Immunohistochemical analysis of extracellular components in the glomerular sclerosis of patients with glomerulonephritis

Immunofluorescence and immunoperoxidase staining was carried out to determine correlations between the progression of glomerular sclerosis and changes in the amount or distribution of glomerular extracellular components, including type I, III, IV, and VI collagens, laminin and fibronectin, in patients with IgA nephropathy, membranoproliferative glomerulonephritis and rapidly progressive glomerulonephritis. Staining of type I and III collagens was not observed in glomeruli from normal individuals or patients with mild glomerulonephritis. In the advanced stages of glomerulonephritis, the staining of type IV and VI collagens, laminin and fibronectin was marked in the glomerular mesangium, and the distribution of fibronectin extended to the glomerular capillary walls in the sclerotic lesions of glomeruli. However, the staining intensity of type IV collagen, laminin and fibronectin was gradually decreased during the progression of glomerular sclerosis. On the other hand, the staining of type I and III collagens was observed focally in sclerotic or hyalinotic glomeruli and around such glomeruli in those patients. Light microscopic examination revealed that patients who showed marked staining of type I and III collagens by immunofluorescence had severe damage of Bowman's capsules. These results suggest that the hyperproduction and/or invasion of interstitial collagens, i.e., types I and III, are closely linked to the progression of glomerular sclerosis and hyalinosis in patients with various types of glomerulonephritis.     

Immunohistochemical studies of glomerular extracellular components in repeated renal biopsies of patients with IgA nephropathy.

Immunohistochemical and light microscopic examinations were carried out to assess the correlation between the progression of glomerular lesions and changes in the intensity of glomerular extracellular components such as type IV and I collagens, laminin and fibronectin, and of IgA deposits in repeated renal biopsies of patients with IgA nephropathy. By light microscopy, the percentage of glomeruli showing glomerular mesangial expansion or sclerosis was found to be significantly higher in the second renal biopsy. Type IV collagen, laminin and fibronectin were also marked in the expanded glomerular mesangium in the second biopsy. Although these components were not observed in the global sclerotic glomeruli, type I collagen was detected in such areas of patients with IgA nephropathy. Patients who revealed high percentages of glomerular sclerosis associated with marked type IV collagen, laminin, fibronectin and/or type I collagen, had high levels of proteinuria and progressive deterioration of renal function. It is concluded that hyperproduction of the above extracellular components mainly in the glomerular mesangium is closely linked to the progression of glomerular lesions in patients with IgA nephropathy.     

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.     

A monoclonal antibody (1G10) recognizes a novel human mesangial antigen.

We have identified a unique mesangial matrix protein of the human glomerulus by using a monoclonal antibody, 1G10, generated against culture human glomerular cells. By immunofluorescence, the antigen recognized by 1G10 (1G10 antigen) is present in mesangium and smooth muscle tissue and cannot be detected in any other tissue examined. Immunoelectron microscopy of glomeruli indicated that 1G10 antigen is present exclusively in the mesangial matrix at the endothelial-mesangial interface. The 1G10 antigen is also expressed by cultured mesangial cells, but not by cultured glomerular epithelial cells, umbilical endothelial cells or fibroblasts. 1G10 did not react with the mesangial matrix proteins [fibronectin (FN), laminin (LAM), collagen types I, III, IV, V, and VI (Col I, III, IV, V, VI), heparin sulfate proteoglycan (HSPG), or thrombospondin (TS)] present under normal and diseased states or smooth muscle antigens (myosin, actin), but did react with a 4 M urea extract of renal cortex and a 0.3% deoxycholate extract of isolated glomeruli. Two dimensional immunoblot analysis using the urea extract demonstrated the binding of 1G10 to an approximately 200 KDa polypeptide with pI 6.0. On one dimensional immunoblot this band did not show cross react with polyclonal antisera to FN, LAM, Col IV, V, VI, HSPG or TS. This mesangial matrix component is trypsin and periodate sensitive, suggesting that it has the character of glycoprotein. In renal biopsy specimens from patients with mesangial proliferative glomerulonephritis (GN) and membranoproliferative GN, the expression of the 1G10 antigen increased along with mesangial hypercellularity or increased accumulation of mesangial matrix, but decreased in completely sclerosed glomeruli. No significant changes in 1G10 antigen expression was observed in membranous GN or minimal change nephrosis compared to normal glomeruli. This study suggests that the 1G10 antigen may not only be a useful marker for the clinical assessment of GN, but may also serve as a potential tool for the study of the pathogenesis of glomerular diseases characterized by cellular proliferation and mesangial matrix expansion.     

