Kyoichi Tashiro

Detection of Serum IL-6 in Patients with Diabetic Nephropathy

Yasuhiko Tomino. MD, Division of Nephrology, Department of Medicine, Juntentto University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113 (Japan) Dear Sir, A study on the detection of serum IL-6 in patients with non-insulin-dependent diabetes mellitus (NIDDM) with or without nephropathy is described. IL-6 is generally regarded as a multifunctional cytokine which has a variety of biological activities, including the ability to stimulate bone marrow stem cell proliferation, B cell differentiation, immuno-globulin secretion, T cell activation, and acute phase protein synthesis [1, 2], IL-6 is also produced by the renal glomerular mesan-gial cells. Cytokines are known to play an important role in autoimmunity and appear to be involved in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). However, Cavallo et al. [3] reported that detectable levels of serum IL-6 were observed in only 10% of IDDM patients. Serum samples were obtained from 9 patients with NIDDM with nephropathy (diabetic nephropathy), 9 patients with NIDDM without nephropathy and 29 patients with chronic glomerulonephritis (CGN). NIDDM was diagnosed with a 75-gram glucose tolerance test. Patients with diabetic nephropathy continuously showed more than 200 mg/24 h. Serum IL-6 levels were measured with ELISA as described previously [4]. Mouse monoclonal anti-IL-6 antibody (HH61-10) and monoclonal horse radish peroxidase-conjugated anti-IL-6 antibody (HH61-2 Fab’) were used in a double-antibody sandwich ELISA [5]. Levels of serum IL-6 of healthy controls were less than 4.0 pg/ml [5]. The mean levels of serum IL-6 in all patients with NIDDM were significantly higher than those in patients with CGN (p < 0.05). The levels of serum IL-6 in patients with diabetic nephropathy were significantly higher than those in cases of CGN or NIDDM without nephropathy (p < O.Ol and p < 0.05, respectively; table 1). It appears that the presence of IL-6 in the patients’ sera may reflect increased localized production of this cytokine at the pancreatic and/or glomerular me-sangial levels. The measurement in serum IL-6 may add

Detection of Apoptotic Cells in Glomeruli of Patients with IgA Nephropathy

The purpose of the present study was to determine the relationship between the existence of apoptotic cells in glomeruli, clinical or histopathological findings and response to treatment in patients with IgA nephropathy. Renal biopsy specimens were obtained from 23 patients with IgA nephropathy. These patients were divided into two groups: mild glomerular damage (12 patients), and severe glomerular damage (11 patients). The nick end-labelling method (TUNEL) and fluorescent staining (Hoechst 33258) were used for the detection of apoptotic cells. Five of 6 patients with apoptotic cells in the glomeruli detected by TUNEL were in the severe glomerular damage group, and only 1 patient was in the mild glomerular damage group. Apoptotic cells in glomeruli were also detected by fluorescent staining in 3 of 5 patients in the severe glomerular damage group who showed apoptotic cells in TUNEL. However, no apoptotic cells were detected in patients in the mild glomerular damage group in fluorescent staining. Mean levels of urinary protein excretion at the time of renal biopsy in the patients with apoptotic cells were significantly higher than those in patients without apoptotic cells (p < 0.01). The mean levels of creatinine clearance (Ccr) in the patients with apoptotic cells were slightly lower than those in patients without such cells. There were no significant differences in the levels of serum creatinine (s-Cr) and BUN in patients with or without apoptotic cells. In the severe glomerular damage group, urinary protein excretion after treatment in the patients with apoptotic cells was significantly improved compared with that in the patients without such cells (p < 0.01). It appears that the levels of proteinuria and renal function tests might be influenced by apoptosis in patients with IgA nephropathy. It is postulated that apoptosis may induce reduction of excess proliferative mesangial cells and/or infiltrated cells, and tissue repair. Thereafter, these histological alterations may improve proteinuria and renal function.

