Evidence against a role of insulin in hypertension in spontaneously hypertensive rats. CS-045 does not lower blood pressure despite improvement of insulin resistance.

Hyperinsulinemia resulting from peripheral insulin resistance has been demonstrated both in spontaneously hypertensive rats (SHR) and in humans with essential hypertension. A new class of antidiabetic drugs, thiazolidinediones, which can improve insulin resistance, may be able to lower not only blood glucose levels but also blood pressure. The present study was therefore designed to clarify the proportion of SHR that are glucose intolerant and to observe the effect on blood pressure of CS-045 (troglitazone) administered at 70 mg/kg per day for 2 weeks to male SHR (n = 13). Among 67 male 8-week-old SHR, 74.6% were glucose intolerant and hyperinsulinemic. Systolic blood pressure did not correlate with plasma glucose or insulin levels before or after glucose loading. Treatment with CS-045 improved insulin resistance, as evidenced by a smaller area under the curve of plasma glucose and insulin levels in response to glucose loading. However, systolic blood pressure was not altered. When the data were reanalyzed according to the presence or absence of glucose intolerance before the treatment, blood pressure in the treated group was the same as in controls despite significant improvement in steady-state plasma glucose levels. These results suggest that hyperinsulinemia and/or insulin resistance may not be involved in the development or maintenance of hypertension in SHR, which is in contrast to models of hypertension such as obese Zucker rats or fructose-fed rats.

Insulin levels during fasting and the glucose tolerance test and Homa's index predict subsequent development of hypertension.

OBJECTIVE: To determine whether there is a longitudinal relationship between hypertension and hyperinsulinemia and to find the most useful parameter(s) for predicting the subsequent development of hypertension. SUBJECTS AND METHODS: The oral glucose (75 g) tolerance test (OGTT) was performed in 313 patients, who were divided into three groups according to glucose tolerance based on the WHO criteria: normal, borderline and diabetes mellitus. The fasting insulin (IRI) levels, sigmaIRI (the sum of the insulin levels 0, 30, 60 and 120 min after the OGTT), insulinogenic index and Homa's index, a candidate for the simple assessment of insulin sensitivity, of the normotensive and hypertensive subjects in each subgroup were compared. In addition, 145 normotensive subjects were followed up for over 3 years and observed for the development of hypertension. RESULTS: Hypertensive diabetic subjects had not only higher fasting IRI levels and sigmaIRI values, but they also had higher Homa's indices than normotensive diabetics. Normotensive subjects with normal glucose tolerance (n = 20) did not develop hypertension. However, 16 out of 94 patients with borderline glucose tolerance and five out of 31 diabetics became hypertensive. The incidence of hypertension in the group with fasting IRI > or = 15, sigmaIRI > or = 150 or Homa's index > or = 4 was between 5 and 9 times higher than that in the group with fasting IRI < 10, sigmaIRI < 100 or Homa's index < 2. This difference was still significant when multivariate analysis, including various factors such as age, body mass index (BMI) and sex, was performed. CONCLUSIONS: These results suggest that higher plasma IRI levels and/or insulin resistance are closely related to the pathogenesis of hypertension in patients with diabetes mellitus. Homa's index, fasting and sigmaIRI may be useful predictors of the subsequent development of hypertension.

Mixed heart cell-lymphocyte reactions and graft survivals in Ag-B-compatible rats.

A method of dissociation of the rat heart cell for the mixed lymphocyte culture purpose is described. The treatment of the young rat heart with 0.1% collagenase-hyaluronidase solution yielded satisfactory heart cell suspension. The hear cells, thus obtained after mitomycin C treatment but not irradiation, stimulated allogeneic lymphocytes in the presence of 2-mercaptoethanol. In the Fischer to Lewis combination compatible at the Ag-B locus, strong reactions of Lewis lymphocytes to the dissociated heart and skin cells of the Fischer rat were related to the acute rejection of heart and skin grafts, while negative reactions by the kidney and spleen cells reflected a prolonged survival of the kidney graft. The role of skin- and hear-specific antigens in the rejection phenomenon is discussed.

Long-term assessment of posttransplant renal prognosis with 31 P magnetic resonance spectroscopy.