Pathophysiological aspects of edema formation in diabetic nephropathy

The present study was undertaken to evaluate some pathophysiological mechanisms of edema formation in diabetic nephropathy. Sixty-three subjects were investigated: 9 normal subjects (I), 9 normoalbuminuric Type 1 (insulin-dependent) diabetic patients (II), 15 microalbuminuric Type 1 diabetic patients (III), 16 Type 1 diabetic patients with nephropathy without edema (IV), and 14 Type 1 diabetic patients with nephropathy and edema (V). Plasma volume (125I-albumin), glomerular filtration rate and extracellular fluid volume (51Cr-EDTA) were measured. Colloid osmotic pressure and albumin concentration were measured in plasma and in subcutaneous interstitial fluid (suction blister technique). The ratio between plasma volume and interstitial fluid volume was reduced in patients with edema compared with group 1 (P less than 0.05). The interstitial colloid osmotic pressure (mm Hg) was significantly reduced (P less than 0.05) in group V compared with the other groups (V: 4.3 +/- 1.1, I: 7.9 +/- 1.7, II: 7.5 +/- 1.8, III: 6.6 +/- 1.5, IV: 6.6 +/- 1.1), but the transcapillary colloid osmotic gradient in patients with edema was comparable with the remaining subjects. The ratio between interstitial and plasma albumin concentration was significantly reduced in group V compared with groups I and II (V: 0.31 +/- 0.1, I: 0.43 +/- 0.06, II: 0.44 +/- 0.06; P less than 0.01; III: 0.41 +/- 0.07, IV: 0.41 +/- 0.08). This reduction was mainly due to enhanced lymph flow. The wash-down of subcutaneous interstitial protein indicated increased capillary filtration, but at the same time limited the increase in net filtration pressure and thereby prevented progressive edema formation in diabetic nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of glomerular diseases in renal biopsies

Summary: The purpose of this study was to determine the pattern of gene expression of type IV collagen alpha chains in several chronic human glomerular diseases using micro dissected glomeruli and assessment of mRNA by competitive polymerase chain reaction (PCR). After showing that the level of a2 type IV collagen mRNA was elevated in sclerotic glomeruli isolated from nephrectomies, we undertook a preliminary cross-sectional study of type IV collagen alpha chain mRNA in renal biopsies in two of the leading causes of glomerulosclerosis: (i) diabetic nephropathy; and (ii) membranous glomerulopathy. We found that glomerular type IV collagen mRNA levels alterations were disease-specific. the relative levels of the individual α-chains of type IV collagen depended on the anatomic site of the glomerular lesions. the α-2IV/α-3IV collagen mRNA ratio was high in diabetes mellitus, but not in membranous glomerulopathy. These data, coupled with that obtained in experimental animals, suggest that a defective basement collagen synthesis is associated with progressive glomerular scarring. If these conclusions are verified in studies of repeat biopsies, the risk of progressive glomerulosclerosis in individual patients could be estimated, leading to the means to assess therapeutic responses.     

