Toshihiko Nagasawa

Gene Transfection of H25A Mutant Heme Oxygenase-1 Protects Cells against Hydroperoxide-induced Cytotoxicity

Heme oxygenase (HO)-1 is a stress-inducible enzyme protecting cells against oxidative stress, and mechanisms have been considered to depend exclusively on its enzyme activity. This study aimed to examine if the protein lacking catalytic activities could also display such resistance against oxidative stress. Stable transfectants of rat wild type HO-1 cDNA (HO-1-U937) and those of its H25A mutant gene (mHO-1-U937) were established using human monoblastic lymphoma cell U937. HO-1-U937 and mHO-1-U937 used in the study exhibited similar levels of the protein expression, while only the former increased enzyme activities. HO-1- and mHO-1 U937 cells became more and less sensitive to H2O2than mock transfectants, respectively; such distinct susceptibility between the cells was ascribable to differences in the capacity to scavenge H2O2 through catalase and to execute iron-mediated oxidant propagation. On the other hand, both cell lines exhibited greater resistance to tert-butyl hydroperoxide than mock transfectants. The resistance of HO-1-U937 to hydroperoxides appeared to result from antioxidant properties of bilirubin, an HO-derived product, while that of mHO-1-U937 was ascribable to increased contents of catalase and glutathione. These results provided evidence that gene transfection of the activity-lacking mutant HO-1 protects cells against oxidative stress through multiple mechanisms involving up-regulation of catalase and glutathione contents.

Alteration of redox state of human serum albumin before and after hemodialysis.

Background:Persistent hypoalbuminemia is a predictor of death in long-term maintenance hemodialysis patients, although cardiovascular diseases remain the leading cause of death. A decreased serum antioxidant activity in maintenance hemodialysis patients may contribute to increased oxidative damage, and may be associated with accelerated atherosclerotic changes. Methods:The aim of this study was to examine the redox state of human serum albumin in maintenance hemodialysis patients by high-performance liquid chromatography (HPLC) using a fluorescence detector. Results: HPLC of human serum albumin on a Shodex-Asahipak ES-502N column at pH 4.85 showed a clear resolution of human mercaptalbumin (HMA) and nonmercaptalbumin (HNA), which are the reduced and oxidized forms of human serum albumin, respectively. The mean ± SD percentage of the HMA fraction of human serum albumin was significantly lower in maintenance hemodialysis patients than in age-matched normal subjects. The percentage of HMA increased 3–5 h after starting the hemodialysis and then decreased to subnormal levels. Conclusion: This suggests that serum albumin may be a major extracellular antioxidant in maintenance hemodialysis patients, and that hemodialysis may rescue serum albumin reduction by inducing intermolecular sulfhydryl-disulfide exchange reaction.

A Panel Set for Epitope Analysis of Myeloperoxidase (MPO)-Specific Antineutrophil Cytoplasmic Antibody MPO-ANCA Using Recombinant Hexamer Histidine-Tagged MPO Deletion Mutants

A major target protein of antineutrophil cytoplasmic antibody with a perinuclear staining pattern (P-ANCA) has been identified as myeloperoxidase (MPO). Recombinant deletion mutants of MPO, eight fragments of the heavy-chain subunit, and two fragments of the light chain subunit were expressed in E. coli using a pQE expression vector. The recombinant hexamer histidine-tagged fragments were partially purified as the denatured proteins on a Ni2+-charged nitrirotriacetic acid column. The recombinant fragments were reacted with a rabbit polyclonal antibody to human MPO in Western blotting. In addition, the reactivities of the proteins with MPO-ANCA-positive sera of four patients with renal diseases were examined by Western blotting. The profile of the reactivity showed that different sera recognized different sets of fragments of the heavy chain, whereas no serum reacted with the fragments of the light chain. These results indicate that the sera of patients with MPO-ANCA-positive diseases showed varied reactivities with the different fragments. Furthermore, an ELISA system using a set of the fragments completely purified by Sephacryl S-200HR column chromatography was established. The panel set is useful for subclassification of MPO-ANCA-related diseases.

