Kiichiro Jinde

In situ hybridization of interleukin 6 in diabetic nephropathy

Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowmans capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN.

Significance of urinary type IV collagen in patients with diabetic nephropathy using a highly sensitive one‐step sandwich enzyme immunoassay

Urinary concentrations of type IV collagen in patients with diabetic nephropathy were measured by a highly sensitive, one‐step sandwich enzyme immunoassay. Samples from 298 patients with non‐insulin‐dependent diabetes mellitus (NIDDM) and 80 healthy controls were examined. In diabetic patients with macroalbuminuria or renal insufficiency, the concentrations of urinary type IV collagen were significantly higher than those of diabetic patients with normoalbuminuria or healthy controls (P < 0.001). Urinary type IV collagen concentration in diabetic patients with microalbuminuria was significantly higher than that in diabetic patients with normoalbuminuria or that in healthy controls (P < 0.001). In contrast, there were no significant changes in the concentration of serum type IV collagen between microalbuminuric patients and normoalbuminuric patients. The area under the receiver operating characteristic (ROD) curve for the urinary type IV collagen concentration was equivalent to that of urinary albumin. It was concluded that urinary type IV collagen concentration determined using this method might be a useful marker for the early detection of diabetic nephropathy. J. Clin. Lab. Anal. 11:110–116.

Follow-up study on urinary type IV collagen in patients with early stage diabetic nephropathy

Type IV collagen is a major component released from the glomerular and tubular basement membranes. To investigate the alteration of renal type IV collagen turnover in early stage diabetic nephropathy, urinary type IV collagen was measured by a highly sensitive one‐step sandwich enzyme immunoassay (EIA). Urinary samples were obtained from 94 diabetic patients without overt proteinuria. Among those patients, 61 were normoalbuminuric and 33 patients were in the microalbuminuric group. Levels of urinary type IV collagen were serially examined at the start of this study and again one year later. The levels of urinary type IV collagen in patients in the microalbuminuric group were significantly higher than those in the normoalbuminuric group (P < 0.01). There was a significant correlation between the concentration of urinary albumin and urinary type IV collagen in both groups (P < 0.05). Twenty‐eight patients (45.3%) in the normoalbuminuric group who showed an abnormal elevation of urinary type IV collagen in comparison to the reference range of normal healthy adults (normal range; less than 3.5 μg/g · Cr). Seven (25%) out of these 28 normoalbuminuric patients with increased urinary type IV collagen progressed to the microalbuminuric group one year later. The levels of urinary type IV collagen in such patients were significantly increased. In the 21 patients who stayed within the normoalbuminuric group, the urinary type IV collagen levels were significantly decreased one year later. It appears that the levels of urinary type IV collagen might reflect ongoing alteration of the extracellular matrix (ECM) turnover and might define more specifically the early stage diabetic nephropathy than the detection of microalbuminuria. It is concluded that the serial measurement of urinary type IV collagen can be a useful marker for detecting renal injury in diabetes. J. Clin. Lab. Anal. 12:378–382, 1998.

Costimulatory molecules CD80 and CD86 in human crescentic glomerulonephritis

BACKGROUND CD80 and CD86, cell-surface molecules found only on antigen-presenting cells (APCs), are required for activation of CD4-postitve (CD4+) T cells by interaction with CD28/cytotoxic T-lymphocyte-associated antigen 4 on T cells. The roles of these molecules in human glomerulonephritis (GN) presently are unknown. METHODS Twelve cases of crescentic GN, thought to be a T helper cell-directed delayed-type hypersensitivity-like injury, and 10 controls with non-immunoglobulin A proliferative GN were used. Expression of CD80, CD86, CD4, CD14, CD68, HLA-DR, and intercellular adhesion molecule-1 was investigated in renal tissues using monoclonal antibodies and compared with clinical data at the time of renal biopsy. RESULTS CD80+ and CD86+ cells were observed significantly more in crescentic GN than in controls. CD86 was expressed in the glomerulus and interstitium, especially in the crescent, and adhesion to Bowmans capsule and periglomerular areas corresponding to these changes. Tubular epithelial cells showed no CD86 expression, but they expressed CD80, and some of them expressed HLA-DR. CD4, CD14, CD68, and CD86 showed similar distribution patterns. Positive correlations were found between CD86+ cells and CD4+, CD14+, and CD68+ cells. The number of interstitial CD86+ cells correlated with deterioration of renal function. Most CD86+ cells were monocyte/macrophages. CONCLUSION This study suggests that the costimulatory molecules CD80 and CD86 have different expressions in human crescentic GN, and CD86 is concerned with crescent formation and CD4+ T-cell accumulations. The majority of APCs are macrophages, and tubular cells also can act as APCs.

