of AVS in a sample of 76 patients with confirmed primary aldosteronism. In 36 primary aldosteronism patients, AVS was performed both under basal conditions and after continuous ACTH infusion, and in 40 primary aldosteronism patients, AVS was performed both under basal conditions and after ACTH IV bolus. Both ACTH protocols determined an increase in the rate of successful cannulation of the adrenal veins. Both ACTH infusion and bolus determined a significant increase in selectivity index for the right adrenal vein and ACTH bolus for the left adrenal vein. Lateralization index was not significantly different after continuous ACTH infusion and IV bolus. In 88% and 78% of the patients, the diagnosis obtained was the same before and after ACTH infusion and IV bolus, respectively. However, the reproducibility of the diagnosis was reduced using less stringent criteria for successful cannulation of the adrenal veins. This study shows that ACTH use during AVS may be of help for centers with lower success rates, because a successful adrenal cannulation is more easily obtained with this protocol; moreover, this technique performs at least as well as the unstimulated strategy and in some cases may be even better. Stringent criteria for cannulation should be used to have a high consistency of the diagnosis.

Objective: We retrospectively examined the outcome of patients who underwent laparoscopic adrenalectomy for Cushing's/subclinical Cushing's syndrome in our single institute., Patients and Methods: Between 1994 and 2008, a total of 114 patients (29 males and 85 females, median age 54 years) with adrenal Cushing's/subclinical Cushing's syndrome were studied. We compared the outcome of patients who underwent laparoscopic adrenalectomy between intraperitoneal and retroperitoneal approaches. Surgical complications were graded according to the Clavien grading system. We also examined the long-term results of subclinical Cushing's syndrome after laparoscopic adrenalectomy., Results: Laparoscopic surgical outcome did not differ significantly between patients with Cushing's syndrome and those with subclinical Cushing's syndrome. Patients who underwent laparoscopic intraperitoneal adrenalectomy had longer operative time than those who received retroperitoneal adrenalectomy (188.2 min vs. 160.9 min). However, operative blood loss and surgical complications were similar between both approaches. There were no complications of Clavien grade III or higher in either intraperitoneal or retroperitoneal approach. We confirmed the improvement of hypertension and glucose tolerance in patients with subclinical Cushing's syndrome after laparoscopic adrenalectomy., Conclusions: Laparoscopic adrenalectomy for adrenal Cushing's/subclinical Cushing's syndrome is safe and feasible in either intraperitoneal or retroperitoneal approach. The use of the Clavien grading system for reporting complications in the laparoscopic adrenalectomy is encouraged for a valuable quality assessment.

Objective: To report a single-center experience with laparoscopic simultaneous bilateral adrenalectomy (LSBA) and to evaluate its safety, surgical outcomes, and potential indications of the procedure., Methods: A total of 21 patients underwent LSBA between 2000 and 2010 at our institution. Four patients had bilateral Cushing's syndrome (CS), two had bilateral pheochromocytoma, and one had a bilateral metastatic tumor. Eleven patients had unilateral or bilateral aldosterone-producing adenoma (APA), associated with CS or subclinical CS. Three patients had unilateral APA with contralateral non-functioning adenoma. Partial adrenalectomy was performed first by using with four ports. After the excision of one gland, the contralateral gland was removed after repositioning of the patient., Results: LSBA was completed in all 21 patients without major complications. Mean operative time was 329.7 min and the estimated blood loss was 94.1 mL. Mean tumor size was 21.8 mm. Of the 16 patients receiving an adrenal-sparing procedure, nine of 11 discontinued glucocorticoid replacement after 2 years. The remaining five patients receiving bilateral total adrenalectomy required 0.5-0.75 mg of dexamethasone permanently. No open conversions, no deaths or no adrenal insufficiencies were encountered., Conclusions:   LSBA represents a safe and viable treatment option for selected patients with bilateral adrenal disease., (© 2011 The Japanese Urological Association.)

Context: 18-Oxocortisol (18-oxoF) is a derivative of cortisol (F) that is produced by aldosterone synthase (CYP11B2). The potential for this steroid as a biomarker for differentiating patients with aldosterone-producing adenoma (APA) from those with idiopathic hyperaldosteronism (IHA) has not been examined., Objectives: We measured 18-oxoF, aldosterone, and F in plasma from adrenal vein sampling (AVS) of patients with primary aldosteronism. We compared 18-oxoF levels and 18-oxoF/F ratios for their potential to differentiate APA from IHA., Design, Setting, and Subjects: This study measured 18-oxoF, F, and aldosterone in AVS obtained from patients with unilateral APA (14 cases) or bilateral IHA (seven cases, 14 samples total) using liquid chromatography-tandem mass spectrometry and RIA analyses., Results: The levels of 18-oxoF and the ratios of 18-oxoF/F, before and after ACTH stimulation, were significantly higher in blood-draining APA than in those from the contralateral adrenal glands and from adrenal glands with IHA., Conclusions: The 18-oxoF levels and ratios of 18-oxoF/F in AVS samples can be a clinically useful biomarker for differentiating APA from IHA and for determining the localization or lateralization of APA in patients with primary aldosteronism.