Pulmonary artery involvement as first manifestation in three cases of Takayasu arteritis

Although the pulmonary artery is often involved in Takayasu arteritis, only a few cases have been reported in which pulmonary artery involvement occurred as the initial clinical manifestation. Based upon history, physical findings, laboratory data, and radiographic studies, we diagnosed Takayasu arteritis in three young females who had pulmonary artery occlusion without apparent lesions in the aortic arch and its main branches. Major symptoms and signs encountered were persistent mild or moderate chest pain, pleural effusion, and an episode of hemoptysis. Pulmonary artery occlusions were confirmed by angiography and perfusion studies. Steroid treatment relieved the symptoms in two cases, but the 3rd case required additional therapy with methotrexate for the suppression of disease activity. We emphasize the importance of considering Takayasu arteritis in cases with the above-mentioned signs and symptoms and pulmonary artery occlusion, despite the absence of aortic lesions.

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.

Influence of hemodialysis on electrocardiographic wave forms

Alterations in Frank lead electrocardiograms induced by hemodialysis were investigated in 19 patients with chronic renal failure. The most prominent findings after hemodialysis were marked increases in the magnitudes of the R wave in Leads X,Y, and Z, and of the maximal QRS vectors in the frontal, sagittal, and transverse planes. Echocardiographic and roentgenographic examinations revealed no evidence of pericardial effusion before and after hemodialysis. Although the true origin of these findings remains undetermined, it appears reasonable to speculate that the decreased intracavitary blood volume due to hemodialysis may cause an increase in the QRS voltage by a short-circuiting effect. It should be emphasized that a sudden increase in QRS amplitude after hemodialysis might lead to an erroneous diagnosis of left ventricular hypertrophy. The clinician should be aware of such voltage increases when examining the ECGs of patients who undergo hemodialysis.

Distinct interleukin-1β-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells

Interleukin-1beta converting enzyme (ICE) family proteases (caspases) are known to be implicated as important effectors of apoptotic pathways. The purpose of this study was to elucidate the role of ICE family proteases in apoptosis of mouse cells derived from the terminal proximal tubule (S3) treated with cisplatin, an anti-tumor drug, or staurosporine, a protein kinase C inhibitor. For this purpose, we measured the activities of ICE family proteases and examined the effects of tetrapeptide and viral ICE family protease inhibitors on the activities of ICE family proteases in and the degree of apoptosis of S3 cells treated with cisplatin and staurosporine. RT-PCR analysis revealed that S3 cells as well as mouse kidney express mRNA for ICE and CPP32, an ICE family protease. Results of enzymatic analysis, determination the degree of DNA fragmentation and cytotoxicity test suggest that CPP32 mediates cisplatin-induced apoptosis of S3 cells, whereas ICE family proteases other than CPP32 mediate staurosporine-induced apoptosis of S3 cells. In conclusion, distinct ICE family proteases mediate apoptosis of mouse proximal tubule cells depending on the stimuli to which the cells are exposed.

Tubulointerstitial nephritis without glomerular lesions in three patients with myeloperoxidase-ANCA-associated vasculitis

BackgroundMyeloperoxidase–antineutrophil cytoplasmic antibody (MPO–ANCA)-associated vasculitis frequently induces crescentic glomerulonephritis. However, a few cases have so far been reported to have only tubulointerstitial (TI) nephritis without any apparent glomerular lesions. We recently treated three similar cases. Therefore, their pathological features as well as clinical manifestations were studied in detail.MethodsThe pathological study was performed with immunohistochemical staining using various antibodies to the vascular endothelial cell surface markers, von Willebrand factor, type IV collagen, cytokeratin, E-cadherin, and MPO in addition to the routine histochemical examination.ResultsThe study disclosed the loss of CD34 endothelial cell surface markers with and without the destruction of type IV collagen (capillary basement membrane) in the peritubular capillaries, even though the glomeruli showed good staining of these factors. Electron microscopy showed breaks in the capillary basement membrane. The loss of CD34 staining was associated with the infiltration of a few mononuclear cells and neutrophils in the lumen of peritubular capillaries and the surrounding interstitial tissues. The cytokeratin staining in the tubular epithelial cells was also diminished around these areas. Tubulitis was demonstrated with or without the destruction of the tubular basement membrane. The clinical manifestations of these three cases were only a few red blood cells and granular casts in the urinary sediment as well as slightly increased β2-microglobulin in the urine, but no proteinuria.ConclusionBased on these findings, the loss of CD34 vascular endothelial markers occurs in the early phase of the disease because of the MPO, which is presumed to have burst out from the infiltrated, activated neutrophils. This MPO, which releases proteolytic enzymes and radical oxygen species, acts on tissue destruction, namely the lysis of endothelial cell membranes as well as vascular basement membranes in the peritubular capillary. This mechanism eventually proceeds to the destruction of the peritubular capillary walls (vasculitis). This pathogenesis is thought to play an important role in the pathogenesis of TI nephritis, which is associated with MPO–ANCA vasculitis.

