Shintaro Yano

Tubulointerstitial mast cell infiltration in glomerulonephritis

Mast cells are involved in chronic inflammation and tissue fibrosis. To determine whether these cells are also involved in tubulointerstitial injury in glomerulonephritis, we assayed mast cell infiltration in the kidneys of 107 patients with primary or secondary glomerulonephritis. Using a monoclonal antihuman tryptase antibody, we detected mast cells in the renal cortical tubulointerstitium, the periglomerular areas, and the medullary interstitium, but not in glomeruli. Renal cortical tubulointerstitial mast cells, including periglomerular area, were estimated as 0.8+/-1.6 cells/mm2 in minimal change nephrotic syndrome (n=7), 1.5+/-0.7 cells/mm2 in minor glomerular abnormalities without nephrotic syndrome (n=7), 6.5+/-7.7 cells/mm2 in membranous nephropathy(n=10), 12.9+/-15.5 cells/mm2 in lupus nephritis (n=15), 13.4+/-8.3 cells/mm2 in focal segmental glomerular sclerosis (n=6), 18.5+/-21.1 cells/mm2 in ANCA-related nephropathy (n=5), 19.8+/-14.2 cells/mm2 in membranoproliferative glomerulonephritis (n=5), 21.3+/-17.7 cells/mm2 in immunoglobulin A (IgA) nephropathy (n=42), and 33.0+/-33.8 cells/mm2 in diabetic nephropathy (n=10). Except for patients with the rapidly progressive glomerulonephritic syndrome (RPGN), the number of infiltrating mast cells significantly correlated with the serum concentration of creatinine at the time of renal biopsy (r=0.59; P < 0.0001) and with the intensity of tubulointerstitial injury as measured by leukocyte infiltration (r=0.72; P < 0.0001) and fibrosis (r=0.75; P < 0.0001). In contrast, mast cell infiltration did not correlate with urinary protein excretion. In relation to serum creatinine concentration, the number of mast cells was fewer in patients with RPGN than in those with chronic glomerulonephritis. These data suggest that mast cells may contribute to the renal deterioration in glomerulonephritis by inducing chronic tubulointerstitial injury.

SULFITE IS RELEASED BY HUMAN NEUTROPHILS IN RESPONSE TO STIMULATION WITH LIPOPOLYSACCHARIDE

Exposure to sulfite, a well‐known air pollutant, induces inflammatory reactions characterized by neutrophil infiltration into the airways. Using a simple and sensitive assay for sulfite concentration in biological fluids, we demonstrate herein that human neutrophils released significant amounts of sulfite (1.0 nmol/h/107 cells) in response to lipopolysaccharide (LPS), a major component of bacterial endotoxin. A large proportion of the sulfite release by neutrophils was dependent on inorganic sulfate contained in culture media, suggesting production via the sulfate reducing pathway in this response. We also show that glucocorticoids and FK506 completely inhibit LPS‐mediated sulfite release by neutrophils. Given the well‐known antimicrobial activities of sulfite, our results suggest that sulfite acts as a neutrophil mediator of host defense. A putative role of sulfite as an endogenous biological mediator is further underscored by the observation that in vivo administration of LPS is associated with a marked increase in the serum concentration of sulfite in Wistar rats. Inhibition of sulfite release by immunosuppressive agents may contribute to increased susceptibility to bacterial infection commonly associated with the administration of these drugs. J. Leukoc. Biol. 64: 595–599; 1998.

Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK.

We have previously shown that integrin‐dependent tyrosine phosphorylation of p130Cas (Cas) could be induced in a mouse fibroblast cell line that does not express focal adhesion kinase p125FAK (FAK). By analyzing FAK‐deficient (FAK−/−) cells transiently expressing Cas mutant proteins, we demonstrate here that the Src homology 3 (SH3) domain of Cas is indispensable for adhesion‐mediated Cas phosphorylation in this mutant cell line. While the FAK directly binds to Cas‐SH3, our findings imply that SH3‐binding molecule(s) other than FAK might regulate Cas phosphorylation, at least in FAK−/− cells. In this regard, we observed that FAK−/− cells expressed cell adhesion kinase β (CAKβ), a protein tyrosine kinase of the FAK subfamily. CAKβ expressed by FAK−/− cells was associated in vivo with Cas in a Cas‐SH3‐dependent manner. Moreover, integrin stimulation induces tyrosine phosphorylation of CAKβ in FAK−/− cells. Thus, our results suggest that CAKβ contributes to integrin‐mediated signal transduction in place of FAK in FAK‐deficient cells.