BACKGROUND: 31P-magnetic resonance spectroscopy (MRS) has been widely used to study pretransplantation renal viability, and although some had discussed posttransplant renal viability, no one has examined long-term posttransplant renal prognosis. We discuss the use of 31P-MRS to assess the long-term prognosis from the time when MRS was performed. METHODS: We studied 20 patients with renal allografts. 1.5 Tesla clinical magnetic resonance imaging (MRI) and 15 cm surface coil was used for 31P-MRS. Localized 31P-MRS was done using image selected in vivo spectroscopy (ISIS) method. Individual peaks were fitted by Lorenzian line-shapes with a least square method and peak area ratios were calculated. RESULTS: A beta-adenosine triphosphate/inorganic phosphate (beta-ATP/Pi) ratio >1.2 had sensitivity of 92.8%, specificity of 100%, and accuracy of 95% for predicting 3-year renal survival; a beta-ATP/Pi ratio >1.2 had sensitivity of 90.9%, specificity of 66.7%, and accuracy of 76.9% for predicting 5-year renal survival. We compared 31P-MRS spectra data between the survived group and failed group. The survived group had significantly higher beta-ATP/Pi, alpha-ATP/Pi, and phosphodiester (PDE)/Pi ratios than the failed group. CONCLUSIONS: We discussed the beta-ATP/Pi value as a parameter for predicting long-term survival of a transplanted kidney from the time when MRS was performed. A value above 1.2 suggests a high probability of 3-year renal survival, whereas a value over 2.5 indicates that the transplanted kidney could survive over 5 years. 31P-MRS may be useful for predicting long-term survival of transplanted kidneys, but additional studies are needed.

Changes in trough levels of whole blood cyclosporine and graft function of a kidney transplant recipient with onset of hypothyroidism after transplantation.

The immunosuppressive and toxic effects of cyclosporine (CsA) are affected by many factors. We present the first case of a kidney transplant patient who had an onset of hypothyroidism about two months after the transplantation. In this case, trough levels of whole blood CsA and total serum cholesterol levels increased at the same time. But the decrease in renal function was not as severe as expected from the very high trough levels of whole blood CsA. Elevation of the trough level of whole blood CsA may be due to a decrease in CsA clearance resulting from the decreased cytochrome P-450 activity in hypothyroidism. Furthermore, a decrease in thyroid hormone level and increase in plasma lipoprotein level might have affected the distribution of CsA, and this change might have influenced the toxic effect of CsA In conclusion, our case suggests that thyroid function and plasma lipoprotein level should be considered important factors that affect the pharmacokinetics and action of CsA.

The prevention and correction of hypocalcemia in the parathyroidectomized rat by portacaval shunt.

Adult rats underwent end-to-side portacaval shunt either 30 days prior to or 12 days following parathyroidectomy. When portacaval shunt was performed initially, the serum calcium failed to decrease following subsequent parathyroidectomy and remained within normal levels up to 110 days. When parathyroidectomy first was done, the significant hypocalcemia was corrected subsequently by portacaval shunt and serum calcium remained close to the normal level up to 75 days. The effect of portacaval shunt depended on the calcium content of the food and was obtained only when rats were fed by a regular diet. Rats on a calcium-deficient diet were hypocalcemic, similar to the parathyroidectomized rats without the portacaval shunt. Prolonged calcium-deficient diet alone, without parathyroidectomy, did not by itself result in hypocalcemia either in the intact rat or in the portacaval shunted rat. The data indicate that portacaval shunt prevents and corrects hypocalcemia in the parathyroidectomized Lewis rat as long as sufficient calcium is available in the diet.

Metformin decreases blood pressure and obesity in OLETF rats via improvement of insulin resistance.

To determine whether improvement of insulin resistance decreases blood pressure as well as obesity, metformin (100 mg/kg/d) or vehicle was administered for 20 weeks to 12-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 10 each), a newly developed animal model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity, hyperinsulinemia, and hypertriglyceridemia. Oral administration of metformin ameliorated glucose intolerance and attenuated the insulin response to glucose loading (2 g/kg, i.p.), as evidenced by a decrease in the area under the curve for glucose and insulin at 24 weeks by 19% and 37%, respectively. At 21 weeks, systolic blood pressure was significantly lower in the metformin group than in controls (130 +/- 1.9 vs. 143 +/- 2.7 mmHg, p < 0.01), despite no difference in body weight. Subsequently, blood pressure tended to be slightly but insignificantly lower in the metformin group, and body weight was significantly lower in the metformin group (532 +/- 9.8 vs. 587 +/- 10.3 g at 31 weeks, p < 0.01). Metformin treatment also lowered the level of serum triglycerides (9.4 +/- 0.6 vs. 13.2 +/- 0.5 mmol/l, p < 0.01) and the plasma norepinephrine concentration (4,222 +/- 373 vs. 7,548 +/- 1,058 pg/ml, p < 0.01). These results suggest that metformin-induced improvement of insulin resistance in obese rats with NIDDM may lower blood pressure, as well as decrease sympathetic activity and reduce body weight.

Immunohistochemical study of human advanced glycation end-products (AGE) and growth factors in cardiac tissues of patients on maintenance dialysis and with kidney transplantation.