Expression of MMP-9 in mesangial cells and its changes in anti-GBM glomerulonephritis in WKY rats

BackgroundMatrix metalloproteinase (MMP)-9, a member of the MMP family with specificity towards type IV collagen, is implicated in the turnover of the extracellular matrix in the kidney. To elucidate its physiological and pathophysiological significance, we examined the expression and localization of MMP-9 in the normal kidney and the changes in these features during the course of anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats, along with the changes in these features of tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-2.MethodsThe expression of MMP-9, TIMP-1 and MMP-2 mRNA was quantified by ribonuclease protection assay, and the gelatinolytic activities of MMP-9 and MMP-2 were evaluated by gelatin zymography. The localization of MMP-9 was visualized by immunohistochemistry and immunofluorescence microscopy.ResultsThe ribonuclease protection assay indicated the almost exclusive expression of MMP-9 mRNA in the glomerulus of normal kidneys. Immunohistochemistry and double-label immunofluorescence microscopy showed that MMP-9 was localized in the mesangial cells. During the course of anti-GBM glomerulonephritis, the expression of MMP-9 mRNA in glomeruli increased on day 1, peaked on days 3 to 7, and then decreased on day 14. The change in MMP-9 mRNA expression was accompanied by parallel changes in the gelatinolytic activity of the active form of MMP-9, TIMP-1 mRNA expression, and MMP-9 immunoreactivity in mesangial cells. In contrast, glomerular MMP-2 mRNA expression and its activity increased after the decline of MMP-9.ConclusionsMMP-9 mRNA was predominantly expressed in the glomerulus in normal rat kidneys and MMP-9 was present in the mesangium. The MMP-9 mRNA expression increased in the glomerulus 3 to 7 days after the induction of anti-GBM glomerulonephritis in WKY rats, in parallel with the development of abnormal glomerular histology and injury, suggesting a role of MMP-9 in proteolysis of the GBM during glomerulonephritis. MMP-2 may participate in the later phase of the nephritis.

Senescence marker protein-30 (SMP30) enhances the calcium efflux from renal tubular epithelial cells

AbstractBackground. Senescence marker protein-30 (SMP30), a calcium binding protein, is preferentially expressed in the renal proximal tubules and hepatocytes and is presumed to play a role in Ca2+ homeostasis. Methods. To explore its physiological functions in the tubular cells, we investigated the effect of SMP30 on Ca2+ efflux via the ATP-dependent plasma membrane calcium pump. LLC-PK1 cells were stably transfected with a cDNA encoding SMP30, and the established transfectants were subjected to ATP responses. Results. Overexpression of SMP30 significantly increased Ca2+ efflux under both basal and ATP-stimulated conditions. Inhibition of calmodulin by trifluoperazine abrogated the enhanced Ca2+ efflux, suggesting that SMP30 activated the calmodulin-dependent Ca2+ pump. It is known that Ca2+ superfluous influx induces cellular injury. Compared with mock-transfected cells, LLC-PK1 cells expressing SMP30 showed resistance to cellular death triggered by Ca2+ superfluous influx. Conclusion. These results suggest the possibility that, in renal tubular cells, endogenous SMP30 participates in Ca2+ efflux via activating the calmodulin-dependent Ca2+ pump and thereby confers resistance of the cells against injury caused by high intracellular Ca2+ concentrations.