Toshiaki Shibasaki

Cadmium transport by human Nramp 2 expressed in Xenopus laevis oocytes.

Using the Xenopus oocyte expression system, human Nramp2, a human intestinal iron transporter, was shown to work as a cadmium transporter. An 1824-bp human Nramp2 cDNA was constructed by PCR cloning from reverse transcription products of human kidney mRNA. When the pH of the extracellular solution was 6.0, human Nramp2 transported (109)Cd(2+). Substitution of external Cl(-) with NO3- had no effect on human Nramp2-dependent cadmium uptake. The concentration-dependent Cd(2+) transport of human Nramp2 indicated Michaelis-Menten type transport with an average K(m) value of 1.04 +/- 0.13 microM and an average V(max) of 14.7 +/- 1.9 pmol/oocyte/h (n = 3). Cd(2+) transport via human Nramp2 was inhibited significantly by Cd(2+), Fe(2+), Pb(2+), Mn(2+), Cu(2+), and Ni(2+), while it was not inhibited by Hg(2+) and Zn(2+). Transport of 0.1 microM Cd(2+) by human Nramp2 was inhibited by metallothionein (IC50 = 0.14 microM). Therefore, human Nramp2 is suggested to function as a pH-dependent cadmium absorption transporter on the luminal membrane of human intestinal cells.

Clinical Characterization of Drug-Induced Allergic Nephritis

To elucidate the clinical characterization of drug-induced allergic nephritis (DIAN), we analyzed the cases attending our department. We now report on the 14 cases of DIAN due to administration of penicillin in 2 cases, cephem antibiotics in 6 cases, nonsteroidal anti-inflammatory drugs in 4 cases and new quinolone anticidal drugs in 2 cases. With 1 exception, all these cases reached the stage of acute renal failure after taking these drugs for 2-13 days, followed by characteristic allergic symptoms such as fever, skin eruptions and serum IgE elevation in 5 of the 14 cases. A lymphocyte stimulation test (LST) with the suspected drugs proved to be positive in 10 of the 12 cases examined, and the uptake of 67Ga in the kidneys was extremely positive in all 6 cases examined, reflecting the natural course of this disease. Furthermore, there were some cases where 67Ga accumulated in the kidneys in spite of the negative result of LST examination. In all of these cases, a needle or open renal biopsy was performed, and acute tubulointerstitial nephritis (AIN) was diagnosed. Almost all cases were treated with glucocorticoid for AIN or dialysis for acute uremic symptoms several times. However, 4 of the 14 cases could not return to normal condition in spite of these forms of treatment. We would therefore like to suggest that LST and 67Ga scintigram are useful diagnostic tools for DIAN as an alternative to renal biopsy.

A randomized open-label comparative study of conventional therapy versus mizoribine onlay therapy in patients with steroid-resistant nephrotic syndrome (postmarketing survey)

BackgroundA previous double-blind 24-week clinical trial of mizoribine (MZ) vs placebo in steroid-resistant primary nephrotic syndrome (SRPNS) showed that MZ was more effective than placebo in reducing the rate of deterioration of renal function. The present study was conducted to evaluate the efficacy and safety of MZ in patients with SRPNS after 2 years’ treatment.MethodsA multicenter randomized open-label controlled trial in patients with SRPNS was conducted as a 2-year prospective postmarketing study.ResultsThere was a significant imbalance in the baseline serum albumin level (s-Alb) between the conventional therapy (CT) and MZ onlay therapy groups. Early dropouts were more frequent in the subset of patients in the CT group having a baseline s-Alb ≤3 g/dl. Therefore, the primary analysis (urinary protein level (UP)-improving effect) was performed using a mixed-effects model, with stratification according to the baseline s-Alb value. The analysis revealed that, in the subset of 34 patients with membranous nephropathy (MN) within the stratum of patients with baseline s-Alb ≤3 g/dl (n = 52), the rate of change (slope of change in the UP level/month), in terms of the log (UP+0.2), was −0.0577 in those allocated to the MZ group and −0.0227 in those allocated to the CT group (P = 0.058). In the stratum of patients with a baseline s-Alb >3 g/dl (n = 97), there were no significant differences in the UP between the two treatment groups. Hence, MZ onlay therapy was not considered to be efficacious in this group of patients. No serious adverse reactions to the drug were observed.ConclusionsThe present study yielded significant results, in that it suggested the possibility that long-term MZ therapy may afford further reduction of the UP, in addition to that obtained following CT, in particular, in MN patients in a severe nephrotic state.

Drug-induced hypersensitivity nephritis: lymphocyte stimulation testing and renal biopsy in 10 cases

The pathomorphological and clinical findings were investigated in 10 cases of drug-induced hypersensitivity nephritis. Hypersensitivity due to drugs was strongly suggested by the lymphocyte stimulation test in all patients. The offending drugs included penicillin, cephem derivatives, nonsteroidal anti-inflammatory drugs, and minocycline. All patients developed acute renal failure shortly after administration of regular doses of the drugs. Allergic symptoms plus a raised level of serum IgE or eosinophilia were seen in 7 patients. The remaining 3 patients receiving nonsteroidal anti-inflammatory drugs had no allergic symptoms, but developed severe proteinuria. Eight patients without severe glomerular damage recovered after withdrawal of the offending drugs and temporal dialysis and/or steroid therapy. Renal biopsies revealed tubulitis and tubular epithelial degeneration with interstitial edema as the common characteristic findings. Granulomatous lesions were occasionally observed. Multinucleated giant cells found in the granulomas were positive for LN-3 which is compatible with HLA-DR antigen. The glomeruli appeared normal, except in 2 cases in whom crescentic glomerulonephritis and thrombotic microangiopathy were seen. Our study suggests that the lymphocyte stimulation test and renal biopsy are the most useful means to confirm the diagnosis and provides further evidence for the participation of cell-mediated immunity in the pathogenesis of drug-induced hypersensitivity nephritis.