Constituents of the extracellular matrices in diabetic glomerulosclerosis

This study was undertaken to elucidate the distributions of laminin, fibronectin, type I, III, IV, V and VI collagen and heparan sulfate proteoglycan (HSPG) in diabetic nephropathy, using immunohistochemical procedures. The pathological features of diabetic glomerulosclerosis were characterized as diffuse and nodular lesions, showing an expanded mesangial matrix associated with a thickened glomerular basement membrane (GBM). In the thickened GBM, laminin was present throughout the whole membrane, type IV collagen occurred along the subendothelial side, and HSPG was present with no change in its amount. On the other hand, the components detected in the slightly expanded mesangial matrix were type IV, V and VI collagen, fibronectin and HSPG, but not laminin. When the matrix was expanded markedly, collagenous components were increased over the other components. In the typical Kimmelstiel-Wilson nodules, the mesangial matrix was occupied mainly by type V and VI collagen with a relative decrease in type IV collagen. When a nodular lesion adhered to Bowman's capsule, type I and III collagen occurred not only in Bowman's space but also within the lesion itself. Furthermore, laminin and HSPG became detectable on the outside of the lesion, but not within it. These results suggested that there was a difference in the distribution and proportion of extracellular matrix components between diffuse and nodular lesions. It appeared that the nodular lesion was not simply an advanced form of the diffuse lesion.     

Effects of candesartan, an angiotensin II type 1 receptor blocker, on diabetic nephropathy in KK/Ta mice

BACKGROUND: Although therapeutic effects of angiotensin II type 1 receptor blocker (ARB) on renal injury in non-insulin dependant diabetes mellitus (NIDDM) have been demonstrated, the beneficial effects and their mechanisms in diabetic nephropathy have not been well evaluated. METHODS: KK/Ta mice were divided into three groups according to the treatment: candesartan 4 mg/kg/day from 6 to 28 weeks of age (group I; early treatment); from 12 to 28 weeks of age (group II; late treatment); only vehicle (group III). BALB/c mice treated with vehicle were used as controls (group IV). Body weight (BW), systolic blood pressure (SBP), blood glucose, urinary type IV collagen and albumin excretion were measured every 4 weeks. Morphometry and immunohistology of albumin, transforming growth factor-beta1 (TGF-beta1) and Smad7 were performed in all groups. RESULTS: BW and blood glucose were higher in groups I, II and III than in group IV from 8 weeks. SBP was markedly reduced in groups I and II compared with group III (p < 0.05, p < 0.005). Urinary type IV collagen and albumin excretion were increased in group III compared to group IV (p < 0.05, p < 0.005), whereas they were reduced in groups I and II when compared to group III (p < 0.05). Morphometric analysis revealed that the whole glomerular area (WGA), glomerular tuft area (GTA), extracellular matrix area (ECMA) and intraglomerular cell nuclei number (NIGCN) were significantly reduced in groups I, II and IV compared to group III at 28 weeks. In immunohistochemistry, TGF-beta1 expression in both glomeruli and tubules of groups I and II decreased compared to that of group III at 28 weeks, while Smad7 in group III glomeruli was reduced compared to that in groups I and II. CONCLUSIONS: It appears that candesartan reduced urinary type IV collagen and albumin excretion, and attenuated glomerular hypertrophy and mesangial matrix accumulation by the TGF-betaS/Smad signaling pathway in KK/Ta mice with diabetic nephropathy.     

Heparan sulfate proteoglycans are lost in patients with diabetic nephropathy.

The pathogenesis of diabetic nephropathy relative to the changes in the glomerular extracellular matrices was investigated. Renal tissues from 10 diabetic patients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This alteration may, in part, be the cause of the proteinuria associated with diabetic nephropathy.     

Urinary excretion of type IV collagen as a specific indicator of the progression of diabetic nephropathy

AIMS AND METHODS: This study was carried out to clarify whether the urinary excretion of type IV collagen (u-IV collagen) detected by specific radioimmunoassay, can be used as an indicator for the progression of diabetic nephropathy. RESULTS: u-IV collagen was higher in diabetic subjects with microalbuminuria and overt proteinuria than those with normoalbuminuria, IgA nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, or control normal subjects. u-IV collagen was positively correlated with serum and urinary beta(2)-microglobulin and negatively with creatinine clearance only in diabetic patients, but not in patients with other glomerular diseases. The serum type IV collagen was not different between all the groups, and not correlated with its urinary excretion. In the advanced diabetic nephropathy, immunoreactive type IV collagen was detected in glomerular basement membrane (GBM), tubular basement membrane and Bowman's capsule much more than that in the normal kidney. CONCLUSION: These findings indicated increased production and degeneration of type IV collagen in diabetic nephropathy. It is suggested that augmented turnover of type IV collagen in GBM and tubular basement membrane results in increased concentrations of free u-IV collagen. Therefore, measurement of u-IV collagen may be a useful, specific indicator of the progression of diabetic nephropathy.     

