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Summary Determination of the urinary steroid profile has been proposed as a sensitive tool for diagnosing adrenocortical tumors. The urinary steroid profiles were determined for patients with adrenocortical tumors. Urinary steroids were extracted, derivatized to form methyloxime-trimethylsilyl ether and analyzed by gas chromatography/mass spectrometry. Patients with adrenal adenomas from primary hyperaldosteronism had increased metabolites of 18-hydroxycorticosterone and aldosterone, and those with Cushing's syndrome had elevated excretion of 11-deoxycortisol, cortisol, 18-hydroxycortisol, and cortisone metabolites. In patients with adrenocortical carcinomas, increased levels of metabolites of 11-deoxycortisol or 3β-hydroxy-5-ene steroids were observed. The urinary steroid profiles of adrenal adenomas and adrenocortical carcinomas were quite different, suggesting the diagnostic validity for discriminating malignant from benign diseases.

Aranidipine, a newly developed calcium antagonist, possesses unique pharmacologic characteristics in that its metabolite (M-1) still has antihypertensive action. We examined the effects of both agents on renal microcirculation using the isolated perfused hydronephrotic rat kidney. During norepinephrine-induced constriction, the addition of aranidipine dilated both afferent and efferent arterioles in a dose-dependent manner; at 10(-6) M, 83 +/- 6% and 90 +/- 6% reversal, respectively. In contrast, its active metabolite exerted dilator action predominantly on the afferent arteriole (79 +/- 4% vs. 44 +/- 17% at 10(-6) M for afferent and efferent arterioles, respectively). We further examined the long-term (8 weeks) effect of these agents on the development of renal injury in salt-loaded subtotally nephrectomized spontaneously hypertensive rats. Both aranidipine and M-1 reduced blood pressure by a similar magnitude. The decreases in proteinuria were observed in the aranidipine-treated group at weeks 6, 8, and 10, whereas in the M-1 group, significant reduction was attained only at week 6. Histopathologic examination revealed that both treatments improved glomerular and arteriolar sclerosis. Glomerular sclerosis, however, was less pronounced in the aranidipine-treated group than in the M-1 group. In conclusion, aranidipine has dilator action on both arterioles, whereas M-1 caused predominant dilation of afferent arterioles. Such metabolic changes may constitute a determinant of efferent arteriolar action of the calcium antagonist.

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.

Platelets might be involved in the pathogenesis of diabetic microangiopathy. Wide interindividual variations in the density of a platelet collagen receptor (2β1 integrin or glycoprotein Ia/IIa) are reportedly associated with polymorphism(s) in the gene encoding the  subunit of the receptor, including a Bgl II polymorphism in intron 7. The aim of the present study was to determine the relationship between the Bgl II polymorphism and the susceptibility to diabetic microangiopathy. A case-control study comparing 227 patients with type II diabetes mellitus (119 with versus 108 without diabetic retinopathy) as well as 169 nondiabetic subjects demonstrated that genotypes with Bgl II (+) allele had a significant increase in the risk for retinopathy. The odds ratio for Bgl II (+/+) to Bgl II (−/−) was 3.41 (95% CI, 1.49-7.78, P = .0036) when analysis was confined to those with a disease duration of diabetes of 10 years or more. The present study suggests that the presence of a Bg II (+) allele is a genetic risk factor for diabetic retinopathy.

Overexpression of truncated IkBα potentiates TNF-α-induced apoptosis in mesangial cells. Background Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-kB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-kB affects TNF-α-induced apoptosis in rat mesangial cells (MCs). Methods To specifically inhibit NF-κ;B activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κ;B. Electrophoretic mobility shift assay was performed to evaluate NF-kB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results TNF-α-induced NF-κ;B activation was blocked by overexpression of IκBΔN. Overexpression of Iκ;BΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α-induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion Overexpression of IκBΔN potentiates TNF-α-induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκ;BΔN to induce apoptosis in MCs under inflammatory conditions.

The important contribution of hypertension to the progression of renal failure is well realized. However, it have been less discussed which drugs are suitable for the different stages of progressive renal failure. The present study examined the effects of timing of antihypertensive therapy using calcium channel blocker and angiotensin converting enzyme inhibitor in 5/6 nephrectomized spontaneously hypertensive rats (SHRs). Forty male 6 week old SHRs were divided into 5 groups (n=8 in each group), and they were placed on a high salt diet after 5/6 nephrectomy. Group 1, high salt diet without any drug. Group 2 received 0.2 mg/kg/day of amlodipine and group 3 received 0.2 mg/kg/day of enalapril mixed in the high salt diet from week 6 respectively. Similarly group 4 received the same doses of amlodipine, and group 5 received the same doses of enalapril from week 10. Each drug protected from increasing blood pressure in 4 groups, and no significant difference was observed between the effects of amlodipine and enalapril. Proteinuria was reduced with both drugs. In histopathological evaluation, glomerulosclerosis was controlled only in group 2, and arterio/olosclerosis was significantly suppressed in all treated groups except group 5. From these results, both amlodipine and enalapril are renal protective in early stage of renal failure with hypertension. However, in advanced stage of renal failure, amlodipine is superior in its renal protective effect.

The significance of increases in the expression of apoptosis-suppressing genes such as bcl-2 and mcl-1/EAT in human adrenal tumors has not yet been fully elucidated. Furthermore the roles of these genes in cell proliferation may involve interaction with steroidogenesis in the tumors via intracellular second messengers. Cyclic AMP (cAMP) caused human adrenocortical H295R cells to overexpress hCYP17 resulting in hypersecretion of cortisol. At the same time, however, expression of bcl-2, which has a cAMP response element (CRE), was not affected. Furthermore, in vivo Bcl-2 protein analysis showed its down-regulation in adrenal hyperplasia of Cushing's disease despite ACTH stimulation. Exogenous addition of glucocorticoid did not affect the expression of bcl-2 family genes. Expressions of Mcl-1/EAT and Bax did not differ markedly among human adrenal glands affected by various pathologies. In conclusion the down-regulation of Bcl-2 in Cushing's disease did not agree with no induction of this gene by cAMP in H295R cells, suggesting that expression of Bcl-2 protein was not regulated mainly by cAMP-protein kinase (PKA) pathways in human adrenal hyperplasia.