Objectives: To assess the clinical effect of the universal use of adrenal venous sampling and to investigate the characteristics of patients with primary aldosteronism undergoing laparoscopic adrenalectomy at a single tertiary care center., Methods: After the screening examination, confirmatory test, and computed tomography (CT) scans were completed, all patients with biochemically diagnosed hyperaldosteronism underwent adrenal venous sampling to differentiate unilateral disease from bilateral idiopathic hyperaldosteronism. A total of 174 consecutive patients with unilateral aldosterone excess underwent unilateral laparoscopic adrenalectomy., Results: The surgically treated cohort was divided into 3 groups according to the CT findings. A total of 129 patients (74.1%) had findings associated with CT-positive macroadenoma (type 1A) and 42 (24.1%) with CT-negative microadenoma (type 2A). Only 3 patients (1.8%) had adrenocortical hyperplasia (type 3). The aldosterone level was normalized in all but 2 patients (98.9%), and the number of antihypertensive agents was significantly reduced within 1 month after adrenalectomy. Of the 174 patients, 155 (89.1%) showed resolution or improvement of hypertension., Conclusions: The routine use of adrenal venous sampling could adequately detect lateralization in patients with unilateral aldosterone excess, which led to satisfactory short-term outcomes after surgery. The results of the present study showed that nearly one fourth of patients with the unilateral form had a CT-negative aldosterone-producing microadenoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Primary aldosteronism (PA) is the most common form of endocrine hypertension affecting ∼8-10% of hypertensive subjects. Aldosterone production in PA occurs under low-renin conditions, and the mechanisms that maintain the production of aldosterone in PA remain unknown. Objective This study was designed to compare the transcript profiles between aldosterone-producing adenoma (APA) and their adjacent adrenal gland (AAG) from the same adrenal., Methods: Total RNA was extracted from ten APA and ten AAG; and subsequently analyzed by microarray and real-time quantitative RT-PCR (qPCR). The microarray data were paired for each APA-AAG, and analyzed by GeneSpring GX 11 with paired t-test and fold change calculations for each transcript. Changes identified by microarray analysis were confirmed by qPCR., Results: Microarray analysis indicated that 14 genes had significantly up-regulated expression in APA compared to AAG. Among the elevated genes were aldosterone synthase (CYP11B2) as well as novel transcription factors, calmodulin-binding proteins, and other genes that have not been previously studied in APA. Selective analysis of 11 steroidogenic enzymes using microarray demonstrated that only CYP11B2 showed a significantly higher transcript level in APA compared to AAG (P<0.001). In contrast, AKR1C3 (17β-hydroxysteroid dehydrogenase type 5), CYP17 (17α-hydroxylase/17,20 lyase), and CYB5 (cytochrome b5) showed significantly lower transcript level in APA (P<0.05)., Conclusion: The transcriptome analysis of APA compared with AAG showed several novel genes that are associated with APA phenotype. This gene list provides new candidates for the elucidation of the molecular mechanisms leading to PA.

Primary aldosteronism (PA) is the most common cause of secondary hypertension, accounting for 10% of all hypertension. Far from being benign, hypertension due to PA is associated with high cardiovascular morbidity and mortality. However, PA is still underdiagnosed in general practice. Recent reports strongly recommend that identifying patients with PA is cost-beneficial based on improved cardiovascular outcomes afforded by specific surgical and medical treatment. This review provides an update of PA including controversial aspects of diagnosis and treatment.

Adrenomedullin 2/intermedin (AM2/IMD) is a new member of the calcitonin/calcitonin gene-related peptide (CGRP) family. CGRP, adrenomedullin (AM), and AM2/IMD share the receptor system consisting of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP). The CRLR/RAMP2 or CRLR/RAMP3 complex forms the AM receptor, whereas the CRLR/RAMP1 forms the CGRP receptor. AM2/IMD binds non-selectively to all three CRLR/RAMP complexes. AM2/IMD has various actions, such as a potent vasodilator action and a protective action against oxidative stress, like AM and CGRP. When administered intracerebroventricularly, AM2/IMD stimulates the sympathetic nervous system and increases blood pressure. In human hypothalamus, AM2/IMD is expressed in the paraventricular and supraoptic nuclei and colocalized with arginine vasopressin. Anterior pituitary cells were diffusely immunostained for AM2/IMD. AM2/IMD stimulates the release of ACTH, prolactin, and oxytocin, but suppresses GH release. Some of these pituitary actions of AM2/IMD have been supposed to be mediated by an unidentified unique receptor for AM2/IMD. In the adrenal gland, immunoreactive (IR)-AM2/IMD and IR-AM were detected in the medulla, while the degree of IR-AM2/IMD and IR-AM in the cortex was relatively weak or undetectable. Furthermore, AM2/IMD and AM were expressed in adrenocortical tumors, such as aldosterone-secreting adenomas, and pheochromocytomas. CRLR and RAMPs are expressed in the hypothalamus, pituitaries, adrenal glands, and adrenal tumors. Thus, AM2/IMD is expressed in every endocrine organ of the hypothalamo-pituitary-adrenal axis together with its receptor. AM2/IMD may act as a neurotransmitter or modulator in the brain and as a paracrine/autocrine regulator in the hypothalamo-pituitary-adrenal axis.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor for the antidiabetic agent thiazolidinedione, which exerts various physiological activities, independent of lowering blood glucose. However, the role of PPARγ in aldosterone production has not been clarified. The objective of this study was to investigate the effect of PPARγ on aldosterone synthase gene (CYP11B2) expression and aldosterone production. Localization of PPARγ expression in normal adrenal cortex was determined by immunohistochemistry. Aldosterone production and CYP11B2 expression levels were determined using human adrenocortical carcinoma H295R cells. Pioglitazone suppressed angiotensin II-induced aldosterone secretion and CYP11B2 expression. PPARγ was expressed in zona glomerulosa in human normal adrenal gland. PPARγ overexpression enhanced pioglitazone-mediated CYP11B2 transrepression. The pioglitazone-mediated suppression of aldosterone secretion and CYP11B2 expression were canceled by PPARγ L466A/E469A mutant. Pioglitazone also suppressed potassium-mediated CYP11B2 induction, but not N6-2'-O-dibutyladenosine-3',5'-cyclic monophosphate stimulation. Rosiglitazone and GW1929 also suppressed CYP11B2 transactivation. Mutation analysis revealed that the Ad1/CRE element in CYP11B2 5'-flanking region was responsible for the pioglitazone-mediated transrepression. Pioglitazone suppressed ionomycin and a truncated constitutively active form Ca(2+)/calmodulin-dependent kinase I (CaMKI)-mediated CYP11B2 transcriptional activation. A CaMK inhibitor KN-93 attenuated pioglitazone-mediated CYP11B2 transrepression. PPARγ suppresses CYP11B2 expression and aldosterone secretion.

