Yoshitaka Yamamoto’s research while affiliated with Akita University and other places

We examined the activity and the characteristics of gelatinolytic enzymes in the urine from normal subjects and the patients with glomerulonephritis (GN). Gelatinolytic activity was assayed by using [3H]gelatin (heat-denatured type I collagen) as a substrate. the activity found to be 2463 ± 204 U/day (mean ± SEM, n = 17) in the urine from the healthy individuals, but was negligible in plasma samples. In GN patients (n=24) urinary gelatinolytic activity (724± 149 U/day, P < 0.001) was significantly lower compared with the healthy individuals. Partial purification (anion exchange column chromatography followed by gel filtration) revealed that two active gelatinolytic enzymes (molecular weight [Mr]92 kDa; high molecular weight [HMW] gelatinase, Mr 40 kDa; low molecular weight [LMW] gelatinase) were present in the urine obtained from the healthy individuals. the activity of these enzymes was inhibited by EDTA, 1,10-phenanthroline and α2-macroglobulin, but not by inhibitors for serine, cystein and aspartic proteinase. As the optimum pH was in the neutral range (pH 6–9), these gelatinases were considered to be neutral endometalloproteinases. These enzymes could degrade acid soluble type IV collagen and native rat glomerular basement membrane (GBM) in addition to gelatin, but not type I collagen. Immunoblotting with antibodies against human metalloproteinases (MP) identified HMW gelatinase with matrix metalloproteinase (MMP)-9 (gelatinase B) and LMW gelatinase with MMP-2 (gelatinase A), respectively. As these enzymes are metalloproteinase (MP) capable of degrading GBM and its component, urinary MP may be a useful subject as a tool searching the metabolic alteration of glomerular extracellular matrix in renal diseases.

A 70-year-old woman with type IIb therapy-refractory hyperlipidemia was diagnosed as having IgA kappa type multiple myeloma. She had neither a family history nor any other disease known to accompany hyperlipidemia. The serum IgA concentration fell from 3.42 g/dl to 1.24 g/dl following chemotherapy with melphalan and prednisolone, and a concomitant decrease in both the serum cholesterol and triglyceride levels was observed. These serum lipids were positively correlated with the serum IgA concentration (p < 0.001) during the three cycles of chemotherapy. These findings suggest the involvement of the monoclonal protein of IgA in the development of hyperlipidemia in the present case.

A 58-year-old woman with kidney-limited recurrence of microscopic polyarteritis (MPA) is described. The patient had a history of histologically-confirmed MPA 7 years previously, which had been in remission with corticosteroid treatment for 30 months followed by no medication thereafter. However, in February 1994, clinical manifestations including leg edema and proteinuria developed, followed by rapidly progressive renal insufficiency. Renal biopsy revealed crescentic glomerulonephritis with necrotizing vasculitis. Furthermore, at the same time antimyeloperoxidase antibody (MPO-ANCA) was detected in plasma. She was diagnosed as having kidney-limited recurrence of MPA without systemic presentation. Corticosteroid therapy was reinstituted, and the renal function improved, with a decrease in the titer of MPO-ANCA.

The histological localization was investigated of adrenomedullin (AM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma. The immunohistological distribution was examined of AM in human, rat, and porcine tissues using a polyclonal antibody to a fragment comprising C-terminal amino acids 40-52 of human adrenomedullin [AM(40-52)NH2]. Almost all of the human pheochromocytoma and normal adrenal medullary cells of all three species were immunostained and found to be intensely positive for AM. Furthermore, AM-immunoreactive cells were present in the pancreatic islets, gastrointestinal neuroendocrine system, anterior pituitary, and choroid plexus with some degree of interspecies heterogeneity. These findings indicate that AM-immunoreactive cells are widely distributed in the endocrine and neuroendocrine system, suggesting that AM plays some important role in the control of systemic and local circulation and also of humoral secretion.

In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca(2+)-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC50 = 50.1 microM) and catecholamines (EC50 = 63.0 microM), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP [1-20]NH2 inhibited carbachol-induced 22Na+ influx via nicotinic receptors (IC50 = 2.5 microM in a noncompetitive manner and thereby reduced carbachol-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50 = 1.0 microM) and catecholamine secretion (IC50 = 1.6 microM). It did not alter high K(+)-induced 45Ca2+ influx via voltage-dependent Ca2+ channels or veratridine-induced 22Na+ influx via voltage-dependent Na+ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca(2+)-dependent exocytosis in response to nicotinic receptor stimulation.