Cardiovascular disease is one of the most common complications of dialysis and renal transplant patients, and high levels of AGE are present in end-stage renal failure. To address the potential involvement of AGE and growth factors in the pathophysiology of cardiovascular complications, we performed immunostaining using cardiac tissues from autopsy cases of patients on maintenance dialysis (10 cases), long-term surviving renal transplant patients with functioning grafts (8 cases), control subjects with normal renal function (7 cases) and non diabetic subjects with mild renal insufficiency (8 cases). We used two types of AGE-antibodies, 6D12 [monoclonal anti-AGE antibody, recognizing N epsilon-(carboxymethyl) lysine(CML)-modified AGE] (oxidative AGE) and non-CML-PA [polyclonal, not recognizing CML], and antibodies against PDGFs, PDGF receptors and TGF beta. Positive 6D12 staining was observed in the coronary arterial walls and in macrophages. The accumulation of 6D12-reactive AGE in the coronary arterial walls of maintenance dialysis patients was significantly greater than that of control subjects (p < 0.05). Renal transplantation significantly reduced this accumulation (p < 0.05). On the other hand non-CML-PA mainly detected AGE in intracardiac arterioles and neural tissues. There was little difference in the accumulation of non-CML-AGE among the four groups. PDGFs and PDGF receptors were mainly detected in vascular endothelial cells and infiltrating cells of cardiac tissues of renal transplant patients, but not of maintenance dialysis patients. TGF beta was not detected in cardiovascular tissue of transplant patients. Our results indicated that the accumulation of oxidative AGE (CML-AGE) in the cardiac vascular tissue is one of the factors for cardiovascular complications of maintenance dialysis patients, and also that renal transplantation has a reducing effect on CML-AGE accumulation. PDGFs may be involved in the cardiovascular complications after renal transplantation.

Piroxicam-induced regression of intestinal adenomatous polyps in APC(delta474) mice.

Mutation of adenomatous polyposis coli (APC) gene results in incidence or development of polyps and colorectal cancer. It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cell growth, cause cell cycle arrest, and induce apoptosis. The aims of this study are to investigate chemopreventive effects of piroxicam and elucidate its mechanism. All APC(delta474) mice have intestinal polyps. Thirty-five APC(delta474) mice were divided into three groups: 0.005% solution of piroxicam in tap water was given for P group (n = 15) and 0.001% solution for P' group (n = 5), and water without piroxicam for C group (n = 15) from 4 weeks of age to 12 weeks, respectively. All mice were sacrificed at the 12th week after birth. Hematoxylin-eosin staining for number and size of polyps, immunohistochemical staining for cyclooxygenase (COX)-1 and -2, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), TUNEL method, and Western blot analysis of COX-2 and VEGF were performed. Polyps were divided into two types of large polyps of >or=300 microm in diameter and small polyps of <300 microm. The number of large polyps in P group decreased significantly compared with C group (p <.0001), but without significant difference in small polyps. There were no significant differences in PCNA index in both of large and small polyps among the three groups. Apoptotic index of polyps in P group increased more than those in C group (p <.05). There was immunohistochemically no significant difference in COX-1 positivity of normal intestinal epithelia and adenomas among three groups. Both numbers of VEGF-positive cells and COX-2 positive cells in the stroma of the small intestine were significantly downregulated in P group (p <.05). COX-2 expression was inhibited in dose-dependent manner without significant difference. There were no significant differences in VEGF expression between P' and C groups. In conclusion, piroxicam suppressed the development of large polyps in APC(delta474) mice by inducing apoptosis and inhibiting VEGF expression in interstitial cells of polyps.

Simplified method for cryopreservation of islets using hydroxyethyl starch and dimethyl sulfoxide as cryoprotectants.

Cryopreservation is an ideal method for long-term storage of human islets. Dimethyl sulfoxide (DMSO) has been used as an intracellular cryoprotectant. However, because of its toxicity, DMSO has to be added stepwise and diluted stepwise with sucrose. We combined hydroxyethyl starch (HES) as an extracellular cryoprotectant with DMSO to simplify the freeze-thawing procedure. Islets were isolated from the pancreas of beagle dogs by an automated digestion method and Ficoll purification. After overnight culture, the islets were cryogeneically stored using cooling by a programmed freezing system. After 4-week storage in liquid nitrogen, the container was rapidly thawed in a 37 degrees C water bath. The function of the islets was assessed upon static incubation immediately after thawing, showing a recovery rate of 71.16% +/- 20.14% and a stimulation index of 1.80 +/- 0.78. In conclusion use of HES allowed a decrease in DMSO concentration and simplified the freeze-thawing procedure for islets.

Adrenal ablation.