The Japan Endocrine Society (JES) attempted to develop guidelines for the diagnosis and treatment of primary aldosteronism (PA). The Task Force Committee (TFC) was composed of a chair, selected by the JES, and additional experts. Systematic reviews of available evidence for Japanese patients were used to recommend the key treatment and prevention. We have evaluated the methods of screening, confirmatory tests and imaging, plus adrenal vein sampling (AVS). Consensus was guided by systematic review of evidence and discussion during each annual meeting of the JES, plus its related meetings, and by e-mail communication. The drafts prepared by TFC were reviewed successively by the members of Research on Intractable Diseases provided by the Japanese Ministry of Health, Labour and Welfare, and in comments from the JES's councilors. At each stage of review, TFC received written comments and incorporated suggested changes. In conclusion, all patients with hypertension should be screened for PA, because of the high prevalence of cardiovascular disease and the current low case-detection rate in Japan. Case detection can be performed in hypertensive patients and those with hypokalemia by determining the aldosterone/renin ratio, and the diagnosis of PA can be confirmed by two of three confirmatory tests. The presence of a unilateral aldosterone-producing adenoma should be established/excluded by AVS by an experienced radiologist, optimally followed by laparoscopic adrenalectomy. In contrast, patients with bilateral adrenal hyperplasia, or those unsuitable for surgery, are optimally treated medically with mineralocorticoid receptor antagonists.

Kisspeptins are biologically active cleavage peptides of the KiSS-1 gene products with important roles in the suppression of tumor metastasis and in the reproduction. The aim of the present study is to clarify changes of the expression of kisspeptins and kisspeptin receptor in the kidney with and without chronic renal impairment. 5/6 nephrectomized rats were used as the rat model of chronic renal failure. Competitive quantitative RT-PCR showed that kisspeptin mRNA levels were decreased in the kidney of 5/6 nephrectomized rats at 56 days compared with sham-operated rats. In contrast, immunoreactive kisspeptin concentrations were increased in the kidney of 5/6 nephrectomized rats at 56 days. On the other hand, kisspeptin receptor mRNA levels were increased in the kidney of 5/6 nephrectomized rats at 14 and 56 days compared with sham-operated rats. Immunocytochemistry showed that kisspeptins and kisspeptin receptor were expressed in renal tubular cells, collecting duct cells, vascular smooth muscle cells in both rats. The intensity of kisspeptin receptor immunostaining was lower in 5/6 nephrectomized rats than in sham-operated rats. Western blot analysis confirmed that kisspeptin receptor protein levels were significantly decreased in the remnant kidney of 5/6 nephrectomized rats (about 23% of sham-operated rats), which is a good contrast to the kisspeptin receptor mRNA expression. The present study has shown that expression of kisspeptins and kisspeptin receptor are altered in the kidney tissues of chronic renal impairment, raising the possibility of their pathophysiological roles in chronic renal failure., (Copyright © 2010 Elsevier Inc. All rights reserved.)

(Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, is a 350 amino-acid protein with a single transmembrane domain and may play important pathophysiological roles in diabetic nephropathy. The aim of the present study is to clarify the expression of (P)RR in the kidney with end-stage renal disease due to diabetic nephropathy. The kidney tissues were obtained at autopsy from patients with and without Type 2 diabetes mellitus (n=5 without diabetes mellitus; and n=8 with diabetes mellitus). Immunocytochemistry showed that (P)RR was mainly expressed in the tubular cells and collecting duct cells of the kidney without diabetic nephropathy. Cells in glomeruli were very weakly and sporadically immunostained for (P)RR. Vascular smooth muscle cells and endothelial cells were very weakly or were not immunostained for (P)RR. Adipocytes in the adipose tissue around the kidney were positively immunostained for (P)RR. Immunostaining pattern of (P)RR in the kidney with diabetic nephropathy was similar to that without diabetic nephropathy. However, most notably, (P)RR immunostaining in the tubular cells and collecting duct cells was clearly and frequently more strongly observed in the kidney with diabetic nephropathy up to the end-stage renal disease. The present study has raised the possibility that (P)RR expressed in the diabetic kidney may play a pathophysiological role in angiotensin I generation and renal fibrosis found in end-stage renal disease., (Copyright 2010 Elsevier Inc. All rights reserved.)

Kisspeptins are neuropeptides which activate the hypothalamo-pituitary gonadal axis and are considered to play important physiological roles in the reproduction. Kisspeptins have also been reported to stimulate the aldosterone secretion from the adrenal cortex. However, the expression of kisspeptins in human adrenal glands and adrenal tumors has not been clarified yet. We, therefore, studied the presence of kisspeptin-like immunoreactivity (LI) in human adrenal glands and adrenal tumors (adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas) by radioimmunoassay and immunocytochemistry. Kisspeptin-LI was detected in all the tissues examined; normal portions of adrenal glands (3.0 +/- 2.3 pmol/g wet weight, n = 21, mean +/- SD), aldosterone-producing adenomas (4.6 +/- 3.3 pmol/g wet weight, n = 10), cortisol-producing adenomas (2.7 +/- 1.4 pmol/g wet weight, n = 14), adrenocortical carcinomas (1.7 +/- 0.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.8 +/- 0.8 pmol/g wet weight, n = 6). There was no significant difference in kisspeptin-LI levels among them. Immunocytochemistry showed positive kisspeptin-immunostaining in normal adrenal glands, with stronger immunostaining found in the medulla. Furthermore, positive kisspeptin-immunostaining was found in all types of adrenal tumors examined; adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas. The intensity of kisspeptin-immunostaining in these adrenal tumors was, however, not so strong as that in normal adrenal medulla. The present study has shown for the first time the presence of kisspeptin-LI in adrenal glands and adrenal tumors.

