Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis.

There is much debate over the origins of fibroblast-type cells that accumulate in interstitial fibrosis. A controversial hypothesis, supported by data from animal and cell-culture studies, is that fibroblast-type cells can derive from tubular epithelial cells by a process of epithelial-mesenchymal transdifferentiation. However, to date, no evidence supports this postulate in human glomerulonephritis. This study sought to provide evidence that tubular epithelial cells can undergo phenotypic change toward a fibroblast-like cell in human glomerulonephritis. One hundred twenty-seven open renal biopsy specimens from patients with minimal change disease (MCD), immunoglobulin A (IgA) nephropathy, and rapidly progressive glomerulonephritis (RPGN) were examined for tubular phenotypic change by two-color immunohistochemistry using the criteria of de novo expression of alpha-smooth muscle actin (alpha-SMA), a myofibroblast marker; loss of the epithelial marker cytokeratin; and collagen production. In normal human kidney and MCD, tubular epithelial cells expressed cytokeratin with no evidence of alpha-SMA staining. However, in 36 of 90 cases of IgA nephropathy and 9 of 18 cases of RPGN, small numbers of tubular epithelial cells in areas of fibrosis showed de novo alpha-SMA expression, accounting for 0.4% +/- 0.2% (IgA nephropathy) and 3.8% +/- 1.5% (RPGN) of cortical tubules. An intermediate stage of phenotypic change was observed in some cuboidal epithelial cells that expressed both cytokeratin and alpha-SMA. Tubules containing alpha-SMA-positive (alpha-SMA(+)) cells also stained for collagen types I and III, suggesting that tubular cells undergoing phenotypic change have an active role in the fibrotic process. There also was a marked increase in transforming growth factor-beta1 (TGF-beta1) tubular expression in areas with interstitial fibrosis, including tubules with phenotypic change. There was a highly significant correlation between tubular alpha-SMA expression and interstitial fibrosis, interstitial alpha-SMA(+) myofibroblast accumulation, deposition of collagen types I and III, tubular TGF-beta1 expression, and renal dysfunction. In conclusion, this study provides evidence that tubular epithelial cells can undergo phenotypic change toward a myofibroblast-like phenotype on the basis of de novo alpha-SMA expression, loss of cytokeratin, and de novo collagen staining. These data, although not conclusive, provide the first support for the hypothesis that transdifferentiation of tubular epithelial cells has a role in progressive renal fibrosis in human glomerulonephritis.

Analysis of prognostic predictors in idiopathic membranous nephropathy.

We studied clinical and histologic parameters at the time of renal biopsy of 19 patients with idiopathic membranous nephropathy (IMN) to investigate the predictors for prognosis of IMN. Nineteen patients diagnosed by open renal biopsy between 1988 and 1993 and followed for at least 5 years were divided into two groups according to the latest follow-up renal function. Group I included 16 patients with normal renal function at the last follow-up point. Group II included three patients with end-stage renal failure at the last follow-up point. Antibodies to CD68, CD45RO, alpha-SMA, collagen IV, and collagen VI were used to investigate glomerular and interstitial changes in biopsy specimens by the indirect enzyme-labeled antibody method. Degree of global glomerulosclerosis, segmental glomerulosclerosis, adhesion to Bowman's capsule, and crescent formation were evaluated by light microscopy (periodic acid-Schiff, periodic acid-metheramine [PAM] staining). The difference between the two groups was analyzed by Mann-Whitney U: test. The number of interstitial cells, the number of interstitial CD45RO-positive cells, and increases of interstitial collagen IV and VI were found to be the most important factors for prognosis of IMN. These findings suggest that the extent of tubulointerstitial changes (cellular infiltration and fibrosis) determines the prognosis of renal function in IMN.

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 microg/g x 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.

In situ hybridization studies of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1 and type IV collagen in diabetic nephropathy.