Detection of cell death of cultured mouse mesangial cells induced by oxidized low-density lipoprotein

The objectives of the present study using cultured mouse mesangial cells (MMC) were (1) to evaluate the type of cytotoxicity induced by oxidized (ox) LDL, i.e. apoptosis, necrosis and types of other cell death and (2) to investigate the pathway of cell death under incubation with antioxidants or scavenger receptor (SR) antagonists. LDH release and a morphological examination were used in this study. Trypan blue staining of MMC was performed to detect dead cells in culture. Cytotoxicity of ox-LDL in MMC was found to be dose- and time-dependent. In the morphological study of electron microscopy, three different types of cell death in ox-LDL-treated MMC were identified. In the morphological study with semithin sections, these three types of dead cells were identified at different dosages of ox-LDL. Type 1 or type 2 dead cells were observed in low dose ox-LDL or in middle-dose ox-LDL-treated MMC, respectively. Type 3 dead cells were marked in high dose ox-LDL-treated MMC. It appears that the cells were apoptotic (type 1), necrotic (type 3) and other types (type 2). The cytotoxicity of ox-LDL was not mediated by cellular internalization of ox-LDL via SRs. On the other hand, the cytotoxicity of ox-LDL was inhibited by antioxidants such as α-tocopherol, probucol, N-acetyl-cysteine or glutathione ethyl ester. It is indicated that the pathways of ox-LDL induced cell death were distinct from the pathway via SRs.

Binding Capacity of Serum IgA to Jacalin in Patients with IgA Nephropathy Using Jacalin-Coated Microplates

Binding capacity of serum IgA to jacalin in 22 patients with IgA nephropathy, 14 patients with diffuse mesangial proliferative glomerulonephritis (non-IgA nephropathy) and 20 age-matched healthy adults was examined by enzyme-linked immunoassay (ELISA) using jacalin-coated microplates. In contrast to previous findings, the binding capacity of serum IgA to jacalin in patients with IgA nephropathy measured by ELISA using jacalin-coated microplates was significantly higher than that in healthy adults. The ratio of serum IgA levels measured by this method to those obtained by single radial immunodiffusion was significantly increased in patients with IgA nephropathy. It appeared that the capacity of serum IgA binding to jacalin was marked in these patients. It is concluded that the binding capacity of serum IgA to jacalin is not ubiquitously impaired in all patients with IgA nephropathy.

A case of primary immunoglobulin light chain amyloidosis with a delayed appearance of Bence Jones protein in urine.

SUMMARY:  We report here a case of a 58‐year‐old man who had nephrotic syndrome and immunoglobulin light chain (AL) amyloidosis. This patient underwent a renal biopsy to confirm the diagnosis. Treatment with permanganate before Congo red staining showed systemic secondary amyloidosis (AA) fibrils, which were sensitive to permanganate oxidation. Although this patient was initially diagnosed as having AA amyloidosis, he did not have any chronic inflammatory disease and/or malignancy. The level of amyloid A protein (7.9 µg/mL) in sera was within the normal range (0–8.0 µg/mL). Therefore, we performed an immunostaining of the precursor protein (amino terminus of constant region: κ and λ light chains, and AA protein) using duodenal biopsy specimens for a precise diagnosis. Immunostaining was positive for the amino terminus of constant region of the λ light chain, and negative for the amino terminus of constant region of the κ light chain and AA protein. No plasma cell proliferation in the bone marrow was observed. We finally diagnosed this patient as having primary AL amyloidosis. It appears that a pathological diagnosis must be performed by immunostaining the precursor proteins with the permanganate digestion technique in tissue of patients with amyloidosis. There were no abnormalities in serum and urine immunoelectrophoresis at the time of renal biopsy in this patient. During the follow‐up period, after discharge, Bence Jones protein appeared in the urine, but not in the serum. It is necessary to observe patients with primary AL amyloidosis carefully to determine if they their condition will progress to multiple myeloma.

Effect of calcium channel blockers, nifedipine and benidipine, on death of cultured mouse mesangi al cells.