The plasma concentration of immunoreactive human brain natriuretic peptide (ir-BNP) was measured in 40 patients on hemodialysis (HD) and in 12 healthy subjects. Immunoreactive human atrial natriuretic peptide (ir-ANP) was also measured. The mean (+/- SE) plasma ir-BNP concentration in the patients before HD (18.4 +/- 3.4 fmol/mL) was markedly higher than that in the control group (0.39 +/- 0.08 fmol/mL). The plasma ir-BNP level was significantly decreased by HD from 18.4 +/- 3.4 fmol/mL to 10.5 +/- 2.2 fmol/mL (P < 0.001), but the latter value was still higher than the upper limit of the normal range for our laboratory. There were significant correlations between the plasma ir-ANP level and the mean blood pressure before HD (P < 0.05) and between the HD-induced changes in plasma ir-ANP level and mean blood pressure (P < 0.001). These correlations were not observed between the plasma ir-BNP level and mean blood pressure. The plasma ir-BNP level correlated with the cardiothoracic ratio and this correlation was closer to that between the plasma ir-ANP level and cardiothoracic ratio. Ultrasound echocardiographic studies in 13 patients revealed that the pre-HD state of high cardiac output was correlated by HD in association with decreases in plasma ir-BNP and ir-ANP levels. Correlations were observed between the pre-HD ir-ANP level and the interventricular septal thickness index (r = 0.68, P < 0.05) and between the change in ir-BNP level and that in left atrial diameter (r = 0.806, P < 0.001). In conclusion, BNP levels were high in HD patients compared with the control subjects and were decreased during HD. In addition, BNP and ANP levels correlated with several parameters of volume change and cardiac status.

Bovine cultured adrenal medullary cells (4 x 10(6)) contained 4266.5 +/- 370.0 fmol of immunoreactive adrenomedullin and 373.4 +/- 32.6 nmol of catecholamines. Nicotinic (but not muscarinic) receptors mediated the Ca(2+)-dependent co-secretion of adrenomedullin and catecholamines, with the molar ratio of adrenomedullin/catecholamines secreted into the medium being equal to the ratio stored in the cells. The concentration-response curve of carbachol for adrenomedullin secretion (EC50 42 microM) was similar to that for catecholamine secretion (EC50 63 microM). Reverse phase HPLC analysis showed that immunoreactive adrenomedullins in the cells and secreted into the medium were both eluted exclusively at the position almost identical to synthetic human adrenomedullin[1-52]NH2.

The pharmacokinetic properties of quinapril were evaluated in 6 hypertensive patients with normal renal function (group A), 4 hypertensive patients with mild-to-moderate renal dysfunction (group B), and 3 hypertensive patients with severe renal dysfunction (group C). Assessments were made after patients received a single oral 10-mg dose of quinapril and after they received 8 consecutive days of treatment. The peak drug level and the area under the curve for quinaprilat (the active metabolite of quinapril) were 2 to 4 times higher in group C than in group A after 8 days of treatment; this difference was statistically significant. A significant correlation between the area under the curve and serum creatinine levels was observed after the single dose and after 8 days of treatment. Based on our results, we recommend that patients with renal dysfunction receive only half the dose of quinapril administered to patients with normal renal function.

Rat brain natriuretic peptide-45 (BNP-45) is a new cardiac hormone secreted into the circulation. In order to evaluate the pathophysiologic role of BNP in the nephrotic syndrome, we investigated the plasma levels and effects of BNP in adriamycin (ADR)-induced nephrotic rats. Plasma levels of BNP rose with time and more than doubled in 3 weeks after injection. Plasma levels of BNP correlated significantly with urinary protein excretion (UPrV) and urinary protein/creatinine ratio (UPrV/UcrV). When rat BNP-45 was injected as a bolus, hypotensive, diuretic, and natriuretic effects were completely abolished in nephrotic animals even at a high dose (2.0 nmol/kg), whereas the peptide produced marked UPrV with an increase in UPrV/UcrV. These results indicate that in ADR-induced nephrosis, BNP secretion from the heart is increased. Remarkable resistance to some hemodynamic and renal effects, while prompt proteinuric effects of BNP, may contribute to the sodium and water retention and urinary protein characteristic of this disorder.

In this study, we attempted to analyze the differences between renal failure associated with methylprednisolone (MP) pulse therapy and natural deterioration of renal function that was unrelated to MP administration. Of 80 patients with renal or collagen disease who received MP pulse therapy at our hospitals, 13 were selected for the study whose serum creatinine levels increased more than 0.5 mg/dl from baseline values following therapy. Somewhat arbitrarily, 7 patients were placed in an MP-associated renal deterioration group (group 1) in which serum creatinine levels returned naturally, or following induced diuresis, to baseline levels, and 6 patients in an MP-independent natural deterioration group (group 2) in which renal function progressively deteriorated. Renal function similarly deteriorated in the two groups following pulse therapy, irrespective of the degree of crescent formation. Our data suggested that hypoproteinemia is the most important index for differentiating MP-associated renal failure from natural deterioration of renal function unrelated to MP pulse therapy. In patients that are nephrotic and have impaired renal function, worsening of renal function following pulse therapy may partly be due to transient MP-associated renal failure. On the other hand, in patients without hypoproteinemia, worsening of renal function is most likely due to active primary disease and is probably not associated with MP pulse therapy.