Microarray analysis of a reversible model and an irreversible model of anti-Thy-1 nephritis.

A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin beta10. Increased expression of thymosin beta10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin beta10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin beta10 may play an important role in the progression of kidney disease.

N-acetyl-cysteine accelerates transfer of diabetes into non-obese diabetic scid mice.

AIMS/HYPOTHESIS: Type 1 diabetes mellitus is caused by autoimmune pancreatic beta cell destruction, and the destructive process involves several molecular mechanisms including oxygen-reactive species. A cysteine derivative, N-acetyl-cysteine, is widely used as an antioxidant, but the role of N-acetyl-cysteine in the protection of pancreatic beta cells in type 1 diabetes remains unclear. The aim of this study was to clarify the effect of N-acetyl-cysteine on beta cells using an adoptive transfer system in a murine model of type 1 diabetes. METHODS: Splenocytes from diabetic female non-obese diabetic mice were transferred into female non-obese diabetic scid/ scid recipients to induce diabetes. Just after transfer, N-acetyl-cysteine was administered to non-obese diabetic scid recipients. Two weeks after transfer, the pancreas of the recipients was examined histologically, and cytokine mRNA expression in the pancreas was analysed. In vitro, CD4-positive splenocytes from diabetic donor mice were stimulated with anti-CD3 and anti-CD28 antibodies with or without N-acetyl-cysteine. RESULTS: Treatment with N-acetyl-cysteine significantly accelerated the transfer of diabetes into non-obese diabetic scid recipients. Treatment with N-acetyl-cysteine accelerated the infiltration of mononuclear cells accompanied by CD8-positive cells into the intra-islet region of the recipient's pancreas, and enhanced interferon-gamma mRNA expression in the pancreas. In vitro, treatment with N-acetyl-cysteine enhanced interferon-gamma and interleukin-2 production by CD4-positive splenocytes of the diabetic donor mice. CONCLUSIONS/INTERPRETATION: N-acetyl-cysteine accelerates the transfer of diabetes into non-obese diabetic scid mice and this effect is accompanied by the promotion of local infiltration and T-helper cell type 1 responses.

Novel resistin promoter polymorphisms: association with serum resistin level in Japanese obese individuals.

OBJECTIVES: Resistin, an adipocyte-secreted cytokine recently discovered in mice, has been proposed as a link between obesity and diabetes. We analyzed resistin gene polymorphisms and examined their association with serum resistin level and obesity phenotypes in humans. SUBJECTS AND METHODS: Sixty young, obese, non-diabetic subjects taking no medication were studied. DNA sequencing and genotyping of identified single nucleotide polymorphisms were performed. Associations between polymorphisms and serum resistin level, BMI, body composition, fat distribution, and several indices of insulin sensitivity were examined. Moreover, single nucleotide polymorphisms in the promoter region were examined for their influence on resistin gene transcriptional activity using luciferase reporter vectors. RESULTS: Ten non-coding single nucleotide polymorphisms were found. The -638G>A, -420C>G, and -358G>A polymorphisms in the promoter region showed marked linkage disequilibrium with each other, and were associated with serum resistin level; however, there was no association between these polymorphisms and parameters related to adiposity or insulin resistance. The results of luciferase assay revealed that -638G>A together with the -420C>G polymorphism influenced resistin gene transcriptional activity. CONCLUSION: We found that variability in the serum resistin level might be related to polymorphic variants of the promoter region of the gene.

Role of genetic factors (CETP gene Taq I B polymorphism and Apo A-I gene Msp I polymorphism) in serum HDL-C levels in women.

BACKGROUND AND AIM: Plasma high density lipoprotein cholesterol (HDL-C) levels are determined by a variety of environmental and genetic factors. The cholesteryl ester transfer protein (CETP) and apolipoprotein A-I (Apo A-I) are considered to be associated with HDL-C metabolism. The aim of this study was to investigate the relationship between the CETP gene Taq I B and Apo A-I gene Msp I polymorphisms and plasma lipid levels taking into account environmental factors, and to determine the combined effects of these polymorphisms on HDL-C levels in Japanese women. METHODS AND RESULTS: The study involved 270 Japanese women aged 30-69 years. We found a significant association between the CETP genotypes and HDL-C levels (p=0.0020), which were also associated with the Apo A-I gene (M1) polymorphism. Stepwise multiple regression analysis revealed that both the CETP Taq I B and Apo A-I gene (M1) genotypes were independent predictive variables. The strength of the association between the Apo A-I (M1) subgroup and HDL-C levels was reduced in the subjects with a high Body Mass Index (BMI). The combination of genotypes provided more detailed information about HDL-C levels. The "high risk" combination of the M1+ (M1+/+) and B1B1 genotypes was associated with the lowest HDL-C level (1.52+/-0.36 mmol/L), and the "low risk" combination of the M1- (M1+/- or M1-/-) and B2B2 genotypes was associated with the highest HDL-C levels (2.06+/-0.34 mmol/L). CONCLUSIONS: Our results suggest that the combination of the two polymorphisms influences HDL-C levels in women, and that the association between genetic factors and HDL-C levels is altered by environmental factors. They may also help to detect individuals with low HDL-C levels at high risk for coronary artery syndrome.