Increased excretion of urinary transforming growth factor beta in patients with focal glomerular sclerosis.

The urinary transforming growth factor beta (TGF-beta) excretion was measured in 33 patients including 10 with systemic lupus erythematosus (SLE), 8 with focal glomerular sclerosis (FGS), 9 with IgA nephropathy (IgAN), and 6 with membranous nephropathy (MN), and in 7 healthy subjects by enzyme-linked immunosorbent assay using a monoclonal antibody specific for TGF-beta 1 + 2 + 3. A significantly increased urinary TGF-beta excretion was observed in FGS patients (555.5 +/- 458.4 ng/mg Cr) as compared with normal controls (46.9 +/- 43.9 ng/mg Cr) (p < 0.05) and a relative increase in SLE patients (96.4 +/- 58.2 ng/mg Cr) and a decrease in MN patients (24.8 +/- 13.3 ng/mg Cr). In contrast, there was no difference in TGF-beta excretion between IgAN patients (54.1 +/- 37.4 ng/mg Cr) and normal controls. A correlation between the amount of proteinuria and TGF-beta was not found. As has been previously demonstrated in experimental studies, TGF-beta may play a similar role in human glomerular diseases. The results obtained in this study raised the possibility that extracellular matrix might be produced by glomerular cells in vivo under the control of TGF-beta and that TGF-beta might act as a stimulator for the development of glomerulosclerosis.

Increased Intraplatelet and Urinary Transforming Growth Factor-β in Patients with Multiple Myeloma

The concentrations of transforming growth factor-beta (TGF-beta) in platelets, plasma and urine from 27 patients with multiple myeloma (MM) and from 22 normal controls were measured by sandwich enzyme-linked immunosorbent assay using a monoclonal antibody specific for human TGF-beta 1+2+3. A significantly increased intraplatelet TGF-beta (24.6 +/- 9.6 vs. 17.8 +/- 8.8 ng/10(5) platelets, p < 0.01) and urinary TGF-beta (1.4 +/- 0.8 vs 1.0 +/- 0.4 ng/mg Cr, p < 0.02) were observed in MM patients compared with normal controls. The mean platelet TGF-beta level in MM patients with osteolytic lesions was more increased than in those without osteolytic lesions (29.6 +/- 7.7 vs 18.4 +/- 8.2 ng/10(5) platelets, p < 0.001). In this study, we evaluated the possible role of TGF-beta in the pathogenesis of myeloma, especially in osteolytic lesions.

Active Inhibitory Effect of Nafamostat Mesylate against the Elevation of Plasma Myeloperoxidase during Hemodialysis

Plasma levels of myeloperoxidase (MPO) were compared between hemodialysis (HD) sessions using heparin and those using nafamostat mesylate (NM) as an anticoagulant by an enzyme immunoassay established in our laboratory. MPO levels were markedly elevated during the entire HD procedure with heparin. In contrast, MPO levels were scarcely elevated during the HD with NM. On the other hand, polymorphonuclear leukocyte-elastase was markedly elevated during both of these HD procedures. These observations indicated that NM selectively attenuated MPO elevation in vivo during HD. This inhibitory effect of NM was further investigated ex vivo. Blood samples from a normal subject were collected with heparin alone, NM alone and a mixture of heparin and NM. Each sample was then circulated in a closed circuit composed of a dialyzer with a cuprophane membrane. MPO levels with heparin alone were shown to markedly rise in the closed system. In contrast, levels of MPO in the blood samples mixed with NM were not elevated even in the presence of heparin. These ex vivo results indicate that NM has an active inhibitory effect on the elevation of plasma MPO induced by granulocyte activation through a dialysis membrane. Our results demonstrate that clinical use of NM as an anticoagulant serves to selectively suppress MPO elevation considered as a consequence of granulocyte activation during HD.

A Case of Cimetidine-Induced Acute Tubulointerstitial Nephritis Associated With Antineutrophil Cytoplasmic Antibody

We present a case of acute tubulointerstitial nephritis (ATIN) that developed in a 63-year-old man who had been taking cimetidine for treatment of a gastric ulcer. The constellation of clinical, laboratory, and histopathologic findings suggested drug-induced ATIN. Of interest, the patient had antineutrophil cytoplasmic antibody (ANCA) in his sera, reactive with myeloperoxidase, elastase, and lactoferrin. Prominent renal histological features included marked plasmacyte infiltration into the renal interstitium. Withdrawal of cimetidine resulted in complete resolution of renal findings, and the titers of ANCA concomitantly declined. Thus, cimetidine may have played a causative role in the development of ANCA-associated ATIN.