Context: The human adrenal gland produces small amounts of testosterone that are increased under pathological conditions. However, the mechanisms through which the adrenal gland produces testosterone are poorly defined., Objective: Our objective was to define the role of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in human adrenal production of testosterone., Design and Methods: Adrenal vein sampling was used to confirm ACTH stimulation of adrenal testosterone production. Adrenal expression of AKR1C3 was studied using microarray, quantitative real-time RT-PCR, and immunohistochemical analyses. AKR1C3 knockdown was accomplished in cultured adrenal cells (H295R) using small interfering RNA, followed by measurement of testosterone production., Results: Acute ACTH administration significantly increased adrenal vein testosterone levels. Examination of the enzymes required for the conversion of androstenedione to testosterone using microarray analysis, quantitative real-time RT-PCR, and immunohistochemistry demonstrated that AKR1C3 was present in the adrenal gland and predominantly expressed in the zona reticularis. Decreasing adrenal cell expression of AKR1C3 mRNA and protein inhibited testosterone production in the H295R adrenal cell line., Conclusions: The human adrenal gland directly secretes small, but significant, amounts of testosterone that increases in diseases of androgen excess. AKR1C3 is expressed in the human adrenal gland, with higher levels in the zona reticularis than in the zona fasciculata. AKR1C3, through its ability to convert androstenedione to testosterone, is likely responsible for adrenal testosterone production.

Adrenocortical carcinoma (ACC) is a rare neoplasm but some of the cases are highly malignant. Clinical outcome of the patients with advanced ACC still remained poor or dismal despite recent development of aggressive antitumor therapies. Target-specific therapies have been developed in a number of human malignancies and resulted in therapeutic benefits in some cancer patients. However, these therapies are only effective in the cases in which corresponding targets are expressed in tumor tissues. Therefore, we evaluated expression of potential surrogate markers using immunohistochemistry in archival materials of adrenocortical carcinoma in order to explore the potential application of target specific therapies in ACC in this study. We immunolocalized ten established or potential surrogate markers of target-specific therapies, located in the Ras/extracellular signal-regulated kinase and phosphatidylinositol-3 kinase/Akt pathways, in 41 ACC cases, 54 adrenocortical adenoma (ACA) cases, and five nonpathological adrenal glands and correlated the findings with clinicopathological factors of the patients. Among these markers examined, only epidermal growth factor receptor (EGFR) was significantly more abundant in ACC than in ACA (P < 0.01). These findings suggest that the agents which specifically inhibit signal transductions through EGFR such as monoclonal antibodies against EGFR are considered to be worthwhile to be attempted in future clinical studies.

Therapeutic angiogenesis has received much attention for its potential benefits in ischemic vascular disorders. Recently, the clinical application of hepatocyte growth factor (HGF) for therapeutic angiogenesis has become well known. Statins have also been reported to promote angiogenesis and ameliorate ischemic conditions. In the present study, we examined the effects of fluvastatin on HGF-induced angiogenesis using a human umbilical vein endothelial cell (HUVEC)/normal human dermal fibroblast (NHDF) co-culture system. The HGF-induced angiogenesis was augmented by fluvastatin at low dose, but it was decreased at high dose. Although fluvastatin increased vascular endothelial growth factor expression in NHDFs, it was observed only at a high dose. Low-dose fluvastatin decreased the HGF-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation (Thr-180/Tyr-182) and HUVEC apoptosis in the presence of HGF. SB203580, a p38 MAPK inhibitor, ameliorated anisomycin (a p38 MAPK activator)-induced angiogenesis suppression in the presence of HGF. Moreover, the augmentation of the HGF-induced angiogenesis by fluvastatin was abrogated by the p38 MAPK inhibitors, SB203580, SB202190, and FR167653. High-dose fluvastatin decreased Akt phosphorylation (Ser-473) and HUVEC proliferation, and it increased p27(kip1) in HUVECs. Interestingly, fluvastatin decreased the mRNA expression of integrins and tissue inhibitor of metalloproteinases (TIMPs) in HUVECs. Our data therefore indicate that the stimulatory effects of low-dose fluvastatin on the HGF-induced angiogenesis are mediated by its inhibitory effects on p38 MAPK phosphorylation induced by HGF, which may result in the suppression of EC apoptosis. High-dose fluvastatin inhibits Akt phosphorylation and HUVEC proliferation, and it increases p27(kip1), which may result in its inhibitory effects on angiogenesis. In addition, integrins and TIMPs are candidates for angiogenesis regulation by fluvastatin. (Hypertens Res 2008; 31: 2085-2096).

Expression of peptide YY (PYY) in the human brain and pituitary tissues was studied by radioimmunoassay, immunocytochemistry, and reverse transcription polymerase chain reaction. The polyclonal antibody raised against human PYY(1-36) in a rabbit was used in the assay, which showed 100% cross-reactivity with PYY(3-36) and no significant cross-reactivity with other peptides including neuropeptide Y and pancreatic polypeptide. The highest concentration of immunoreactive PYY was found in the hypothalamus (0.44+/-0.060 pmol/g of wet weight, mean +/- SEM, n=8), followed by the pituitary (0.41+/-0.16 pmol/g of wet weight, n=3). Reverse-phase high performance liquid chromatography of tissue extracts of human rectum and cortical brain showed a peak eluted in the position of authentic PYY(1-36) and PYY(3-36). Immunocytochemistry showed positive immunostaining for PYY in neurons of the paraventricular, arcuate, and supraoptic nuclei of the human hypothalamus. Moreover, reverse transcription polymerase chain reaction analysis showed expression of mRNA for PYY in human brain and pituitary tissues. The present study has shown for the first time expression of PYY in the human brain and pituitary tissues, suggesting a role for PYY as a neurotransmitter, in the neuroendocrine physiology, such as regulation of appetite and energy expenditure and modulation of pituitary hormone secretion.