Glomerular expression of biglycan and decorin and urinary levels of decorin in primary glomerular disease.

AIMS: Recent studies have suggested that small leucine-rich proteoglycans (SLRP) of the extracellular matrix play a major role in modulating the activity of growth factors and in regulating the deposition of collagens. In this study, the expression of the SLRPs biglycan and decorin in the glomeruli of patients with primary glomerular disease (minimal change disease, IgA nephropathy, and membranous nephropathy) and urine immunoreactive levels examined. METHODS: Renal biopsy specimens were obtained from patients with minimal change disease, IgA nephropathy and membranous nephropathy. Immunohistochemical staining was performed on fresh-frozen samples using anti-biglycan and anti-decorin antibodies. Examination of urine proteoglycan excretion from a total of 26 patients and 8 normal volunteers was performed by indirect ELISA. RESULTS: In normal kidney samples, biglycan and decorin expression was found predominantly in the intrarenal arteries and tubulointerstitium, with only minimal expression in the glomeruli. Glomerular expression of these proteoglycans in glomerular disease was unchanged in all of the 4 patients examined with minimal change disease. In the case of IgA nephropathy or membranous nephropathy, some of the patients showed minimally increased immunostaining of either biglycan or decorin, but there were no signs of simultaneous upregulation of both proteoglycans. To further examine the changes in proteoglycan expression, ELISA was performed on urine samples. Urine biglycan levels were below detection levels, but high values of urine decorin immunoreactivity were found in the patients with glomerular disease. A significant negative correlation was found between urine decorin and creatinine clearance. CONCLUSION: These results suggest that distinct changes in the expression of the SLRPs biglycan and decorin may be seen in patients with primary glomerular disease. Moreover, the negative relationship between urine decorin levels and renal function supports the hypothesis that decorin may be involved in the pathophysiology of renal dysfunction in humans.

Ubc9 interacts with chicken ovalbumin upstream promoter-transcription factor I and represses receptor-dependent transcription.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.

Interactions of HLA-DRB4 and CTLA-4 genes influence thyroid function in Hashimoto's thyroiditis in Japanese population.

Hashimoto's thyroiditis (HT) is an autoimmune disease of the thyroid gland, and like many other autoimmune diseases, it is associated with the HLA and CTLA-4 gene. We have examined the distribution of the HLA DRB4*0101 allele and a CTLA-4 exon 1 A/G polymorphism in Japanese HT patients and controls and investigated possible interactions of these genes with thyroid function. Seventy Japanese HT patients and 105 controls were included in this study. HT was diagnosed on the basis of positivity for thyroid peroxidase (TPO) autoantibodies and the presence of a palpable diffuse goiter. Genotyping was performed by polymerase chain reaction (PCR)-based methods. CTLA-4-GG or -AG was more prevalent in the patients, and the odds ratio for the G allele was 4.95. The frequency of DRB4*0101-positive individuals was significantly higher in HT (odds ratio=2.17). The TSH values of HT patients at the time of diagnosis were compared to CTLA-4 genotype and HLA-DRB4*0101 positivity. They were slightly higher in the CTLA-4-AG group than in the -GG group and significantly higher in the HLA-DRB4*0101-positive group than in the -negative group (p<0.01). When the TSH values were compared in 4 groups based on positivity or negativity for HLA-DRB4*0101 and CTLA-4 GG or AG, they were found to be significantly higher in the CTLA-4-AG and HLA-DRB4*0101-positive group than in the 3 other groups (F=5.75, 3 degrees of freedom, p<0.01). These findings suggest that the interaction between the HLA-DRB4 and CTLA-4 genes determines the thyroid function of TPO-positive goitrous Japanese HT patients.