Immunofluorescence staining of renal biopsy samples in patients with diabetic nephropathy in non-insulin-dependent diabetes mellitus using monoclonal antibody to reduced glycated lysine

This is the first report on immunofluorescence staining of renal biopsy samples in human diabetic nephropathy (DN) using monoclonal antibodies to reduced glycated lysine. In order to detect the localization of glycated lysine in the mesangial matrix and/or the glomerular basement membrane (GBM), we examined immunofluorescence staining using antibodies against reduced glycated lysine in the glomeruli of 16 patients with DN and ten age-matched patients with diffuse mesangial proliferative glomerulonephritis without IgA deposition (DPGN) as controls. In the early stage of DN, immunofluorescence microscopy revealed the presence of intense staining for reduced glycated lysine in the GBM as well as in part of the tubular basement membrane, but not in the mesangial area. In contrast, immunofluorescence microscopy revealed less staining for glycated lysine in the GBM in the advanced stage of DN, and no reaction with any part of the renal tissue in patients with DPGN. It was concluded that detection of reduced glycated lysine in GBM in the early stage of DN might be associated with the initial pathogenesis of this disease.

Value of pathological grading in prediction of renal survival in IgA nephropathy

Summary: In order to clarify the most reliable risk factor to predict renal outcome, 206 patients with IgA nephropathy were studied for mean period of 9.2 years. the histopathological changes of this disease using light microscopy were divided into four grades (grade 1–4). These grades included glomerular, interstitial and vascular lesions. the cumulative rate of kidney survival progressing to end stage renal failure (ESRF) in all patients was 94% at 5 years, 87% at 10 years and 80% at 15 years after renal biopsy. None of the patients in grade 1 reached ESRF. the cumulative rate of kidney survival in grade 2 was 99% at 5 years, 98% at 10 years and 89% at 15 years after renal biopsy. In grade 3, it was 94% at 5 years, 79% at 10 years and 75% at 15 years. In grade 4 it was 53% at 5 years, 33% at 10 years and 22% at 15 years after renal biopsy. Forward stepwise multivariate regression analysis revealed that, in addition to the histopathological findings, three more risk factors were found to influence actuarial renal survival rate. These factors were: (i) the levels of serum creatinine; (ii) the level of serum albumin; and (iii) the amount of proteinuria at the time of renal biopsy. In parallel studies, forward stepwise multivariate regression analysis isolated three risk factors that influenced the progression of the reciprocal of serum creatinine. These factors were: (i) the levels of total protein; (ii) the degree of our pathological grading; and (iii) the amount of proteinuria. It was concluded that our pathological grading was useful as a prognostic parameter because of its simplicity and availability in routine clinical activities.

Significance of high levels of serum IgA and IgA-class circulating immune complexes (IgA-CIC) in patients with non-insulin-dependent diabetes mellitus

Significance of serum IgA and IgA-class circulating immune complexes (IgA-CIC) elevation in patients with non-insulin-dependent diabetes mellitus (NIDDM) was described. Seventeen patients with NIDDM and 17 patients with diffuse mesangial proliferative glomerulonephritis without deposition of IgA (DPGN) as controls were examined. The levels of serum IgA in patients with NIDDM were significantly higher than those in patients with DPGN (p < or = 0.01). The levels of IgA-CIC in patients with NIDDM were also significantly higher than those in patients with DPGN (p < or = 0.01). Production of IgA derived from B cells and the proportion of IgA bearing B cells in patients with NIDDM were not significantly higher than those in patients with DPGN. Furthermore, the levels of IgA in pharyngeal washings from diabetic patients were not significantly higher than those for DPGN patients. Duration of diabetes, the level of HbA1c, and the presence of hypertension, microalbuminuria, or retinopathy showed no significant correlations with the levels of serum IgA or IgA-CIC in patients with NIDDM. It was postulated that the elevations of serum IgA and IgA-CIC were based on subclinical infection of the mucosa and/or deterioration of IgA clearance in patients with NIDDM.