Effect of cadmium on gap junctional intercellular communication in primary cultures of rat renal proximal tubular cells.

Cadmium mainly accumulates in the kidney and causes renal injury. To clarify the mechanism of Cd nephrotoxicity, we investigated the effects of this element on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Sixty minutes after exposure to 100 microM Cd, dye coupling experiments showed that gap junctional intercellular communication (GJIC) was significantly inhibited. This inhibition occurred before the appearance of cytotoxicity. Intracellular calcium concentrations [Ca2+]i, which modulate the function of gap junctions, gradually increased after exposure to Cd and reached a maximum after 60 minutes. These results suggest that the inhibition of GJIC as a result of Cd exposure is related to an increase in [Ca2+]i, and that GJIC inhibition may be an indicator of nephrotoxicity.

Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

Long-term exercise down-regulates leptin receptor mRNA in the arcuate nucleus.

The purpose of this study was to investigate the effects of exercise on blood leptin concentrations and expression of leptin receptor subtype-b (Ob-Rb) mRNA in the arcuate nucleus of hypothalamus (ARC). Male Wistar rats (26 weeks old) underwent regular wheel exercise for 12 weeks. The expression of Ob-Rb mRNA in the ARC decreased at the end of the study period despite reductions of abdominal fat-pad weight and serum leptin concentration. Serum 1,5-anhydroglucitol levels were higher in exercising rats, suggesting lower serum insulin levels in exercising rats. Our results suggested that 12-week wheel exercise reduced the expression of Ob-Rb mRNA in the ARC probably through improvement in insulin resistance.

A Case of Renal Amyloidosis Associated with Hepatic Adenoma: The Pathogenetic Role of Tumor Necrosis Factor-α

We report a case of a 35-year-old man with secondary amyloidosis chiefly involving the kidney and heart. The patient showed severe proteinuria and ischemic heart damage and had hepatic adenoma at the age of 33. Biopsy specimens from the kidney, heart, stomach and rectum showed extensive deposition of amyloid. After the surgical resection of a 300-gram hepatic adenoma, highly elevated c-reactive protein (CRP) levels decreased and the serum amyloid A (SAA) level was completely normalized. Normal liver cells were immunostained with rabbit anti-SAA antibody, but the cells in adenoma tissue and kidney were not. Electron microscopic examination revealed extracellular deposition of amyloid fibrils in the hepatic adenoma and kidney tissue. The concentration of tumor necrosis factor-alpha (TNF-alpha) (312 pg/mg tissue protein) was 7-fold higher in adenoma tissue than in normal liver tissue. Moreover, SAA (2.8 ng/mg tissue protein) was 2-fold higher in normal liver tissue than in adenoma tissue. Since TNF-alpha has been known to induce SAA production in target cells, the present results suggest that the hepatic adenoma produced TNF-alpha, which then caused mainly secondary amyloidosis in the kidney and heart. Currently, 2 years after surgical resection, urinary excretion of protein has been markedly reduced (from 3.5 to 0.8 g/day) and renal and cardiac functions are normal without specific medical treatment.

A Case of Pheochromocytoma of the Urinary Bladder in a Long-Term Hemodialysis Patient

The development is reported of a pheochromocytoma of the urinary bladder in a 33-year-old patient with systemic lupus erythematosus who had undergone long-term hemodialysis. A hypertensive episode followed by defecation was demonstrated by the monitoring of blood pressure with a portable automated sphygmomanometer without elevation of plasma catecholamines. The tumor was surgically removed and normalization of the patients blood pressure followed. Thus, monitoring of blood pressure for 24 h proved useful for the diagnosis of pheochromocytoma and, in particular, of paroxysmal hypertension.

Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells.

A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH‐DXR) inhibited glutathione S‐transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH‐DXR induced caspase‐3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 μM GSH‐DXR, GST‐P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase‐3 activation and DNA fragmentation were induced following the suppression of GST‐P expression by treatment with GSH‐DXR. When the cells were treated with 100 μM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase‐3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 μM) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST‐P, and consequent prevention of DXR‐ and GSH‐DXR‐induced DNA fragmentation. Overexpression of GST‐π (placental type of human GST isozymes) by transfection of GST‐π sense cDNA into AH66 cells decreased sensitivities to DXR and GSH‐DXR, and the suppression of GST‐P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site‐directedly mutated GST‐P in which the active‐site residue Tyr39 was replaced with His (W39H) or the substrate‐binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST‐P in AH66 cells treated with GSH‐DXR must play an important role in the induction of apoptosis.