Effects of intestinal fatty acid-binding protein gene Ala54Thr polymorphism and beta3-adrenergic receptor gene Trp64Arg polymorphism on insulin resistance and fasting plasma glucose in young to older Japanese men.

The present study was performed to investigate the effects of the intestinal fatty acid-binding protein (FABP2) gene Ala54Thr polymorphism and the beta(3)-adrenergic receptor (beta3AR) gene Trp64Arg polymorphism on body mass index (BMI), blood pressure, heart rate, glucose and lipid profiles, and serum leptin level in 196 young men aged 21 to 39 years, 186 older normoglycemic men (fasting plasma glucose [FPG] < 110 mg/dL) aged 40 to 65 years, and 122 older hyperglycemic men, including 77 type 2 diabetic patients. Genomic DNA was extracted from the peripheral blood, and these polymorphisms were assessed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. In the older groups, the beta3AR Arg64-allele frequency tended to be lower and the FABP2 Thr/Thr54 genotype frequency tended to be higher in hyperglycemic patients, although these differences did not reach statistical significance. Also, there were no significant differences in the genotype or allele frequency of either variant between the 27 hyperlipidemic and 204 normolipidemic subjects. In the younger group, there were no significant differences in any of the parameters measured between the genotypes of beta3AR or FABP2. In the older normoglycemic subjects, heart rate was significantly lower (P =.037) in beta3AR Arg64-positive subjects, and FPG was significantly higher in subjects with the FABP2 Thr/Thr genotype than the other genotypes (99.8 +/- 5.6 v 96.5 +/- 5.6 mg/dL, P =.010). In the older hyperglycemic group, the beta3AR Arg64-positive group had significantly lower high-density lipoprotein (HDL) cholesterol and free fatty acid (FFA) levels (P =.024 and P =.043, respectively). There were no synergistic effects of these 2 variants on any measured parameter, but only the FABP2 Thr/Thr genotype was related to a higher FPG in the older normoglycemic men. In conclusion, no major difference was associated with the beta3AR Trp64Arg or FABP2 Ala54Thr polymorphism in terms of type 2 diabetes or hyperlipidemia in young to older Japanese men. However, a slight but significant increase in FPG was observed in older Japanese men with the FABP2 Thr/Thr genotype.

Expression profiles of COUP-TF, DAX-1, and SF-1 in the human adrenal gland and adrenocortical tumors: possible implications in steroidogenesis.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF), DAX-1, and steroidogenic factor-1 (SF-1) are orphan members of the nuclear hormone receptor superfamily. COUP-TF and DAX-1 have been shown to negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic cytochrome P450 genes. We therefore examined the expression levels and immunolocalization of COUP-TF, DAX-1, and SF-1 in human adrenal gland (NL) and adrenocortical adenomas, and compared the results with CYP17 expression levels and its enzyme activities to study their potential correlation with adrenocortical steroidogenesis. In NL (n = 10), expressions of COUP-TF, DAX-1, and SF-1 were detected in the nuclei of adrenocortical cells, but not in the medulla. In cortisol-producing adenomas causing Cushing syndrome (CS, n = 20), CYP17 expression was upregulated (298 +/- 2% vs NL 98 +/- 4%), whereas expression levels of both COUP-TFs (COUP-TFI, 52 +/- 5% vs NL 98 +/- 4%; COUP-TFII, 18 +/- 4% vs NL 98 +/- 4%) and DAX-1 (42 +/- 4% vs NL 100 +/- 4%) were reduced. In deoxycorticosterone-producing adenomas (DOC, n = 2), on the other hand, CYP17 expression was extremely reduced (8 and 12% vs NL 98 +/- 4%), whereas DAX-1 expression increased markedly (350 and 360% vs NL 100 +/- 4%). Expression levels of SF-1 did not differ between NL (100 +/- 8%) and CS (106 +/- 10%), but its expression appeared to be decreased in DOC (25 and 20%). These results showed CYP17 expression to be upregulated and downregulated in CS and DOC, respectively, in a manner reciprocal to that of its repressors, COUP-TF and/or DAX-1. In summary, the results indicate that co-localization of COUP-TF, DAX-1, and SF-1 in NL was lost in adrenocortical tumors and that these orphan receptors play an important role in the regulation of steroidogenesis in human adrenals.

Secretion of GIP in responders to acarbose in obese Type 2(NIDDM) patients.