CYP17 gene transcription is activated by SF-1 binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and its transcription is inhibited by COUP-TF binding to the sequence. Another orphan receptor, DAX-1, is shown to act as a suppressor of SF-1-mediated transcription. We examined the expression level of these orphan receptors in adrenocortical tumors and compared the results with CYP17 expression. CYP17 was highly expressed in cortisol-producing adenomas, whereas COUP-TF and DAX-1 expression levels were very low. In deoxycorticosterone-producing adenomas, on the other hand, CYP17 expression was extremely low, whereas DAX-1 was highly expressed and SF-1 expression was slightly decreased. In conclusion, the reciprocal expression of CYP17 and the transcriptional repressors COUP-TF and DAX-1 indicates that these orphan receptors have a pathophysiologic role in the excessive hormone production in cortisol- and deoxycorticosterone-producing adrenocortical tumors.

It has been demonstrated that antihypertensive treatment of hypertensive diabetic patients is quite effective in preventing macrovascular and microvascular complications and improving prognosis. Nevertheless, the target blood pressure level of antihypertensive treatment in hypertensive diabetic patients with microalbuminuria (i.e., with early diabetic nephropathy) remains to be established. In this study, we evaluated the effect of intensive blood pressure control (diastolic blood pressure

Numerous epidemiological studies have shown a close relationship between obesity and hypertension. However, there have been few reports on the relationship between changes in the body weight and blood pressure of lean to normal-weight young subjects. The purpose of this study was to investigate the effects of body weight control on blood pressure in lean to obese young Japanese individuals in a 3-year follow-up study. University students (3, 558 males and 1, 418 females, aged 18.6±0.8 in 1994) were classified into 4 groups according to the baseline body mass index (BMI), and were followed up for 3 years. Among male students, changes in body weight were significantly correlated with changes in blood pressure during the 3 years in all 4 BMI groups, and the correlation coefficient was larger in the group with higher baseline BMI. Positive correlations between changes in body weight and changes in heart rate were noted only in the obese and mildly-obese groups. Also in female students, positive correlations were observed between changes in body weight and changes in blood pressure in lean to obese groups. However, no correlations between changes in body weight and changes in heart rate were noted in any of the female groups. To summarize, close correlations were observed between changes in body weight and those in blood pressure during the 3 years in both male and female university students. These findings suggest the importance of body weight control not only in obese but also in normal to mildly-obese young subjects in reducing or preventing an increase in blood pressure. There could be, however, a gender difference in the effects of body weight change on heart rate. (Hypertens Res 2000; 23: 421-426)

Gitelman's syndrome (GS) is an autosomal recessive disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia, and hypocalciuria that has recently been reported to be linked to thiazide-sensitive Na-Cl cotransporter (TSC) gene mutations. In this study, possible mutations in the TSC gene of six Japanese patients clinically diagnosed with GS were investigated. Twenty-six exons encoding TSC were am- plified by PCR and then completely sequenced by the direct sequencing method. Patient A showed a missense mutation of Arg 642 to Cys on the paternal allele and a missense mutation of Val 578 to Met and a 2-bp deletion (nucleotide 2543-2544) on the maternal allele. This deletion results in a frameshift that alters codon 837 to encode a stop signal rather than phenylal- anine, and it is predicted to lead to loss of the latter half of the intracellular carboxy terminus. In the second family, two af- fected sisters, patients B and C, had a homozygous missense mutation of Thr 180 to Lys. Both of their parents, who are consanguineously married, have a heterozygous Thr180Lys mutation. Patient D has a homozygous mutation Thr180Lys, which is the same as the second family. Haplotype analysis indicates that patients B and C are not related to patient D. In patients E and F, we could identify only one mutant allele; Ala569Glu and Leu849His, respectively. All of the mutations identified are novel except for the Arg642Cys mutation, which has been found in a Japanese GS patient. Although further in vitro study is required to prove that the mutations are respon- sible for GS, it is possible that Thr180Lys and Arg642Cys mutations might be common mutations in Japanese GS. Gitelman's syndrome (GS), a recognized variant of Bartter's syndrome, is characterized by hypokalemic metabolic alkalosis in combination with hypomagnesemia, hypocalciuria, and pre- sentation in early adulthood (1). The measurement of urinary calcium excretion is useful in distinguishing between GS and "classical" Bartter's syndrome; hypocalciuria is present in the former, whereas normo- or hypercalciuria has been reported in the latter (2). Because the electrolyte disturbances in GS resemble the effects of chronic thiazide administration, GS was suggested to be due to a defect in distal thiazide-sensitive sodium-chloride transport (3). Recently, Simon et al. demonstrated complete linkage of GS to the locus encoding the thiazide-sensitive Na-Cl cotransporter (TSC), and identified mutations in the TSC gene in GS patients (4). Thereafter, several investigators reported a wide variety of TSC gene mutations in cases of GS (5- 8). To date, 46 distinct TSC mutations have been found. In this study, we have investigated possible mutations in the TSC gene of six Japanese patients clinically diagnosed with GS. We identified six TSC gene mutations, including five novel mutations.

The effects of belonging to sports clubs on male high school students was evaluated. The relationships between the type and extent of school-based exercise were examined in conjunction with percent body fat, blood pressure (BP), and other key metabolic parameters. A total of 264 male Japanese high school students (age range: 17-18 years old) were studied. Percent body fat was measured and blood was collected in the fasting state during a routine health check. Subjects were divided into two groups. The exercise (E) group (n=150) included students who had belonged to a sports club during the past 2 years. The non-exercise (NE) group (n=114) included students who did not belong to a sports club during the past 2 years. The body mass index was significantly greater in group E (21.7 +/- 2.3 (SD) kg/m2) than in group NE (20.7 +/- 2.6 kg/m2, p < 0.01). However, the percent body fat in group E (13.6 +/- 3.4%) was significantly lower than that in group NE (14.9 +/- 3.8%, p < 0.01). The diastolic BP and heart rate in group E (64 +/- 7 mmHg, 70 +/- 11/min) were significantly lower in group E than in group NE (66 +/- 8 mmHg, p < 0.05; 76 +/- 14/min, p < 0.01). The serum triglyceride level was significantly lower, and the HDL cholesterol level was higher in group E than in group NE. The homeostasis model assessment (HOMA) index, used as an index of insulin resistance, was similar in the two groups. However, the level of the HOMA index was significantly lower among the 62 subjects in group E who preferred highly dynamic exercise (1.50 +/- 0.46) than it was among those in group NE (1.66 +/- 0.49, p < 0.05). Results indicate that belonging to sports clubs influences the BP and lipid profiles of adolescent males, as well as their percent body fat. In view of the reduction of cardiovascular risk factors, it is recommended that even young males practice regular exercise, especially aerobic exercise.