Progressive expansion of the mesangial matrix is one of the most characteristic histological features of diabetic nephropathy (DN). To determine the balance between the turnover and degradation of extracellular matrix (ECM) in renal tissue of patients with DN, we examined the expression of matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type IV collagen (IV-C) mRNAs using a high-resolution in situ hybridization. Patients were divided into three grades: mild (grade I), moderate (grade II) and severe (grade III) mesangial expansion and tubulointerstitial injury. The relationship between the expression of these mRNAs and degree of glomerular mesangial expansion and interstitial injury was also examined. Cells positive for each mRNA were observed in glomerular resident cells, including glomerular mesangial, epithelial and endothelial cells and cells of Bowman's capsule. A number of tubular epithelial cells and some infiltrating cells in the interstitium also expressed these mRNAs. The expression of MMP-3 mRNA and TIMP-1 mRNA was strongest in glomeruli of grade I and inversely correlated with mesangial expansion. In contrast, the expression of all three types of mRNA was correlated with the degree of interstitial injury. Our results indicate that IV-C, MMP-3 and TIMP-1 mRNAs are expressed in glomerular resident cells, tubular epithelial cells and infiltrating cells in renal tissue of DN, and suggest that their expression changes with the degree of mesangial expansion and interstitial injury. Altered expression of MMP-3 and TIMP-1 may be associated with the progression of DN.

A diabetic case with hemoglobin J-Meerut and low HbA1C levels.

A diabetic patient with hemoglobin (Hb) J-Meerut and low HbA1C levels is reported. An automatic glycohemoglobin analyzer used for the determination of HbA1C revealed an abnormal peak of the peripheral blood obtained from a Japanese female with diabetes. She showed a lower HbA1C level (3.7%) than expected from her fasting plasma glucose (172 mg/dl). High performance liquid chromatography and isoelectric focusing indicated that her abnormal hemoglobin was Hb J-Meerut [alpha 120(H3)Ala-->Glu] and it accounted for 28.3% of the total hemoglobin. Abnormal hemoglobinemia should be considered when a major discrepancy between the levels of HbA1C and fasting plasma glucose is observed.

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 noninsulin-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 (ROC) 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.

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.

Localization of glycated proteins in the glomeruli of patients with diabetic nephropathy.

Glycation of proteins is regarded as one of the major causes of the development and progression of diabetic nephropathy. Based on the numerous reports on experimental models and on our own newly developed techniques, we planned to localize Amadori products and advanced glycation end-products (AGEs), as well as the mRNA expression of cytokines, enzymes and their inhibitors, which are responsible for the expansion of the mesangial areas of the glomeruli. Ten patients with diabetic nephropathy were examined. Patients with immunoglobulin (Ig) A nephropathy and normal portions of the surgically removed kidneys served as controls. Amadori products and AGEs in biopsy specimens were stained by specific monoclonal antibodies, and mRNA expression of the above substances was detected by in situ hybridization. There was a parallel progression in the degree of staining with anti-Amadori product antibody or anti-AGE antibody with the severity of tissue damage in patients with diabetic nephropathy. Patients with IgA nephropathy and normal renal tissues did not show any positive staining with these antibodies. The expression of transforming growth factor beta 1, stromelysin and tissue inhibitor of matrix proteinase 1 in the glomeruli was decreased in diabetic patients with advanced tissue damage, but they were progressively expressed in the advanced stage of IgA nephropathy. It is concluded that Amadori products and of AGEs were formed in parallel in diabetic kidneys. The decrease in the expression of the cytokine and enzymes might be due to altered protein formation associated with glycation.

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 Bowman's 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.

Urinary albumin fragments as a new clinical parameter for the early detection of diabetic nephropathy.

Urinary albumin fragments (uAF) from patients with NIDDM were analyzed as a possible factor in the early discovery of diabetic nephropathy before emergence of microalbuminuria. SDS-PAGE and immunoblot assay were employed for detection of uAF. Samples from 252 patients with NIDDM, 158 patients with non-diabetic diseases, and 48 healthy volunteers were examined; uAF were detected in 139 (55.2%), 94 (59.5%), only one (2.1%), respectively. In diabetic patients with normoalbuminuria, uAF were detected in 48 out of 159 cases (30.2%). Two years after the initial study, 3 of the 17 diabetic patients (17.6%) with normoalbuminuria and uAF showed micro- or macroalbuminuria. It was concluded that detection of uAF might be useful for the early detection of diabetic nephropathy in NIDDM.

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.