Acarbose has been shown to reduce postprandial hyperglycemia and to improve lipid parameters in diabetics via its inhibitory effects on intestinal alpha-glucosidases. Response to acarbose may therefore be dependent upon gastric or pancreatic hormone function. To test this hypothesis, we treated 27 mild type 2 (NIDDM) Japanese diabetics who were mildly obese with low-dose acarbose (150 mg/day) for 3 months. We then performed a responder analysis to determine specific hormonal responses that may be associated with a good response to acarbose. At the end of the treatment period, a total of 15 evaluable patients was grouped as responders (n=6) and nonresponders (n=9) based on an effective decrease in postprandial glucose levels (>30 mg/day) and glycosylated hemoglobin (HbA1c) levels (>0.5%). There were no differences between the two groups in demographic variables or mean postprandial glucose levels at baseline. There was a small but significant increase in postprandial cholecystokinin (CCK) in responders, and fasting gastric inhibitory peptide (GIP) levels were significantly increased in responders and all patients after treatment. Serum leptin levels were reduced by treatment in our mildly obese responders and this was associated with a significant decrease in body weight. These results suggest that treatment with low-dose acarbose may reduce hyperglycemia in mild type 2 Japanese patients and may improve metabolic control by regulating hormones involved in glycemic control and digestive absorption. Acarbose may provide a safe adjunct to help treat insulin resistance in type 2 patients.

Effects of renal sorbitol accumulation on urinary excretion of enzymes in hyperglycaemic rats.

We studied the effects of epalrestat, a specific inhibitor of aldose reductase, on renal sorbitol accumulation and the resulting urinary enzyme excretion in hyperglycaemic rats. The activities of proximal tubule-derived enzymes such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), gamma-glutamyltranspeptidase (GGT) and dipeptidyl aminopeptidase IV (DAPIV) in urine were determined in five groups of male Wistar rats (each n = 7): (a) 0.9% saline-loaded, (b) 10% glucose-loaded, (c) 10% glucose-loaded with epalrestat pretreatment, (d) 10% mannitol-loaded and (e) 10% mannitol-loaded with epalrestat pretreatment. Epalrestat was given mixed in chow at a dose of 50 mg/kg body weight. Urinary NAG, AAP, GGT and DAPIV activities were significantly increased (P<0.005, P<0.05, P<0.01, P<0.01, respectively) by the induction of hyperglycaemia. In contrast, enzyme excretion was not increased in the mannitol- or saline-loaded groups. Pre-treatment with epalrestat completely prevented the increased urinary excretion of NAG, AAP and GGT. At the end of the infusion study, renal cortical glucose concentrations of the glucose-loaded groups with and without epalrestat pretreatment were approximately fivefold higher than those of the mannitol- or saline-loaded groups (P<0.005 each). Renal cortical sorbitol concentrations of the glucose-loaded group was also approximately twofold higher than those of the mannitol- or saline-loaded groups (P<0.01 each). However, in the group that received both glucose and epalrestat, renal cortical sorbitol concentration was not increased. These results suggest that accumulation of intracellular sorbitol leads to proximal tubular cell dysfunction and abnormal enzymuria.

Adult T-cell leukemia with hypercalcemia-induced metastatic calcification in the lungs due to production of parathyroid hormone-related protein.

A 60-year-old man was diagnosed as adult T-cell leukemia with severe hypercalcemia because of production of parathyroid hormone-related protein. After admission, the patient had respiratory insufficiency with an infiltrative shadow in his lungs suggestive of pneumonia. However, neither improvement in respiratory function nor disappearance of the abnormal chest shadow was observed with administration of various antibiotics. An autopsy demonstrated the chest shadow had been caused by metastatic calcification associated with hypercalcemia due to production of parathyroid hormone-related protein. The possibility of metastatic calcification should be considered in patients with adult T-cell leukemia and hypercalcemia who have an abnormal chest shadow.

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression.

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.

Mild renal dysfunction is associated with insulin resistance in chronic glomerulonephritis.