Background. Some pharmacokinetic data for vancomycin (VCM) during continuous arteriovenous hemofiltration have been reported, but reports on the effect of continuous venovenous hemodiafiltration (CVVHDF), which is more commonly performed in patients with multiple organ failure (MOF), on VCM pharmacokinetics are scanty. Method. We selected five patients with MOF with serious infection with methicillin-resistant Staphylococcus aureus who needed treatment with VCM during CVVHDF. Blood flow rate was 80 ml/min, and dialysis fluid flow rate and filtration flow rate were both 0.5 l/h. A hemofilter made of polysulfon was used. After administration of 0.5–1.0 g of VCM, serial samples of blood and dialysate/filtrate outflow were obtained during CVVHDF. Pharmacokinetic parameters were calculated by a standard model-independent method. Results. Mean ± SE values for the pharmacokinetic parameters of VCM were: elimination rate constant, 0.0369 ± 0.0124/h; systemic clearance, 25.6 ± 5.0 ml/min; CVVHDF clearance, 15.9 ± 3.4 ml/min; non-CVVHDF clearance, 9.6 ± 8.4 ml/min; and distribution volume, 51.1 ± 21.6 l. The harmonic mean of half-life was 18.9 h. Conclusion. The systemic clearance of VCM during CVVHDF was relatively higher than anticipated. The pharmacokinetic parameters obtained in the present study will be useful to optimize the dose schedule of VCM in patients with MOF being treated with CVVHDF.

The present study examined the role of intrarenal bradykinin in angiotensin-converting enzyme inhibitor (ACEI)-induced dilation of renal afferent (AFF) and efferent arterioles (EFF) in vivo, and further evaluated whether ACEI-stimulated bradykinin activity differed in superficial (SP) and juxtamedullary nephrons (JM). Arterioles of canine kidneys were visualized with an intravital charge-coupled device camera microscope. E4177 (an angiotensin receptor antagonist, 30 microg/kg) dilated AFF and EFF in SP (15 +/- 3% and 19 +/- 5%) and JM (15 +/- 3% and 18 +/- 4%). Subsequently, cilazaprilat (30 microg/kg) caused further dilation of both AFF (29 +/- 4%) and EFF (36 +/- 4%) in JM, whereas in SP it dilated only EFF (29 +/-3%). Similarly, in the presence of E4177, cilazaprilat caused further increases in sodium excretion. This cilazaprilat-induced vasodilation and natriuresis was abolished by a bradykinin antagonist (N(alpha)-adamantaneacetyl-D-Arg-[Hyp3,Thi5,8,D-Phe7]b radykinin). In parallel with these results, cilazaprilat increased renal bradykinin content, more greatly in the medulla than in the cortex (5.7 +/- 0.4 versus 4.6 +/- 0.1 ng/g). Similarly, cilazaprilat elicited greater bradykinin-dependent increases of nitrite/nitrate in the medulla. In conclusion, zonal heterogeneity in renal bradykinin/nitric oxide levels and segmental differences in reactivity to bradykinin contribute to the diverse responsiveness of renal AFF and EFF to ACEI. ACEI-enhanced kinin action would participate in the amelioration of glomerular hemodynamics and renal sodium excretion by ACEI.

There is increasing evidence about important cardiovascular effects of aldosterone through classic mineralocorticoid receptors in the heart. It is now clear that aldosterone/excess salt administration has been shown to produce both cardiac hypertrophy and interstitial cardiac fibrosis in rats. In clinical studies, it has been reported that aldosterone seems to play an important role in cardiac hypertrophy. However, it has still not been established whether aldosterone is involved in cardiac hypertrophy in patients with end-stage renal failure treated with hemodialysis. In the present study, we have analyzed the association between cardiac hypertrophy and aldosterone in 29 patients (18 patients with nondiabetic nephropathy and 11 patients with diabetic nephropathy) who developed end-stage renal disease and received hemodialysis. Among the nondiabetic patients, left ventricular mass index correlated significantly with plasma aldosterone concentrations during both before and after hemodialysis, but it did not correlate with plasma renin activity. Furthermore, left ventricular mass index also correlated with mean blood pressure. In contrast, these correlations were not seen in the diabetic patients, despite similar age distribution, duration of hemodialysis, and several echocardiographic parameters between two groups. In conclusion, our study provides new evidence for a relation between left ventricular hypertrophy and plasma aldosterone concentrations that seems to be independent of blood pressure in nondiabetic patients with end-stage renal failure treated with hemodialysis.

We investigated the efficacy of additional administration of 400 mg troglitazone (+T), which became available as a treatment for type 2 diabetes following the demonstration of its ability to reduce insulin resistance, in combination with diet (D + T) or sulfonylurea (S + T) therapy. Body fat area as determined by computed tomographic (CT) scanning at the umbilical level, as well as several clinical and biochemical parameters of glycemic control and lipid metabolism, were compared before and after 3 months of additional treatment with troglitazone. The body mass index (BMI) tended to increase in both groups (22.7 +/- 0.6 v 23.2 +/- 0.6 kg/m2 in D + T, nonsignificant [NS]; 22.2 +/- 0.5 v 22.3 +/- 0.5 kg/m2 in S + T, NS), while it tended to decrease in the control group (only diet therapy, 23.6 +/- 0.6 v 23.1 +/- 0.8 kg/m2, NS). Mean blood pressure ([BP] 96 +/- 3 v 89 +/- 4 mm Hg, P.05) decreased significantly in the D + T group. Changes in the glycemic and lipid profile and leptin did not reach statistical significance. The D + T group showed a significant decline in immunoreactive insulin ([IRI] 12.4 +/- 1.2 v 8.0 +/- 1.0 microU/mL, P.05), reflecting markedly reduced insulin resistance, as well as a significant increase in plasma insulin-like growth factor-1 ([IGF-1] 175.7 +/- 14.2 v 189.8 +/- 12.6 ng/mL, P.05). A slight weight gain was associated with a tendency for subcutaneous fat to increase, while visceral fat decreased in both troglitazone-treated groups. The decrease in the visceral to subcutaneous fat ratio (V/S ratio) was statistically significant in the D + T group (1.09 +/- 0.11 v 0.94 +/- 0.09, P.05), while the V/S ratio in the control group did not change. A notable finding of this study is the difference in the response to troglitazone between subcutaneous and visceral adipose tissue. It is suggested that troglitazone may exert beneficial effects by reducing visceral fat.