Of 135 patients who had bilateral adrenalectomies for metastatic breast cancer, 110 could be evaluated and 63 patients (57.3%) responded (43 objective, 20 subjective responders). Patients aged 31 to 45 years had a 56% response rate; 58% of patients aged 46 to 70 responded. Oophorectomy responders benefited from adrenalectomy 62% of the time and oophorectomy failures reponded in 38% of the cases. Patients with a disease-free interval of zero to 2.5 years responded to adrenalectomy at a rate of 46%, whereas patients with a free interval greater than 2.5 years responded at a rate of 73%. The median survival rate of 63 adrenalectomy responders was 28 months; it was ten months for 47 nonresponders.

[Clinical effect of miconazole gel against upper digestive tract mycosis].

A total of 43 patients, comprising 41 patients with oral candidiasis and 2 with esophageal candidiasis, were treated with miconazole (MCZ) gel to assess its efficacy and safety in treating upper digestive tract mycosis. The efficacy of the drug was evaluable in 33 of them, consisting of 32 patients with oral candidiasis and 1 with esophageal candidiasis. The clinical efficacy rate of the drug against oral candidiasis was 87.5% (28/32 patients), and the clinical response was good in the 1 evaluable patient with esophageal candidiasis. The safety of the drug was assessed in 40 patients. In 3 (7.5%) of them, nausea occurred as an adverse event, but was not particularly serious in any of them. No abnormal laboratory test values caused by the drug were observed. The results suggested that MCZ gel would be a very useful drug in treating oral and esophageal candidiasis.

[Analysis of genomic instability by fluorescence DNA sequencer].

Within the recent four years, there have been substantial advances in understanding the molecular pathogenesis of HNPCC. As one of the result of investigation, microsatellite instability has been observed in hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Moreover, there is strong supporting evidence that mismatch repair genes play a role in HNPCC. Here, we present our investigational results and discuss possible molecular mechanisms governing DNA mutation and genomic instability, leading to the development of neoplasm. We investigated replication error (RER) of 4 microsatellite markers (dinucleotide repeats) in 131 patients with colorectal cancer (10 met the criteria of HNPCC group B), using fluorescence-based DNA sequencer. We detected RER positivity (at more than two loci) in 12 of 131 patients (9.2%). PCR-SSCP and direct sequencing of the mismatch repair genes, hMSH2 and hMLH1, revealed that two patients in HNPCC group B had germline mutations of hMSH2. The fluorescence-based techniques, such as the present RER analysis do not require radioactive materials and specialized rooms, and can easily be performed in a clinical laboratory.

[Pharmacokinetic study of UFT in cancer patients receiving maintenance dialysis].

Six post-operative cancer patients receiving chronic dialysis (4 colorectal and 2 gastric cancer) were given daily UFT (300 mg/day tid). To determine the clearance of UFT with dialysis, the plasma concentration of tegafur, 5-FU and uracil were measured on the day with dialysis and without dialysis. The plasma concentrations of tegafur at 2 hours after oral administration of UFT showed no difference between the day with dialysis and without dialysis. The concentration of 5-FU on the next day with dialysis was almost same as that of the patients with normal renal function because of the clearance of 5-FU with dialysis, although that on the 2nd day after dialysis was higher than that with dialysis. It was difficult to compare the plasma concentrations of uracil, because they are different in each patient. Therefore, it was considered to be difficult to maintain the suitable plasma concentration of uracil. However, clearance of UFT with dialysis was good and no severe side effect was observed in administration of UFT. These data suggest that UFT can be useful drug for gastrointestinal cancer patients receiving dialysis in a post-operative adjuvant chemotherapy.

The interaction of calcitonin gene-related peptide with angiotensin II on blood pressure and renin release.

Calcitonin gene-related peptide (CGRP) has been shown to be a potent vasorelaxant. We tested the interaction of CGRP with angiotensin II (Ang II) in conscious, unrestrained Wistar rats. Rat CGRP (rCGRP, 0.1 and 1.0 nmol/kg per min) dose-dependently lowered mean arterial blood pressure and increased the heart rate. The effects continued throughout the infusion period of 30 min. Moreover, rCGRP significantly attenuated the pressor responses to Ang II (100 ng/kg per min). High-dose rCGRP (1.0 nmol/kg per min) almost abolished the pressor action of Ang II, and a much higher dose of Ang II (1000 ng/kg per min) was needed to restore the pre-infusion pressure. Plasma renin activity was dose-dependently increased by rCGRP, but was attenuated by simultaneous Ang II infusion. Rat CGRP also increased the plasma aldosterone concentration, as did Ang II. These results suggest that CGRP may have a neuromodulatory role in cardiovascular regulation.