BACKGROUND: Insulin resistance is associated with advanced and moderate chronic renal failure (CRF). However, insulin resistance in chronic glomerulonephritis (CGN) before onset of frank renal dysfunction is not fully evaluated. We attempted to investigate the association of insulin resistance with mild renal dysfunction and with abnormal calcium homeostasis. PATIENTS AND METHODS: Eighteen young, lean non-diabetic male patients with biopsy-proven CGN (age 30 +/- 7 years, body mass index 23.0 +/- 2.5 kg/m2) were enrolled. Insulin sensitivity was estimated by the glucose infusion rate (M value) during euglycemic hyperinsulinemic clamping for 60 to 120 min. Calcium-related parameters including intracellular calcium concentrations ([Ca2+]i) in platelets were also measured. Renal function was normal or slightly impaired (serum creatinine, 1.0 +/- 0.2 mg/dl; glomerular filtration rate (GFR), 68 to 131 ml/min/1.48 m2). We divided subjects into an insulin-sensitive (IS) group (M value > 7.3 mg/kg/min, the overall mean) and an insulin-resistant (IR) group (M value < 7.3 mg/kg/min). RESULTS: During a 75 g oral glucose tolerance test, the plasma glucose concentration at 120 min after glucose loading and the immunoreactive insulin concentration at 60 min were significantly higher in the IR group. GFR was notably lower in the IR group than in the IS group (p = 0.0003), and was significantly correlated with insulin sensitivity (p < 0.02, r = 0.58). The basal [Ca2+]i was significantly higher in the IR than in the IS group (39 +/- 9 vs. 30 +/- 9 nM, p < 0.05). CONCLUSION: Mild renal dysfunction and elevated basal [Ca2+]i are associated with insulin resistance in CGN.

The effects of calcium channel blockers on nuclear factor kappa B activation in the mesangium cells.

It has been reported that calcium channel blockers (CCBs) have an inhibitory action on cell growth and transcriptional changes induced by cytokines and hormones. In this study, we examined the effects of CCBs on nuclear factor kappa B (NFkappaB), which plays a key role in the intracellular signaling of various growth factors and cytokines. The activity of NFkappaB was determined by luciferase assay with the transfection of the reporter gene, which has six NFkappaB-recognizing sequences in the upstream of herpes simplex virus thymidine kinase promoter. In cultured human mesangial cells, increased intracellular calcium concentration by calcium ionophore, A23187, showed a stimulatory effect on the phorbor 12-myristate 13-acetate (PMA)-induced activation of NFkappaB, while L-type calcium channel agonist, Bay K 8644, did not have any significant effects on either basal or PMA-stimulated activity of NFkappaB. At a higher concentration (10 microM), nifedipine, verapamil, or efonidipine showed an inhibitory effect on the activation of NFkappaB by PMA and A23187, while at a lower concentration (1 microM), only efonidipine showed a significant inhibitory effect. From these results, we conclude that CCBs have an inhibitory effect on NFkappaB via the independent pathway of an L-type calcium channel and that the potency of this effect is variable among L-type calcium channel blockers.

A 35-year-old man with cerebral hemorrhage and pheochromocytoma: the second brain-dead organ donor in Japan.

A 35-year-old man was brought into the emergency room of Keio University Hospital by ambulance because of a sudden onset of coma. His Glasgow Coma Scale was 3 and his blood pressure 150/100 mmHg. CT scanning revealed a subcortical hemorrhage 8 cm in diameter. His respiration deteriorated rapidly, and an emergency craniotomy was performed for hematoma removal and cerebral decompression. Postoperatively the patient remained in a deep coma (GCS = 3) requiring respiratory support. The family presented an organ donor card previously signed by the patient, and brain death was confirmed in accordance with Japan's transplant law. As a result of two tests conducted six hours apart brain death was confirmed on the 5th postoperative day. With the family's consent, the donor's heart, kidneys and skin were removed for organ transplantation to be performed in other institutions. An autopsy was performed after the removal of the organs and skin. An extensive subgaleal hemorrhage was found in the left cerebral hemisphere, and microscopic examination revealed extensive necrosis with karyolysis of neuronal cells, but no viable neuronal cells were found in the cerebrum. The brain stem was marked by edema, hemorrhage, infarction necrosis and neuronal cell loss. The cerebellum was swollen and congested and showed autolysis of the granular layer. These findings suggested brain death syndrome with respirator brain. Other autopsy findings included a huge pheochromocytoma in the right adrenal gland, bilateral bronchopneumonia, liver congestion and fatty metamorphosis with four cavernous hemangiomas, and mild chronic lymphocytic thyroiditis. This patient was the second brain-dead organ donor and the first brain-dead patient to undergo postmortem examination in Japan.

Tubular osteopontin expression in human glomerulonephritis and renal vasculitis.