Pressure control of renin secretion involves a complex integration of shear stress, stretch, and transmural pressure (TP). This study was designed to delineate TP control of renin secretion with minimal influence of shear stress or stretch and to determine its mechanism. Rat juxtaglomerular (JG) cells were applied to a TP-loading apparatus for 12 h. In cells conditioned with atmospheric pressure or atmospheric pressure + 40 mmHg, renin secretion rate (RSR) averaged 29.6 ± 3.7 and 14.5 ± 3.3% ( P < 0.05, n = 8 cultures), respectively, and active renin content (ARC) averaged 47.3 ± 4.6 and 38.4 ± 3.4 ng of ANG I ⋅ h−1⋅ million cells−1( P < 0.05, n = 10 cultures), respectively. Total renin content and renin mRNA levels were unaffected by TP. The TP-induced decrease in RSR was prevented by Ca2+-free medium and the Ca2+channel blocker verapamil and was attenuated by thapsigargin and caffeine, which deplete intracellular Ca2+stores. Thapsigargin and caffeine, but not Ca2+-free medium or verapamil, prevented TP-induced decreases in ARC. The adenylate cyclase activator forskolin did not modulate TP-induced decreases in RSR or ARC. These findings suggest that TP not only stimulates Ca2+influx but also inhibits prorenin processing through an intracellular Ca2+store-dependent mechanism and thus inhibits active renin secretion by JG cells.

Background. Angiotensin II (Ang II) is involved in the direct inhibition of renin release from juxtaglomerular (JG) cells in the kidney as the negative feedback loop of the renin-angiotensin system. Ang II also modulates renin release via the sympathetic nervous system, since the renal sympathetic nerves stimulate renin release, and the interaction of the sympathetic nervous system with Ang II has been demonstrated to occur at multiple levels.

The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.

1. The effect of cicletanine, a novel antihypertensive agent with natriuretic activity, on blood pressure and progression of renal failure in 5–6 nephrectomized spontaneously hypertensive rats with salt loading was examined. 2. All nephrectomized rats were randomly assigned to one of four groups and their diet was changed from a normal- to a high-salt (5.5% NaCl) diet for the next 10 weeks. Either 10 or 50 mg/kg per day cicletanine (low- and high-dose cicletanine, respectively) or 10 mg/kg per day trichlormethiazide were administered to rats during this period once a day. During the experimental period, urine volume, urinary excretion of sodium, protein, prostaglandin (PG) E2 and 6-keto-PGF1α and systolic blood pressure (SBP) were measured every 2 weeks. 3. Systolic blood pressure was significantly reduced by the administration of trichlormethiazide and the higher dose of cicletanine, but not by the lower dose of cicletanine. 4. In contrast with changes to SBP, levels of serum creatin- ine in rats treated with both doses of cicletanine were significantly lower than in controls (0.57±0.12, 0.78±0.12 and 1.68±0.26 mg/dL for high- and low-dose cicletanine and control, respectively). 5. Urinary excretion of both PGE2 and 6-keto-PGF1α were significantly increased in groups treated with high and low doses of cicletanine compared with control. In rats treated with trichlormethiazide, PGE2 and 6-keto-PGF1α levels were significantly decreased compared with control. 6. In contrast with changes in SBP, marked glomerular sclerosis with hyalinosis found in the control group was not ameliorated by trichlormethiazide treatment. These changes were not observed in rats treated with low- and high-dose cicletanine, particularly those treated with the higher dose of cicletanine. 7. These data suggest that administration of cicletanine has a beneficial protective effect regarding the progression of renal failure, regardless of the level of blood pressure, through a direct and/or indirect action on the glomerulus.

Antibodies against glutamic acid decarboxylase (GADA) is known to be a good predictive marker for insulin-dependency among adult diabetic patients. However, since not all of the GADA-positive patients will develop insulin requirement, we investigated whether other markers, that is, antibodies against IA-2 (IA-2A), insulin autoantibodies (IAA) and HLA class II type, would affect its predictive value for insulin requirement. Adult diabetic patients in the non-insulin-requiring stage were screened for GADA and registered in the study if positive. At the end of the follow-up period, 15 of the 43 GADA-positive patients required insulin. Among GADA-positive patients, the GADA titers of the insulin-requiring patients were significantly higher (199 U vs. 5.8 U, P

Although calcium antagonists are believed to exert preferential vasodilator action on the renal preglomerular afferent arteriole, we recently demonstrated that efonidipine, a novel calcium antagonist, vasodilates both afferent and efferent arterioles. Nilvadipine also is reported to increase renal blood flow and reduce filtration fraction, suggesting indirectly afferent and efferent arteriolar vasodilation. No direct investigation, however, has been conducted examining the renal microvascular action of nilvadipine. We therefore characterized the renal microvascular reactivity to nilvadipine, by using the isolated perfused rat hydronephrotic kidney. The administration of angiotensin II (0.3 nM) caused marked vasoconstriction of afferent (from 13.5 +/- 0.6 to 9.2 +/- 0.6 microm, p0.01, n = 6) and efferent arterioles (from 11.5 +/- 1.0 to 7.4 +/- 0.7 microm, p0.01; n = 5). The subsequent addition of nilvadipine (10 nM, 100 nM, and 1 microM) caused 37 +/- 5%, 91 +/- 4%, and 95 +/- 8% reversal of afferent arteriolar constriction, respectively. Similarly, efferent arterioles manifested 59 +/- 12% reversal by 1 microM nilvadipine. Thus unlike nifedipine, which we previously reported to cause modest efferent arteriolar dilation (21 +/- 1% reversal at 1 microM), nilvadipine possesses the greater ability to dilate efferent arterioles (p0.01 vs. nifedipine), although both antagonists cause similar magnitudes of afferent arteriolar vasodilation. Variable effects on the efferent arteriole suggest the heterogeneity in the calcium antagonist with regard to the renal microvascular action of this agent.