Objective: The purpose of our study was to determine how frequently the right adrenal vein could be unequivocally identified on MDCT and the spectrum of anatomic variations seen in the right adrenal vein., Materials and Methods: Contrast-enhanced MDCT was performed in 104 patients with thoracoabdominal vascular disease using an 8-MDCT scanner. Both axial and multiplanar images were reviewed by two radiologists. The following points regarding the right adrenal vein were evaluated: degree of visualization; relationship to accessory hepatic or other veins; anatomy, including location of the orifice in relation to the surrounding structures; direction from the inferior vena cava; and length and diameter., Results: The right adrenal vein was detected in 79 (76%) of 104 patients. The right adrenal vein formed a common trunk with the accessory hepatic vein in six (8%) of the 79 patients. The orifice was craniocaudally located between the level of vertebrae T11 and L1. Among the 73 patients, the right adrenal vein joined the inferior vena cava in the right posterior quadrant in 71 patients (97%) and in the left posterior quadrant in two (3%). The transverse direction from the inferior vena cava was posterior and rightward in 56 patients (77%) and posterior and leftward in 17 (23%); the vertical direction from the inferior vena cava was caudal in 65 (89%) and cranial in eight (11%) patients. The length and diameter averaged 3.8 and 1.7 mm, respectively., Conclusion: MDCT enabled the identification of the right adrenal vein and delineation of its anatomy, including its position and relationship to surrounding structures.

A highly sensitive quantification method of aldosterone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using recently developed picolinyl derivatization. Aldosterone was smoothly and quantitatively converted to the ethyl ether-picolinyl derivative by treatment with HCl-ethanol followed by the esterification with picolinic acid in the presence of 2-methyl-6-nitrobenzoic anhydride and 4-dimethylaminopyridine. The positive ion-ESI mass spectrum of the ethyl ether-picolinyl derivative was characterized by an appearance of protonated molecule ([M+H](+)) as a base peak. The ethyl ether-picolinyl derivatization gave a successful result in a separation of aldosterone from corticosterone, dehydrocorticosterone and cortexolone, and also provided an approximately 10-fold higher ESI response in the positive-LC-ESI-MS/MS (selected reaction monitoring; SRM) when compared to that of underivatized molecule (negative mode). The limit of quantification of aldosterone by SRM using ethyl ether-picolinyl derivatization (m/z 494-->m/z 448) was 1 pg/0.2 ml serum with accuracy and precision of 92.6% and 5.6%, respectively.

Adrenomedullin 2/intermedin (AM2/IMD) is a new member of calcitonin/calcitonin gene-related peptide family. AM is expressed in various tumors including adrenocortical tumors and modulates tumor growth. The AM2/IMD expression has not been studied, however, in adrenal tumors. The expression of AM2/IMD and AM was therefore studied in human adrenal tumors and attached non-neoplastic adrenal tissues by immunocytochemistry (ICC). Immunoreactive (IR)-AM2/IMD was measured by RIA. Furthermore, the expression of AM2/IMD and its receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity-modifying proteins (RAMPs) 1, 2, and 3 mRNA in these tissues was studied by reverse transcription PCR (RT-PCR). ICC showed that AM2/IMD and AM immunoreactivities were localized in adrenocortical tumors and pheochromocytomas. AM2/IMD and AM immunoreactivities were detected in medulla of attached non-neoplastic tissues, while the degree of immunoreactivity for AM2/IMD and AM in cortices of attached adrenals was relatively weak or undetectable. RIA detected IR-AM2/IMD in adrenal tumors (0.414+/-0.12 to 0.786+/-0.27 pmol/g wet weight, mean+/-S.E.M.) and attached adrenal tissues (0.397+/-0.052 pmol/g wet weight). Reverse-phase high-performance liquid chromatography showed one broad peak eluted in the similar position to synthetic AM2/IMD with several minor peaks. RT-PCR showed expression of AM2/IMD, CRLR, and RAMP1, RAMP2, and RAMP3 mRNA in tissues of adrenal tumors and attached adrenal glands. In conclusion, AM2/IMD is expressed in human adrenal tumors and attached non-neoplastic adrenal tissues and may play (patho-)physiological roles in normal and neoplastic adrenals as an autocrine/paracrine regulator.

Urotensin II (UII), first identified from goby urophysis, is a potent vasoactive peptide hormone and an endogenous ligand for an orphan G protein-coupled receptor GPR14, now named urotensin II receptor (UT-R). In addition to its vascular actions, UII has been shown to have mitogenic effects on tumor growth and some regulatory effects on adrenal steroidogenesis. In the present study, we examined expression of UII and UT-R in human adrenal tumors and attached non-neoplastic adrenal tissues by immunohistochemistry. Both UII and UT-R were immunolocalized in tumor cells of all adrenal tumors examined: 8 cases of cortisol-producing adenomas, 8 cases of aldosterone-producing adenomas, 2 cases of non-functioning adenomas, 17 cases of adrenocortical carcinomas, and 8 cases of pheochromocytomas. In attached adrenals, immunoreactivity for UII was detected in medulla, but much weaker in the cortex than in cortical tumors, suggesting that expression of UII was up-regulated in neoplastic adrenocortical tissues. No significant differences were found in the degree of immunoreactivity for UT-R between the tumors and the attached adrenal tissues. The present study showed that both UII and UT-R were expressed in the adrenal tumors and attached non-neoplastic adrenal tissues, and suggests possible roles of UII and UT-R in tumor growth and/or secretory activities of these tumors.