Activation of mRNA expression of collagen, collagenase and its inhibitor on renal biopsy specimens in patients with IgA nephropathy

Summary: In situ hybridization of mRNA for collagen IV, collagen VI, stromelysin (MMP‐3) and TIMP1 was examined in renal biopsy specimens from patients with IgA nephropathy (IgAN) or diabetic nephropathy with various degrees of tissue damage. The majority of cells in the glomeruli expressed these mRNA almost simultaneously, but a few cells demonstrated positive expression for only one of these probes. There was a parallel relationship between the degree of tissue damage and that of mRNA expressions of these probes in patients with IgAN, while patients with diabetic nephropathy showed a reverse relationship between these two parameters. It is concluded that patients with mesangial proliferative glomerulonephritis expressed mRNA for collagen collagenase and its inhibitor in the glomeruli in parallel with the progress of tissue damage. In contrast, glomerular samples from patients with diabetic nephropathy showed that there was an inverse relationship between tissue damage and expression of mRNA. It is concluded that expression of collagen, collagenase and its inhibitor parallels the progression of glomerular changes in IgAN, but such parallel expression was not observed in patients with diabetic nephropathy.

The roles of costimulatory molecules CD80 and CD86 in IgA nephropathy

Background: IgA nephropathy (IgAN) is a most common disease in human nephritis. Renal dysfunction is considered relating to the extent of interstitial infiltrating cells. These cells are mainly activated T cells and macrophages. T cells activation requires not only the binding of antigen/MHC-II with T cell receptor (TCR) but also costimulatory signals. Although many surface molecules on antigen presenting cells (APC) can provide these signals such as ICAM-1, the most important costimulatory molecules are CD80 (B7-1) and CD86 (B7-2). They can activate T cells by binding their receptor CD28 on T cells and then restrain T cells activation by binding their other receptor CTLA-4 on T cells. In this study, we investigated the roles of costimulatory molecules CD80 and CD86 in IgAN and verified the types of APC. Methods: Open renal biopsies obtained from 33 patients who were diagnosed IgAN were used in this study. The histopathological changes were divided into four grades of grade 1 to grade 4 according to the mild to serious tissue damage. Three patients were grade 1, while grades 2 to 4 each had 10 patients. Ten biopsies from non-IgA mesangial proliferative glomerulonephritis (PGN) patients with minor tissue damage were used as a control. The expressions of monoclonal antibodies of CD80, CD86, CD45RO (activated T cells’ marker), CD68 (macrophages’ marker), CD14 (monocytes’ marker), HLA-DR and ICAM-1 were investigated in all renal tissues and the results were expressed as a percentage of positive tubuli, the number of intraglomerular positive cells per glomerulus, the number of periglomerular positive cells per glomerulus and the number of interstiThe roles of costimulatory molecules CD80 and CD86 in IgA nephropathy

Laminin in the interstitium indicates prognosis in patients with IgA nephropathy

Laminin is implicated in cell migration, remodelling of the extracellular matrix and basement membrane. Therefore, laminin deposition could be of concern in the progression of some renal diseases. In the present study, immunohistochemical and in situ hybridization studies were performed for laminin in IgA nephropathy (IgAN) specimens in order to reveal the importance of laminin deposition for their prognosis. Fifteen patients were used as the stable group who maintained stable renal function for more than 10 years after biopsy. Thirteen patients formed the progressive group who reached end‐stage renal failure within 10 years after biopsy. Immunohistochemically, laminin was specifically deposited in damaged glomeruli and strong immunoreactivity was particularly shown in the expanded interstitial area containing infiltrating mononuclear cells. Although the staining score in glomeruli was not significantly different between these two groups (P = 0.181), deposition in the expanded interstitium was extensive and significantly greater in the progressive group (P < 0.01). In situ hybridization revealed that most of the interstitial cells and some of the tubular cells expressed laminin messenger ribonucleic acid. In conclusion, the extent of interstitial laminin deposition was an important prediction of adverse renal outcome. This laminin could be produced by interstitial cells and/or tubular cells.