To compare the incidence of cough between two angiotensin converting enzyme (ACE) inhibitors, imidapril and enalapril, comparative crossover study was performed in 489 patients (228 men and 261 females) with essential or renal parenchymal hypertension. Patients were randomly assigned to one of two treatment groups, a group receiving imidapril for 12wk (Period I) followed by enalapril for 12wk (Period II), and a group in which the order of drugs was reversed. The occurrence of cough during treatment was monitored by questionnaire in all cases. There were no differences in background characteristics between the two groups. The incidence of cough during Period I was 15.2% (32/210) in the group initially treated with imidapril (Group IE) and 38.6% (85/220) in the group initially treated with enalapril (Group EI), the difference being statistically significant (p

The effect of pravastatin on renal function in hypertensive patients with mild renal dysfunction and hyperlipidemia was examined. A total of 57 subjects given dihydropyridine calcium blockers were randomly assigned to placebo (n = 25) and pravastatin groups (n = 32). The period of study was 6 months. In the placebo group, lipid metabolism did not change throughout the study period, but the serum creatinine concentration (Scr) increased from a baseline of 1.6+/-0.07 mg/dl to 2.1+/-0.2 mg/dl in the 6th month of study and blood urea nitrogen (BUN) increased from 26.2+/-1.1 mg/dl to 32.4+/-30.1 mg/dl. In the pravastatin group, the serum total cholesterol decreased from a baseline of 251.4+/-7.3 mg/dl to 218.2+/-6.5 mg/dl in the 6th month of study, while Scr (1.3+/-0.07 mg/dl vs. 1.3 +/-0.09 mg/dl) and BNU (20.5+/-1.2 mg/dl vs. 21.0+/-1.4 mg/dl) did not change. The change in Scr in the placebo group was significantly different from that in the pravastatin group (F = 3.75, p = 0.05). The slope of the change in 1/Scr was 0.02+/-0.07 dl x mg(-1) x month(-1) in placebo group and -0.01+/-0.03 dl x mg(-1) month(-1) in pravastatin group (P

Sustained hypertension alters vasomotor regulation in various vascular beds. We studied whether nitric oxide (NO)-dependent and NO-independent vasodilator mechanisms are altered in renal microvessels in hypertension. To directly visualize the renal microcirculation, the isolated perfused hydronephrotic rat kidney model was used. After pretreatment with indomethacin (100μmol/l), afferent arterioles were constricted by norepinephrine (NE) or by increasing renal arterial pressure (i.e., myogenic constriction; from 80 to 180mmHg). Acetylcholine (ACH) was then added, and the renal microvascular response was assessed by computer-assisted video image analysis. A similar protocol was conducted in the presence of nitro-L-arginine methylester (L-NAME; 100μmol/l). During NE constriction, ACH caused dose-dependent and sustained vasodilation of the afferent arteriole, similar in magnitude in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In the presence of L-NAME, ACH (0.01-1μmol/l) elicited only transient dilation, and the degree of vasodilation was very low in SHR. During myogenic constriction, afferent arterioles from WKY and SHR kidneys responded to ACH with only transient vasodilation, which was unaffected by NO inhibition; the transient vasodilative responses elicited by ACH (0.1-1μmol/l) were smaller in SHR than in WKY. In conclusion, ACH has both sustained and transient vasodilative effects on the afferent arteriole. Sustained vasodilation is attributed to NO generation, which is similar in WKY and SHR. In contrast, transient vasodilation, mediated by NO-independent vasodilator factors, is impaired in SHR. Deranged vasodilatory mechanisms in hypertension may disturb the renal microcirculation, which may result in renal injury. (Hypertens Res 1999; 22: 31-37)

Two novel 1α-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type I. Background Vitamin D dependency rickets type I (VDDR-I) is an autosomal recessive disorder in which 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase) activity in renal proximal tubules is deficient. VDDR-I is recognized throughout the world, but occurs more frequently in a subset of the French-Canadian population. We and others have recently cloned the human 1α-hydroxylase cDNA and gene, making it possible to screen for mutations. The first VDDR-I mutations were reported in one American and four Japanese patients. In this study, we screened for 1α-hydroxylase mutations in French-Canadian patients with VDDR-I. Methods The nine exons of the 1α-hydroxylase gene were amplified by polymerase chain reaction (PCR) from genomic DNA of four unrelated French-Canadian patients with VDDR-I and their parents, and sequenced. Results Three of the patients were homozygous for a single base-pair deletion (G) at position 262 in the cDNA that lies in exon 2, and causes a premature termination codon upstream from the putative ferredoxin- and heme-binding domains. The fourth patient was homozygous for a 7-bp insertion (CCCCCCA) at position 1323 of the cDNA that lies in exon 8, and causes a premature termination upstream from the putative heme-binding domain. In each family, obligate carriers have one copy of the mutant allele. These mutations, which could be detected by PCR-restriction fragment length polymorphism and polyacrylamide gel electrophoresis of the PCR products, were not found in 25 normal French-Canadians. Conclusion We describe two novel 1α-hydroxylase mutations that are consistent with loss of function in four French-Canadian patients with VDDR-I and suggest that the 1α-hydroxylase mutations arise from more than one founder in this population.

Interleukin (IL)-1 and IL-4 synergistically stimulate NF-IL6 activity and IL-6 production in human mesangial cells. Background While interleukin (IL)-4 inhibits pro-inflammatory cytokine expression by human monocytes, we have observed that it potentiates IL-6 production by IL-1-activated human mesangial cells (MC). To study the mechanism of this cell-type specific interaction between IL-1 and IL-4 in MC, we examined the effect of both cytokines on the activities of nuclear factor κB (NF-κB) and nuclear factor IL-6 (NF-IL6), transcription factors that are essential for IL-6 gene expression. Methods We evaluated IL-6 synthesis, mRNA expression, and mRNA stability by ELISA, Northern analysis, and the actinomycin D method, respectively. Activities of NF-κB and NF-IL6 were analyzed by gel shift assay. Results IL-4 augmented the IL-1 stimulated IL-6 mRNA levels by about threefold without altering mRNA stability. IL-1 treatment rapidly induced the binding activity of NF-κB. In contrast, IL-4 did not affect basal and IL-1-induced NF-κB activities. Both IL-1 and IL-4 stimulated NF-IL6 activity as early as 30minutes after treatment. When MC were treated with both cytokines together, marked activation of NF-IL6 was observed at five hours. Conclusions These results suggest that simultaneous activation of NF- κB and NF-IL6 is essential for IL-6 gene expression and that IL-1 and IL-4 cooperatively stimulate MC IL-6 production through their synergistic activation of NF-IL6.

OBJECTIVE Leptin, the obese gene product, is secreted exclusively by adipocytes and is thought to act as a lipostatic signal that regulates body weight homeostasis. We previously reported that thyroid hormone is one of the up-regulating factors of leptin in vitro. T3, at physiological concentrations, stimulates leptin mRNA expression and leptin secretion by 3T3-L1 adipocytes. The aim of this study was to explore the role of thyroid hormone in the regulation of leptin in humans. DESIGN AND PATIENTS A total of 59 non-obese women aged 38.4 +/- 1.8 years (mean +/- SEM) were studied: 19 patients with hyperthyroidism, 17 patients with hypothyroidism, and 23 normal control subjects. The correlation between serum leptin concentrations and body mass index (BMI) was analyzed, and serum leptin levels were compared among the three groups. MEASUREMENTS Serum leptin concentrations were measured by radioimmunoassay. RESULTS Serum leptin concentrations after logarithmic transformation were correlated significantly (P < 0.05) with BMI in the hyperthyroid (r = 0.46), the hypothyroid (r = 0.84), and normal (r = 0.63) groups. Even though age, body weight, and BMI were similar in all groups, serum leptin levels in the hypothyroid patients (5.30 +/- 1.12 micrograms/l) were significantly (P < 0.05) lower than in the hyperthyroid and normal groups (6.87 +/- 0.66 and 6.58 +/- 0.68 micrograms/l, respectively). CONCLUSIONS These results indicate that thyroid hormone may play an important role in the appropriate secretion of leptin in humans.