Adrenocortical carcinoma (ACC) is a rare, highly malignant tumor. The aim of the present study is to evaluate the prognostic relevance of a proliferation marker Ki67/MIB1 by immunohistochemistry in 17 cases who underwent resections of the primary tumors and diagnosed to have ACC at Tohoku University Hospital based on the criteria of Weiss during the period from 1976 to 2005. The follow-up periods ranged from 221 days to 10659 days (about 29 years) with the median of 1895 days. The median age at diagnosis was 46 years old, and the mean size of the primary tumors was 7.1 cm with the minimal of 3.5 cm. Ki67/MIB1 labeling index (Ki67/MIB1LI) ranged from 1% to 26%. Kaplan-Meier analysis revealed that patients with Ki67/MIB1LI of 7% or more were associated with significantly shortened disease-free survival (P = 0.0037). The evaluation with Weiss criteria revealed that the median score of Weiss criteria was five, and 13 patients (76.5%) presented positive findings in the criteria of mitotic rate. The survival analysis with Weiss score showed that patients with the scores of 6 or more had both significantly shortened disease-free survival (P = 0.0001) and overall survival (P = 0.0063). The present study has suggested that Ki67/MIB1LI, as well as Weiss score, is a useful predictor for tumor recurrence after resection of the primary tumors in patients with ACC.

Accurate localization of aldosterone-producing adenoma (APA) is essential for the treatment of primary aldosteronism (PA). In order to confirm the clinical usefulness of adrenal venous sampling (AVS), we retrospectively studied 87 cases of PA in whom AVS was conducted. We collected right and left adrenal venous effluents simultaneously before and after adrenocorticotropic hormone (ACTH) stimulation for measurements of aldosterone concentration (A) and cortisol concentration (C). Based on AVS results, we judged 66 cases as having unilateral aldosterone hypersecretion and the remaining 21 cases as having no apparent laterality. Of the above 66 subjects, 61 underwent laparoscopic removal of the adrenal gland through a retroperitoneal approach. The presence of APA was histopathologically confirmed, and blood pressure decreased significantly with normalization of plasma aldosterone concentration (PAC) in all cases. The receiver operator characteristics (ROC) curve analysis between the operated and no-apparent-laterality groups revealed that the ratio of A/C on the higher side to A/C on the lower side (A/C ratio) after ACTH stimulation is a useful index, with a cutoff value of 2.6, a sensitivity of 0.98 and a specificity of 1.0. The ROC curve analysis between the APA side and contralateral side within the operated patients revealed that the cutoff value of A was 1,340 ng/dL, with a sensitivity of 0.92 and a specificity of 1.00. Our results indicate the usefulness of simultaneous AVS and ACTH stimulation for localizing APA.

Primary aldosteronism is defined as secondary hypertension accompanied by aldosterone hypersecretion that leads to suppressed plasma renin, hypokaremia and hypertension. Primary aldosteronism occurs in 3-15% of hypertensive patients and is commonly caused by aldosterone-producing adenoma (APA) or bilateral idiopathic hyperaldosteronism (IHA). APA is usually treated by adrenalectomy, whereas IHA is best managed medically. Therefore, it is important to distinguish between unilateral and bilateral disease. Computed tomography (CT) is used to differentiate the subtypes. Even when multidetector CT is used, the detection rate of APAs of 1 cm or less is lower than 60% for the following reasons: (a) aldosterone secretion does not depend on tumor size, (b) a tumor may exist without lipid-rich cells, and (c) non-functioning unilateral adenomas are not uncommon, especially in older patients. Adrenal venous sampling is the gold standard for the differentiation of unilateral from bilateral disease in patients with primary aldosteronism. It is important to compare the laterality of aldosterone secretion by performing simultaneous bilateral blood collection 15 min following adrenocorticotropic hormone stimulation. The value of (aldosterone/cortisol)(side)/(aldosterone/cortisol)(contralateral side) provides the best discrimination between patients with and without APA.

We describe a case of mesangial proliferative glomerulonephritis (MesPGN), the clinical symptoms of which were exacerbated by aldosterone-producing adenoma (APA). A 45-year-old man, who had had a history of hypertension for several years, presented with renal derangement, with serum creatinine at its upper normal limit and with microhematuria and proteinuria. He also presented with hypokalemia, with a plasma aldosterone concentration at its upper normal limit and plasma renin activity at its lower normal limit. After the administration of spironolactone, we resected his left adrenal gland, which had a nodular lesion as well as aldosterone hypersecretion. The treatment normalized his arterial blood pressure and serum potassium concentration. Although his proteinuria disappeared with the reduction in arterial blood pressure, the microhematuria continued. The administration of losartan because of the histological finding of MesPGN reduced the amount of hematuria. A dissociated response of hematuria and proteinuria to antihypertensive treatment indicated that MesPGN was coincidental with APA.

Adrenomedullin2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. In the present study, we developed a specific radioimmunoassay of human AM2/IMD. Expression of AM2/IMD was studied in the human brain, pituitary, heart and kidney obtained at autopsy by radioimmunoassay and immunocytochemistry. Immunoreactive-AM2/IMD was detected by radioimmunoassay in human brains (range; 0.163-1.495 pmol/g wet weight), pituitaries (4.46+/-0.689 pmol/g wet weight, mean+/-S.E.M, n=3), left ventricles of hearts (0.251+/-0.0321 pmol/g wet weight, n=4), kidneys (3.49+/-1.18 pmol/g wet weight, n=5), and plasma obtained at healthy subjects (24.7+/-1.78 pmol/l, n=3). Reverse-phase high performance liquid chromatography showed that immunoreactive-AM2/IMD in human brain, kidney and plasma extracts were eluted in the position of authentic AM2/IMD. Additional peaks eluted earlier were found in the brain tissue and plasma. Immunocytochemistry showed that immunoreactive-AM2/IMD was localized in paraventricular and supraoptic nuclei of hypothalamus, anterior and posterior lobes of pituitary, cardiomyocytes, pericardial adipocytes, vascular endothelial cells of pericardial veins, and vascular smooth muscle cells of coronary arteries and renal arterioles as well as in renal tubular cells. The present study has shown expression of AM2/IMD in various types of cells in the central nervous system and the cardiovascular system, and suggested possible (patho)physiological roles of AM2/IMD in these systems.

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.