Tubulointerstitial change is a common histopathologic feature of acute and chronic glomerular diseases and is more closely correlated than glomerular damage with renal function and subsequent outcome. Monocyte infiltration is presumed to be initiated by chemoattractants and has a pivotal role in tubulointerstitial changes. Osteopontin (OPN) is a candidate as such a chemoattractant and has been shown to recruit monocytes into the interstitium of animal models of renal diseases. In this study, we investigated OPN expression by immunostaining and its correlation with clinical and histopathologic parameters in patients with immunoglobulin A (IgA) nephropathy, diffuse proliferative lupus nephritis (DPLN), and myeloperoxidase-antineutrophil cytoplasmic autoantibody-associated microscopic polyangiitis (MMP). Twenty patients with IgA nephropathy, 12 patients with DPLN, and 14 patients with MMP were studied. OPN expression, which was constitutively observed on the apical membrane of distal tubules, was upregulated in the cytoplasm of proximal and distal tubular epithelium parallel to the degree of interstitial mononuclear cell infiltration in patients with IgA nephropathy, as well as those with DPLN. CD68(+) monocyte infiltration significantly correlated with the degree of OPN expression in the tubular epithelium. Conversely, there was no apparent induction of OPN in the proximal and distal tubular epithelium of patients with MMP despite remarkable monocyte infiltration. In conclusion, these data suggest that inducible expression of OPN in the tubular epithelium seems to be associated with interstitial monocyte infiltration and subsequent tubulointerstitial changes in some forms of human renal diseases.

Oestrogen attenuates the increases in blood pressure and platelet aggregation in ovariectomized and salt-loaded Dahl salt-sensitive rats.

OBJECTIVE: To investigate the effects of oestrogen supplementation after ovariectomy on systolic blood pressure and platelet aggregation on different sodium content diet in the female Dahl salt-sensitive rats. METHODS: At 12 weeks of age, rats were ovariectomized or sham-operated and were fed either a high NaCl (8%) or low NaCl (0.3%) diet Ovariectomized rats were treated with either 17beta-oestradiol or placebo for 8 weeks, whereas sham-operated rats received placebo alone. After 8 weeks, the systolic blood pressure and platelet aggregation were measured and analysed by two-way analysis of variance. RESULTS: The systolic blood pressure of ovariectomized rats was significantly higher than that of sham-operated rats, and this increase in systolic blood pressure was suppressed by oestrogen supplementation. Systolic blood pressure was inversely correlated with plasma 17beta-oestradiol levels (r= -0.77, P< 0.01) and with the uterus weight to body weight ratio (r = -0.47, P < 0.01). Platelet aggregation was significantly enhanced by salt loading. Salt loading and female hormonal manipulation significantly interacted on platelet aggregation. Only in Dahl salt-sensitive rats fed a low sodium diet, ovariectomy increased platelet aggregation, whereas hormone replacement did not improve it. In Dahl salt-sensitive rats fed a high sodium diet, hormone replacement reduced platelet aggregation. CONCLUSIONS: Oestrogen replacement suppresses the development of hypertension and attenuates platelet aggregatory function in the salt-loaded ovariectomized Dahl salt-sensitive rats. It has a potential to inhibit the atherosclerotic process in postmenopausal hypertension.

Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages.

BACKGROUND: The 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) is almost exclusively expressed in the kidney. However, 1alpha-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1alpha-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1alpha-hydroxylase at a molecular level. METHODS: We used the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells by treatment with phorbol ester. The expression of 1alpha-hydroxylase in THP-1 cells was examined by Northern blotting and immunoblotting using an antibody raised against a synthetic peptide corresponding to the 14 C-terminal amino acids of 1alpha-hydroxylase. We investigated the regulation of 1alpha-hydroxylase mRNA expression by RNase protection assay. RESULTS: Northern blot and immunoblot analyses confirmed the expression of 1alpha-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1alpha-hydroxylase mRNA in THP-1 cells (198 +/- 9%). 1,25-(OH)2D3, known as a suppressor of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-gamma (2000 IU/mL) increased the expression of 1alpha-hydroxylase mRNA in differentiated THP-1 cells (922 +/- 25%). CONCLUSIONS: The present results suggest that 1alpha-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney. Interferon-gamma treatment increases macrophage 1alpha-hydroxylase levels via directly increasing gene expression of this enzyme.

Urinary steroid profile in adrenocortical tumors.

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 33-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.