Acute methotrexate toxicity resulting from methotrexate-induced renal failue is a medical emergency requiring extracorporeal removal of methotrexate. The optimum method of methotrexate removal has not yet been established. We report a case of osteosarcoma with lung metastasis that was treated with high-dose methotrexate as adjuvant chemotherapy. Although no problems occurred after the first 5 courses of methotrexate, methotrexate-induced renal failure and methotrexate toxicity appeared after the sixth course. The patient was treated, either with hemoperfusion plus high-flux hemodialysis, or hemoperfusion alone, and pre- and post-treatment serum methotrexate concentrations were monitored. The reduction in methotrexate by hemoperfusion alone for 2 hours was 54%, compared to a mean reduction of 59% by hemoperfusion combined with high-flux hemodialysis, for 3 hours. Rebound increases in methotrexate levels were small (less than 1 μmol/L) with either method. The combination of hemoperfusion and hemodialysis resulted in good control of volume status, as well as improvement in serum chemistry values.

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

Using the method of vector autoregressive modeling (VAR) analysis of frequently sampled oral glucose tolerance test (OGTT) results, we evaluated abnormalities in the feedback relationships between plasma glucose and insulin in gastrectomized patients to assess insulin secretion capacity and insulin resistance following gastrectomy. VAR modeling analysis was applied to the plasma glucose and insulin level data from the frequently-sampled 75g-OGTT results of 38 subjects who had undergone total or subtotal gastrectomy and 977 controls without gastrectomy. After gastrectomy, the predicted response of insulin to a glucose challenge was excessive in normal subjects and those with slightly impaired glucose tolerance. Furthermore, the glucose response to insulin was clearly positive in gastrectomized subjects with moderately to severely impaired glucose tolerance, i.e., diabetics, indicating strong insulin resistance. The insulin resistance in this situation cannot be explained by decreased peripheral glucose disposal. Our results suggest that the lowered glucose tolerance which follows gastrectomy results from disturbance of the hormonal relationship between pancreas and intestine (entero-insular axis), which causes increased intestinal glucose absorption, and the insulin resistance which occurs in response to hyperinsulinemia in patients with normal fasting plasma glucose. Disturbance of the entero-insular axis may cause not only increased glucose absorption but also hyperglucagonemia, both of which contribute to hyperglycemia in diabetic patients after gastrectomy.

A 38-yr-old female with a TSH- and GH-secreting pituitary adenoma is described, who had both overt symptoms, hyperthyroidism and acromegaly. Her serum TSH was not suppressed despite high concentrations of free T3 and free T4, and her alpha-subunit/TSH molar ratio was high. Her serum GH was consistently high, and was not suppressed by an oral glucose tolerance test. Preoperative testing revealed that, although the TSH response was impaired, TSH, alpha-subunit and GH were increased by TRH injection, and that these hormones were reduced by bromocriptine or somatostatin analog. Although she did not have hyperprolactinemia, the in vitro culture and immunohistochemical studies revealed that the adenoma cells produced and released PRL, in addition to TSH, alpha-subunit and GH. Immunohistochemical studies showed the presence of GH in the cytoplasm of many adenoma cells. TSH beta-positive adenoma cells were less frequently seen than GH-positive adenoma cells. No cells showed the coexistence of GH and TSH beta, and a few cells were positive for PRL. By electron microscopy, the adenoma was found to be composed of a single cell type resembling thyrotrophs, and did not have any characteristics of somatotrophs. This case was considered to be of interest, because the adenoma was ultrastructurally monomorphous, but immunohistochemically polymorphous.

We investigated the effects of a calcium antagonist, efonidipine, which was reported to dilate not only afferent arterioles but also efferent alterioles, on progression of renal failure in salt-loaded partially nephrectomized spontaneously hypertensive rats (SHR). Forty-four SHR's with 5 of 6 nephrectomy were divided into four groups: group 1 as control (n=20); group 2, efonidipine-treated (n=8); group 3, enalapril-treated (n=8); and group 4, nifedipine-treated (n=8). The rats were given these drugs and a high-salt diet (5% NaCl) for 8 weeks. During the experiment, systolic blood pressure (SBP) and daily urinary protein excretion were measured every 2 weeks. At the end of the study, serum creatinine was determined, and renal tissues were obtained for light microscopic examination. SBP was markedly reduced by 8-week antihypertensive treatment. (control, 267+/-7 mmHg; efonidipine, 181+/-7 mmHg; enalapril, 200+/-12 mmHg; nifedipine, 184+/-6 mmHg). Glomerular sclerosis developed markedly in the control group, but was partially prevented in all treated groups. Similarly, urinary protein excretion (UPE) was suppressed by efonidipine (180+/-16 mg/day) and enalapril (186+/-16 mg/day vs. 301+/-28 mg/day for control). In contrast, nifedipine failed to prevent the increase in urinary protein excretion (258+/-22 mg/day). In conclusion, efonidipine attenuates SBP increase and ameliorates glomerular injury as well as nifedipine and enalapril. Furthermore, beneficial effects of efonidipine, but not nifedipine, on proteinuria suggest that different mechanisms mediate the improvement of proteinuria; one possible mechanism could be efferent arteriolar dilation, not reported in nifedipine.

It is hard to achieve antihypertensive goal in daily clinical practice. Combination therapy is mostly applied since antihypertensive therapy with single agent has limitations. Ca channel blocker (CCB), angiotension II receptor blocker (ARB) and angiotensin-converting enzyme (ACE) inhibitor are current mainstream antihypertensives and combination therapy of CCB and ARB is especially applied. CCB shows good antihypertensive effect and has fewer side-effects. On the other hand, ARB inhibits renin-angiotensin system and excels at cardioprotective and renoprotective effect. These combination therapies are vital to achieve antihypertensive goal. In the years ahead, it is necessary to review whether or not there are differences on the effect and medical cost economy even between antihypertensive agents in the same class.