Metabolism of Transforming Growth Factor-β in Patients Receiving Hemodialysis Especially Those with Renal Osteodystrophy

We evaluated the intraplatelet and plasma levels of transforming growth factor beta (TGF-beta) in patients with or without renal osteodystrophy (ROD) who were undergoing hemodialysis (HD). Intraplatelet and plasma levels of TGF-beta were examined before and after HD, and compared with those from healthy controls. Patients undergoing HD had significantly higher mean intraplatelet levels of TGF-beta before and after HD than did the healthy subjects (22.7 +/- 7.8 and 29.5 +/- 15.8 vs. 18.7 +/- 7.9 ng/10(5) platelets; p < .05). The mean intraplatelet and plasma levels of TGF-beta in patients after HD were significantly increased than those before HD and in healthy subjects (p < .05). Moreover, patients with ROD showed a significantly higher mean intraplatelet and plasma levels of TGF-beta than that without ROD (p < .05). To investigate the effects of TGF-beta on ROD in HD patients, we evaluated such parameters as parathyroid hormone (PTH) and alkaline phosphatase (ALP), which reflect the lesions of ROD. The mean intraplatelet level of TGF-beta was not correlated with either para-meter. Meanwhile, no correlation was observed between the intraplatelet level of TGF-beta and the hematocrit (Hct). Similarly, no correlation was observed between the intraplatelet levels of TGF-beta and the dose of erythropoietin (EPO) administered. These findings indicate that metabolism of TGF-beta occurs during HD and overproduction of TGF-beta may play an important role in the pathogenesis of ROD.

Effects of posture on creatinine clearance and urinary protein excretion in patients with various renal diseases

To investigate the effects of a change in posture on renal function, we measured Ccr and the urinary excretion of protein, albumin, immunoglobulin G (IgG) and transferrin in 80 patients with renal disease and in 9 healthy controls. Patients and controls were studied serially while supine for 60 min; then after standing upright for 60 min. Almost all subjects showed a drop in the Ccr with standing (p < 0.01). The percent change in Ccr after standing was more remarkable in patients with glomerulonephritis vs the healthy subjects (74.0 +/- 21.9% vs 89.9 +/- 12.3%, p < 0.01). The change in urinary excretion of protein and albumin after standing in patients with membranous nephropathy (MN-N) significantly exceeded that in patients with IgA nephropathy (IgA-N) (182.1 +/- 89.3% vs 108.1 +/- 59.2% in urinary protein and 181.7 +/- 98.7% vs 113.3 +/- 40.9% in urinary albumin, p < 0.01). Urinary excretion of IgG and transferrin tended to increase after standing in those two groups, but not significantly. Results indicate that posture affects urinary protein excretion, probably via an increase of glomerulocapillary hydrostatic pressure and/or change in the permeability of the glomerular capillary walls. We recommend that comparable postures should be used when protein excretion is used as a diagnostic tool and in monitoring structural damage to glomeruli, particularly in patients with membranous nephropathy.

Role of serotonin in nephrotoxic serum nephritis in WKY rats

Our objective was to determine whether serotonin is involved in inducing nephrotoxic serum nephritis in WKY rats. After injection of antiglomerular basement membrane antiserum, urinary protein excretion was significantly decreased in rats treated with the serotonin receptor antagonist, MCI-9042, and in rats treated with p-chlorophenylalanine. Similarly, severe necrotizing lesions and crescent formation were inhibited in a dose-dependent manner by treatment with MCI-9042 and p-chlorophenylalanine. The number of intraglomerular ED-1-positive cells was increased on day 3 and thereafter in the placebo group. A significant increase in the number of crescent lesions was observed in the placebo group on day 7 and thereafter. Neither adenosine diphosphate- nor collagen-induced platelet aggregations were inhibited in platelet-rich plasma from rats treated with MCI-9042. No significant differences were observed in the production of circulating antibody and antibody deposition in rat glomeruli among the study groups. These results indicate that pathologic changes and urinary protein excretion are closely related to the presence of serotonin in nephrotoxic serum nephritis of WKY rats. Thus serotonin may play a key role in the glomerular injury in this model. Studies on the mode of action of MCI-9042 on platelet aggregation in vivo indicate that the antiplatelet effect of this drug did not contribute to the inhibition of renal injury in this experimental model. This study suggests that serotonin participates in macrophage-mediated immune injury in nephrotoxic serum nephritis of WKY rats.