We describe a case of secondary hypertension in which aldosterone-producing adenoma (APA) and renal artery stenosis (RAS) coexisted. RAS caused a significant pressure gradient, and successful angioplasty of it improved the affected renal function but did not reduce the systemic blood pressure (SBP). Surgical resection of APA performed several months later reduced SBP. In cases of suspected secondary hypertension, other cause(s) of hypertension should be considered, if hypertension persists after correction of one possible cause.

We describe a case of renovascular hypertension that was caused by renal artery dissection after an angiographic evaluation of the lower extremities. Retention of contrast medium in the affected kidney even 1 day after the procedure caused us to suspect renal artery dissection. Magnetic resonance angiography revealed irregular streaks in the lumen of the affected artery, a sign of dissection. Treatment by a transluminal angioplasty with stent insertion improved renal function and reduced systemic blood pressure with normalization of plasma renin activity. In patients whose systemic blood pressure increases suddenly after any conceivable physical traction on the abdominal aorta, evaluation of the renal arteries should be considered. Appropriate endovascular treatment can preserve renal function and reduce systemic blood pressure.

Balloon angioplasty is an established intervention to treat renovascular hypertension. Conventional balloon angioplasty is usually effective in cases of renovascular hypertension caused by fibromuscular dysplasia. In the present report, we describe two cases of renovascular hypertension caused by fibromuscular dysplasia in which stenotic lesions were resistant to conventional balloon angioplasty but were successfully managed by a cutting balloon. After cutting balloon angioplasty, systemic blood pressure was normalized in both patients, without the use of antihypertensive agents. Angioplasty by using a cutting balloon is a new therapeutic choice against renal artery stenosis that is resistant to dilation by a conventional balloon.

We describe a case of renovascular hypertension with renal artery stenosis concealed by aneurysms. Arteriography demonstrated no apparent renal artery stenosis, but did reveal aneurysms on the left renal artery. Captopril-loaded renoscintigraphy could not detect disturbed renal perfusion. High basal and exaggerated plasma renin activity after captopril administration were the only clues indicating renovascular hypertension. A reduction of the systemic blood pressure and normalized plasma renin activity after resection of the aneurysms confirmed preoperative renovascular hypertension. Fibromuscular dysplasia was an underlying cause of the arterial deformity. In cases of hypertension accompanied by renal artery aneurysms, the captopril-challenge test can be a useful tool to detect renal artery stenosis concealed by the aneurysms.

Atherosclerotic renal artery stenosis (ARAS) is a significant cause of end stage renal dysfunction (ESRD) among the elderly. Although early detection of ARAS and induction of adequate treatment could reduce the incidence of ESRD, there have been few reports about parameters predictive of ARAS among Japanese. In this study, we investigated the clinical indicators that predict ARAS among Japanese with risk factors of atherosclerosis (> 40 years of age plus hypertension, dyslipidemia or diabetes mellitus). After eliminating the patients who had already been diagnosed with renal artery stenosis, 202 patients were enrolled. The renal arteries of all 202 patients were evaluated by magnetic resonance arteriography (MRA), and the stenoses with > 50% reduction in diameter at the ostium of the renal artery were defined as ARAS. MRA detected ARAS in 42 patients (31 hemilateral and 11 bilateral). Between the patients with and without ARAS there was no significant difference in gender distribution, detection of abdominal vascular bruits or smoking habit. The prevalences of diabetic, hypertensive and cerebrovascular comorbidity were also not significantly different. The mean blood pressure, body mass index and total serum cholesterol values were similar between the two groups. However, age, pulse pressure, serum uric acid, serum creatinine, amount of urinary protein, and coronary artery comorbidity were significantly higher, while estimated creatinine clearance was significantly lower in the patients with ARAS than in those without ARAS. A high prevalence of hypertensive retinopathy was also noted among patients with ARAS. Multivariate analysis revealed that older age and renal impairment were independent predictors of ARAS in Japanese patients with atherosclerotic risk factors.

Digoxin, which is one of the most commonly prescribed drugs for the treatment of heart failure, is mainly eliminated from the circulation by the kidney. P-glycoprotein is well characterized as a digoxin pump at the apical membrane of the nephron. However, little is known about the transport mechanism at the basolateral membrane. We have isolated an organic anion transporter (OATP4C1) from human kidney. Human OATP4C1 is the first member of the organic anion transporting polypeptide (OATP) family expressed in human kidney. The isolated cDNA encodes a polypeptide of 724 aa with 12 transmembrane domains. The genomic organization consists of 13 exons located on chromosome 5q21. Its rat counterpart, Oatp4c1, is also isolated from rat kidney. Human OATP4C1 transports cardiac glycosides (digoxin, K(m) = 7.8 microM and ouabain, K(m) = 0.38 microM), thyroid hormone (triiodothyronine, K(m) = 5.9 microM and thyroxine), cAMP, and methotrexate in a sodium-independent manner. Rat Oatp4c1 also transports digoxin (K(m) = 8.0 microM) and triiodothyronine (K(m) = 1.9 microM). Immunohistochemical analysis reveals that rat Oatp4c1 protein is localized at the basolateral membrane of the proximal tubule cell in the kidney. These data suggest that human OATP4C1/rat Oatp4c1 might be a first step of the transport pathway of digoxin and various compounds into urine in the kidney.