Susceptibility to Graves' disease (GD) is determined by environmental and genetic factors. The genetic susceptibility to GD is conferred by genes in the human leukocyte antigen (HLA), and several other genes unlinked to HLA are thought to contribute to the development of GD. Three recent papers described the association of GD with the CTLA-4 gene. CTLA-4 is a candidate gene for T-cell mediated autoimmune diseases because it is a negative regulator of T-cell proliferation. As CTLA-4 association with GD may be influenced by the racial composition of the population, it is important to study it in other ethnic groups. We investigated the distribution of CTLA-4 gene polymorphism in 153 Japanese patients with GD (35 males and 118 females) and 200 controls (96 males and 104 females). An A/G transition at position 49 of exon 1 was analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The distribution of genotype frequencies differs between GD and controls (chi2 = 9.46, 2 degrees of freedom, p < 0.01). The presence of at least one G allele (GG or AG) conferred an odds ratio of 2.64 (95% CI = 1.92-3.36). The present study supported the association of the CTLA-4 gene with GD in Japanese and showed that the CTLA4 gene could be one of the non-HLA linked susceptibility genes for GD.

Our purpose was to determine the effect of ovariectomy on intracellular Ca2+ mobilization and platelet aggregation in sodium induced hypertension. At the age of 12 weeks ovariectomy or sham operation was performed in female Dahl-Iwai salt sensitive rats on a 0.3% NaCl diet. Four weeks later we assessed the effects of ovariectomy and an 8% NaCl diet on agonist induced intracellular Ca2+ mobilization in fura-2 loaded platelets and platelet aggregation. Ovariectomy enhanced the increase of systolic blood pressure and heart to body weight ratio on an 8% NaCl diet. However, thrombin evoked intracellular Ca2+ was not correlated with systolic blood pressure (r = −0.338, P = .17), and was lowered by sodium loading and ovariectomy (360 ± 23 to 285 ± 9, 296 ± 10 nmol/L, P < .05). Furthermore, the ionomycin induced intracellular calcium fraction in the absence of external Ca2+ that reflected internal Ca2+ discharge capacity was reduced in ovariectomized rats compared with sham operated rats on an 8% NaCl diet (648 ± 15 v 768 ± 35 nmol/L, P < .05). The internal Ca2+ discharge capacity was inversely correlated with systolic blood pressure (r = −0.506, P = .03). In addition to the decreased internal Ca2+ discharge capacity, intracellular Ca2+-independent platelet aggregation by phorbol 12-myristate 13-acetate, a protein kinase C activator, was significantly enhanced in hypertensive rats. We concluded that ovariectomy enhanced sodium induced hypertension associated with the decreased internal Ca2+ discharge capacity and increased platelet aggregation in Dahl-Iwai salt-sensitive rats.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D 3 -1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D 3 -1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D 3 -25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D 3 -24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D 3 (rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.

It is well known that alpha 2-adrenergic agonism inhibits insulin secretion and stimulates glucagon secretion in both animal and human studies. Recently, alpha 2-adrenergic blockers (DG-5128, MK-912, and SL 84.0418) have been studied as antihyperglycemic agents in human subjects. To clarify the action mechanism(s) of these agents, we investigated the effects of alpha 2 agonists and antagonists (10(-10) to 10(-4) mol/L) and pretreatment by pertussis toxin (PTX) on glucose-stimulated insulin secretion using the hamster insulinoma cell line HIT-T15. The imidazoline-derivative alpha 2-adrenoceptor agonists clonidine and oxymetazoline at concentrations as low as 10(-8) mol/L significantly inhibited glucose-stimulated insulin secretion by 63% and 65%, respectively (P.01 for both). These inhibitory effects were abolished by 20-hour preincubation of these cells with PTX 100 ng/mL. The imidazoline-derivative alpha 2-adrenoceptor antagonist DG-5128 at a concentration of 10(-4) mol/L doubled insulin secretion with or without pretreatment by PTX (P.01 for both). Furthermore, both clonidine and oxymetazoline at a high concentration of 10(-4) mol/L stimulated insulin secretion with pretreatment of the cells by PTX (P.05 for both). These results indicate that glucose-stimulated insulin secretion is inhibited by alpha 2-adrenoceptor agonists through PTX-sensitive G-protein in HIT-T15 cells. It is also suggested that imidazoline compounds at high concentrations directly stimulate insulin secretion.

Restoration of bone loss associated with thyrotoxicosis follows normalization of thyroid function. However, the extent of bone loss and restoration remain controversial. To clarify whether bone recovery is complete, we examined lumbar and femoral bone mineral density (BMD) by dual-energy x-ray absorptiometry (DXA) in 14 thyrotoxic premenopausal women with Graves' disease and 31 premenopausal women treated for Graves' disease by subtotal thyroidectomy who had been in remission for at least 3 years. In the remission group, to exclude the influence of subclinical hyperthyroidism, thyrotropin (TSH) levels were followed and subjects with low levels excluded. Thus, all 31 subjects had normal thyroid hormone levels with transiently or persistently elevated TSH levels post-thyroidectomy. Data from the two study groups were compared with those from healthy premenopausal controls matched for age, height and weight. Mean lumbar (anterior-posterior and lateral), femoral neck, and trochanter BMDs were significantly lower in the thyrotoxic group than in controls (p.05, all four BMDs). Mean lumbar (anterior-posterior), femoral neck and trochanter BMDs were significantly higher in the remission group than in controls (p0.05, all three BMDs). At the time of DXA, the 31 remission subjects showed a significant positive correlation between lumbar BMD and TSH (p0.05) and a significant negative correlation between femoral neck BMD and free triiodothyronine (FT3) (p0.05). These observations suggest: (1) in premenopausal women, bone loss associated with thyrotoxicosis due to Graves' disease is present but is fully restored when remission is reached after subtotal thyroidectomy; (2) subclinical hypothyroidism after subtotal thyroidectomy may result in higher BMD than that of controls.