We examined the effects of the short–acting calcium channel blocker (CCB) nifedipine and the long–acting CCB benidipine on the death of mouse cultured mesangial cells induced by tumor necrosis factor alpha (TNF–α) and/or cycloheximide (CHX). Cell death was evaluated by a morphological study using semithin sections. The dead cells were divided into three types, i.e., apoptotic cells (type 1), necrotic cells (type 3) and other types of dead cells, the so–called ‘secondary necrotic cells‘ or ‘postapoptotic necrotic cells’ (type 2). In the morphological study with semithin sections, cells in the presence of TNF–α or CHX and nifedipine or benidipine showed low percentages of all dead cell types with 24 h incubation. Both nifedipine and benidipine have protective effects against TNF–α or CHX. It is postulated that CCB might inhibit the apoptotic or necrotic processes by TNF–α or CHX with 24 h incubation. With 36 h incubation, CCB increased the percentages of all types of dead cells except for treatment with 1×10–5 M benidipine and CHX. It appears that these cell–protective effects might be decreased after treatment with TNF–α or CHX and CCB for 36 h. In conclusion, the short–acting CCB nifedipine and the long–acting CCB benidipine have protective effects on mouse cultured mesangial cells against TNF–α or CHX . However, nifedipine and benidipine did not inhibit specific types of cell death using semithin sections in this study.

Tubulointerstitial Nephritis and Uveitis Syndrome Associated with Renal Tryptaseand Chymase-positive Mast Cell Infiltration

We report the clinical course and immunohistochemical analysis of a patient who presented with tubulointerstitial nephritis and uveitis syndrome (TINU syndrome). The patient, a 40-year-old woman, was referred to our hospital with general fatigue and a slight fever from another hospital. Mast cells are closely related to the development of renal interstitial fibrosis in patients with glomerulonephritis. To determine the role of mast cells in renal interstitial injury in TINU patients, we performed immunohistochemical studies on renal biopsy specimens using anti-human tryptase and anti-human chymase antibodies specific for mast cells. Double immunostaining of tryptase and chymase was also performed in renal tissues. In double immunofluorescence, cells with both chymase and tryptase (MCtc) were marked in the regions of interstitial fibrosis in this patient. It appears that mast cells are one of the constitutive cells of interstitial fibrosis in patients with TINU syndrome.

Apoptotic cells in six patients with IgA nephropathy in repeat biopsies

SUMMARY: Programmed cell death is a selective process of physiological cell deletion and is known as apoptosis. The purpose of the present study was to determine the relationship between the existence of apoptotic cells in glomeruli and the clinical or histopathological findings obtained in repeat renal biopsies of patients with IgA nephropathy. Repeat renal biopsy specimens were obtained from six patients with IgA nephropathy. The nick end labelling method (TUNEL) was used for the detection of apoptotic cells. Clinical laboratory data, i.e. urinary protein excretion, creatinine clearance (Ccr), blood urea nitrogen (BUN) and serum creatinine, (s‐Cr) were obtained from these patients. At the first renal biopsy, apoptotic cells in the glomeruli were observed in three out of six patients using TUNEL. These patients were classified as the severe glomerular damage group. The other three patients without apoptotic cells were in the mild glomerular damage group. Mean levels of urinary protein excretion at the first renal biopsy in the patients with apoptotic cells were slightly higher than those in patients without apoptotic cells. Levels of Ccr in patients with apoptotic cells were lower than those in patients without apoptotic cells. There were no significant differences in the levels of BUN and s‐Cr in patients with or without apoptotic cells. Two patients with apoptotic cells in glomeruli at the first renal biopsy did not show apoptotic cells at the second renal biopsy. These two patients showed improvement not only in clinical laboratory findings but also in histological findings at the second biopsy. Only one patient with apoptotic cells at the first and second biopsies exhibited deterioration at the second biopsy. All three patients without apoptotic cells at the first renal biopsy also showed deterioration of the clinical laboratory and histopathological findings. It is postulated that various factors other than apoptosis might induce progression of renal injuries in such patients. It appears that the clinical laboratory data, i.e. proteinuria, renal function and histopathological findings, might be influenced by apoptosis in patients with IgA nephropathy. It is postulated that apoptosis may induce reduction of excess proliferative glomerular mesangial cells and/or infiltrating cells and tissue repair.