We have recently demonstrated that aldosterone causes nongenomic vasoconstriction by activating phospholipase C (PLC) in the preglomerular afferent arteriole (Af-Art). In the present study, we tested the hypothesis that endothelium modulates this vasoconstrictor action by releasing nitric oxide (NO). In addition, to study the post-PLC mechanism, we examined possible contributions of phosphoinositol hydrolysis products. Rabbit Af-Arts were microperfused at 60 mm Hg in vitro, and increasing doses of aldosterone (10(-10) to 10(-8) mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (P<0.01) constriction was observed from 5x10(-9) mol/L, and at 10(-8) mol/L, intraluminal diameter decreased by 29%+/-3% (n=9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10(-10) mol/L, and at 10(-8) mol/L, the diameter decreased by 38%+/-2% (n=6). NO synthesis inhibition reproduced this augmentation (n=7). Pretreatment with chelerythrine (10(-6) mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10(-8) mol/L now decreased the diameter by 18%+/-3% (n=7). However, in Af-Arts treated with thapsigargin (10(-6) mol/L) or dantrolene (3x10(-5) mol/L), which blocks inositol 1,4,5-triphosphate (IP3)-induced intracellular calcium release, aldosterone at 10(-8) mol/L decreased the diameter by only 9%+/-1% (n=6) or 9%+/-2% (n=5), respectively. These results demonstrate that in the Af-Art endothelium-derived NO modulates vasoconstrictor actions of aldosterone that are mediated by the activation of both IP3 and PKC pathways. Such vasoconstrictor actions of aldosterone may contribute to the development or aggravation of hypertension by elevating renal vascular resistance in cardiovascular diseases associated with endothelium dysfunction.

We have isolated and characterized a novel human and rat organic anion transporter subtype, OATP-D. The isolated cDNA from human brain encodes a polypeptide of 710 amino acids (Mr 76,534) with 12 predicted transmembrane domains. The rat clone encodes 710 amino acids (Mr 76,821) with 97.6% amino acid sequence homology with human OATP-D. Human and rat OATP-D have moderate amino acid sequence homology with LST-l/rlst-1, the rat oatp family, the prostaglandin transporter, and moatl/MOAT1/KIAA0880/OATP-B. Phylogenetic tree analysis revealed that OATP-D is branched in a different position from all known organic anion transporters. OATP-D transports prostaglandin E1 (Km 48.5 nM), prostaglandin E2 (Km 55.5 nM), and prostaglandin F2,, suggesting that, functionally, OATP-D encodes a protein that has similar characteristics to those of the prostaglandin transporter. Rat OATP-D also transports prostaglandins. The expression pattern of OATP-D mRNA was abundant mainly in the heart, testis, brain, and some cancer cells. Immunohistochemical analysis further revealed that rat OATP-D is widely expressed in the vascular, renal, and reproductive system at the protein level. These results suggest that OATP-D plays an important role in translocating prostaglandins in specialized tissues and cells.

Three patients who suffered from congestive heart failure caused by severe hypertension were treated with a combination therapy consisting of angiotensin converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB). Before initiation of treatment, all three patients showed elevations of serum creatinine concentration (sCr), plasma renin activity (PRA), and plasma aldosterone concentration (PAC), which indicated insufficient blood supply to the kidney during exacerbation of hypertension. All three cases successfully recovered from hypertensive heart failure with the combination therapy. sCr gradually decreased during continuation of the therapy, although one patient showed an increase in sCr at an early stage of the combination therapy. Blockade of the renin-angiotensin-aldosterone system (RAAS) by the combination of ACEI and ARB was well tolerated in patients with severe hypertension with renal damage and showed a beneficial effect in protecting against further renal damage. This result suggests that combination therapy with ACEI and ARB should be considered as a candidate treatment in cases of severe hypertension.

Aldosterone (Aldo) accelerates hypertension, proteinuria, and glomerulosclerosis in animal models of malignant hypertension or chronic renal failure. Aldo may exert these deleterious renal effects by elevating renal vascular resistance and glomerular capillary pressure. To test this possibility, directly examined were the action of Aldo on the afferent (Af) and efferent (Ef) arterioles (Arts). Examined were the effect of Aldo added to both the bath and lumen on the intraluminal diameter (measured at the most responsive point) of rabbits. Aldo caused dose-dependent constriction in both arterioles with a higher sensitivity in Ef-Arts. Vasoconstrictor action of Aldo was not affected by a mineralocorticoid receptor antagonist spironolactone and was reproduced by membrane-impermeable albumin-conjugated Aldo, suggesting that the vasoconstrictor actions are nongenomic. Pretreatment with neomycin (a specific inhibitor of phospholipase C) abolished the vasoconstrictor action of Aldo in both arterioles. In addition, the vasoconstrictor action of Aldo on Af-Arts was inhibited by both nifedipine and efonidipine, whereas that on Ef-Arts was inhibited by efonidipine but not nifedipine. The results demonstrate that Aldo causes nongenomic vasoconstriction by activating phospholipase C with a subsequent calcium mobilization thorough L- or T-type voltage-dependent calcium channels in Af- or Ef-Arts, respectively. These vasoconstrictor actions on the glomerular microcirculation may play an important role in the pathophysiology and progression of renal diseases by elevating renal vascular resistance and glomerular capillary pressure.

We have isolated three novel organic anion transporter cDNAs designated rat GST-1 (gonad-specific transporter), rat GST-2, and human GST, expressed at high levels in the testis. Rat GST-1, GST-2, and human GST consist of 748, 702, and 719 amino acids, respectively, and all molecules possess the 12 predicted transmembrane domains, which is a common structure of organic anion transporters. Northern blot analyses and in situ hybridization revealed that both of the rat molecules are highly expressed in the testis, especially in Sertoli cells, spermatogonia, and Leydig cells. Weak signals are also detected in the epididymis and ovary in adult rat. The exclusive expression of human GST mRNA in the testis was confirmed by RT-PCR. The pharmacological experiments of Xenopus laevis oocytes injected with the respective rat GST-1- and GST-2-cRNAs revealed that both rat GST-1 and GST-2 transport taurocholic acid, dehydroepiandrosterone sulfate, and T4 with Michaelis-Menten kinetics (taurocholic acid, Km = 8.9 and 2.5 microm, dehydroepiandrosterone sulfate, Km = 25.5 and 21.microm, and T4, Km = 6.4 and 5.8 for rat GST-1 and GST-2, respectively). T3 was also transported by rat GST-1 and GST-2. These data suggest that rat GST-1 and GST-2 might be one of the molecular entities responsible for transporting dehydroepiandrosterone sulfate and thyroid hormones involved in the regulation of sex steroid transportation and spermatogenesis in the gonad.