Effects of insulin on rat renal microvessels: Studies in the isolated perfused hydronephrotic kidney. Although insulin is demonstrated to decrease vascular tone, the role of insulin in renal microcirculation has not been fully determined. In the present study, the effect of insulin on renal microvascular tone was assessed using the isolated perfused hydronephrotic rat kidney. Insulin (300 µ U/ml) had no effect on the basal renal microvessel diameter. In addition, insulin did not alter myogenic (that is, pressure-induced) constriction of preglomerular microvessels, with similar magnitude of constriction observed in response to elevated renal perfusion pressure from 80 to 180mm Hg (interlobular artery, -23 ± 3% vs. -19 ± 4%; afferent arteriole, -22 ± 3% vs. -21 ± 4%, for control and insulin, respectively). In striking contrast, insulin dose-dependently reversed the norepinephrine (NE)-induced tone of interlobular arteries, afferent arterioles, and efferent arterioles, with 94 ± 9%, 104 ± 6%, and 86 ± 10% reversal at 300 µ U/ml, respectively. These vasodilator actions were markedly inhibited by N-Arg; in the presence of N-Arg, insulin (300 µ U/ml) exerted only a modest dilator action on interlobular arteries (24 ± 9% reversal), afferent arterioles (23 ± 10% reversal), and efferent arterioles (14 ± 9% reversal). A similar renal microvascular responsiveness to insulin was also observed during angiotensin II (Ang II)-induced constriction. In conclusion, the ability of insulin to dilate the renal microvasculature differs, with marked inhibitory action during NE/Ang II-induced constriction and almost no inhibition during myogenic constriction. Furthermore, the present study suggests that the insulin-induced renal vasodilation is mediated by nitric oxide.

It has been demonstrated that intramuscularly injected expression plasmid DNA is taken up by myofibers and subsequently expresses exogenous genes. In the present study, we assessed intramuscular DNA injection as a means of systemically delivering interleukin-5 (IL-5). We constructed an IL-5 expression plasmid, pCAGGS-IL-5, containing murine IL-5 cDNA under the control of the CAG promoter. The soleus muscle of mice was pretreated with bupivacaine. Two days later, mice were injected with pCAGGS-IL-5 or a control pCAGGS plasmid DNA at the same site. At 2 weeks after DNA injection, eosinophils had increased from 2-3% to 8-29% of peripheral white blood cells in 9 of 10 mice injected with pCAGGS-IL-5, while eosinophils never exceeded 3% in control mice injected with pCAGGS. IL-5 mRNA was present in the muscle area injected with pCAGGS-IL-5. IL-5 was also detectable by ELISA in the sera of most mice injected with pCAGGS-IL-5, but in none of the control mice. These results demonstrate that intramuscular plasmid injection serves as a useful method of systemically delivering cytokines by combining the strong CAG promoter and bupivacaine pretreatment.

The present study was undertaken to determine whether the non-peptide V2 arginine vasopressin (AVP) antagonist 5-dimethylamino- 1[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepi ne hydrochloride (OPC-31260) produces water diuresis and improves hyponatremia in patients with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Eleven patients (9 males and 2 females, 64 +/- 3.5 yr) with SIADH were included in the present protocol, which was comprised of 3 successive days. Day 1 was a control day, and on days 2 and 3 OPC-31260 was administered intravenously. Five blood and urine collections were made at 1-2 h intervals during the 6 h observation period each day. A single administration of 0.25 and 0.5 mg/kg OPC-31260 increased the 4 h cumulative urine volume and decreased urinary osmolality to below 225 mOsm/kg H2O. Such a diuretic effect was independent of an increase in urinary solute excretions. This aquaresis by 0.5 mg/kg OPC-31260 caused a significant increase in serum sodium level by approximately 3 mEq/L. The antagonistic effect of OPC-31260 lasted for 4 h when it was given intravenously. These results indicate that OPC-31260 is an effective therapeutic agent for hyponatremia in patients with SIADH.

Leptin, the obese gene product, is secreted exclusively by adipocytes and regulates energy balance. We examined the effects of thyroid hormones on the regulation of leptin in 3T3-L1 adipocytes. Fully differentiated adipocytes were incubated with thyroid hormones, and the expression of leptin mRNA was measured by quantitative reverse transcription-polymerase chain reaction. After a 24-h incubation, triiodothyronine (T3) at 10-1000 nmol/l significantly increased the expression of leptin mRNA (237-337%). These stimulatory effects were not observed in preadipocytes. By contrast, thyroxine (T4) at 1-1000 nmol/l did not affect leptin mRNA expression in adipocytes. We also measured the levels of secreted leptin in conditioned media using radioimmunoassay. T3 at 10 and 1000 nmol/l significantly increased the levels of secreted leptin (132% and 126%, respectively) after a 24-h incubation. Our present data suggest that thyroid hormone is a novel regulator of leptin mRNA expression and protein secretion in 3T3-L1 adipocytes.

Based on our earlier animal study, we became interested to investigate if thromboxane A2 (TXA2) is involved in angiotensin converting enzyme (ACE) induced cough in man. To 11 patients with hypertension, who had developed cough induced by ACE inhibitors, a TXA2 synthetase inhibitor, ozagrel was given for 1 to 2 months together with the ACE inhibitors. One patient developed headache induced by ozagrel and was eliminated from the study after 3 weeks. In other 10 patients, no obvious drug attributable abnormality was observed in subjective and objective symptoms or laboratory tests. In ten patients, cough scores were taken just before and after the administration of a combination of an ACE inhibitor with ozagrel. Median values of cough scores after the combination was significantly (p = 0.012) lower than before the combination. Ozagrel reduced cough scores in 5 patients, completely suppressed cough in 3 patients and in 2 of 10 patients, ozagrel did not affect cough scores. Our observations suggest that TXA2 may somehow, mediate coughing induced by the ACE inhibitors. Further, patients on ACE inhibitors who develop cough may benefit from TXA2 synthetase inhibitors.

Hypertension is characterized by a complex mode of inheritance, consisting of the accumulation and interaction of major and minor genes. The existence of a single major gene locus (ht) has been demonstrated in the backcross analysis of spontaneously hypertensive rats (SHR) and normotensive Donryu rats. Intracellular Ca2+ concentration ([Ca2+]i) determines the tonus of vascular smooth muscle. It has been hypothesized that abnormal Ca2+ transport is an inheritable trait with profound influence on the development of hypertension. Backcross analysis between SHR and Donryu rats was performed to demonstrate ht and to dissect polygenic hypertensive traits through ht and abnormal intracellular Ca2+ metabolism. Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2+]i in platelets were significantly greater in SHR than in Donryu and F1 rats. The backcrossed rats were distributed into two clusters on a scattergram of blood pressure versus [Ca2+]i, demonstrating the existence of ht. The blood pressure level was correlated with thrombin-stimulated [Ca2+]i in each cluster. Increased [Ca2+]i in platelets was not coinherited with ht and was considered to be a minor inheritable hypertensive trait discriminated from ht. Therefore, [Ca2+]i in platelets is an inadequate marker for searching ht.