Renal distribution of collagen types III, IV and V in various glomerular diseases

The purpose of this study is to examine the immunochemical changes of the glomerular basement membrane (GBM) and the mesangium, in pretreated paraffin-embedded sections with trypsin by utilizing monoclonal antibodies to type III (anti-III), type IV (anti-IV) and type V (anti-V) collagens. We observed 6 normal kidneys and 44 kidneys with various renal diseases. In normal human kidney the staining with anti-IV demonstrated GBM, mesangium, Bowman's BM, tubular BM and capillary BM. Anti-V was also seen in the interstitium. On the other hand, anti-III stained only interstitium. Thickened GBM in membranoproliferative glomerulonephritis (MPGN) and diabetic nephropathy, and irregular GBM in Membranous Nephropathy and Alport's syndrome were also evident in anti-IV stain, while widened mesangial area was seen in anti-V rather than anti-IV stain. In severely proliferative GN, anti-III as well as anti-IV and anti-V was detected in the mesangium in spite of existence of neither adhesion nor Bowman's gap. In MPGN type II, anti-III was observed along the GBM. In obsolescent glomeruli, anti-IV was not always detected although anti-V was constantly seen. On the other hand, anti-III was markedly positive in the crescents and obsolescent glomeruli. These results suggest that it is possible for mesangial, endothelial and epithelial cell to produce several types of collagens and type III collagen is closely related to the process of the glomerular obsolescence.     

Immunohistochemical study on the extracellular matrix components in various renal diseases]

In order to clarify considerable alterations of the extracellular matrix components in various renal diseases, monoclonal antibodies against type IV collagen (IV col), 200-KD laminin (200-KD Lam), 400-KD laminin (400-KD Lam) and heparan sulfate proteoglycan (HSPG) were applied to 142 renal biopsy specimens for the indirect immunofluorescence. These subjects included 5 cases with 1 hour specimen in the transplanted kidney, 32 minimal change nephrotic syndrome (MCNS), 30 mesangial proliferative glomerulonephritis (PGN), 2 focal segmental glomerulosclerosis (FGS), 21 membranous nephropathy (MN), 9 membranoproliferative glomerulonephritis (MPGN), 2 poststreptococcal acute glomerulonephritis (PSAGN), 13 diabetic nephropathy (DN), 16 lupus nephritis (LN), 9 diffuse sclerosing glomerulonephritis (DSGN), 2 amyloid kidney and 1 granulomatous nephropathy in sarcoidosis. In the transplanted kidney, the staining intensity of IV col was stronger than that of 200-KD Lam, 400-KD Lam and HSPG in general. IV col was predominantly distributed throughout the mesangium area and less along the glomerular basement membrane (GBM). The positive stainings with 200-KD Lam along the GBM and that with 400-KD Lam in the mesangium area were weakly recognized. HSPG was mainly detected along the GBM in the linear fashion. In MCNS, the distribution of the extracellular matrix components was mostly identical to that in the transplanted kidney. In the group of the glomerulonephritis showed the proliferations of mesangial cells, such as PGN and MPGN, the staining intensity of both IV col and 400-KD Lam, particularly in the latter, was remarkably increased in the sclerotic lesions. In MN, the thickened GBM was strongly stained with both IV col and 200-KD Lam, and the stainings of 200-KD Lam were more intensive. And still more, by the double labelling method performed for the couple of IV col and immunoglobulins, the correlation between glomerular capillary walls and/or mesangial areas and immunoglobulins deposits became more clear. These findings suggest as follows: (1) both IV col and 400-KD Lam, in particular 400-KD Lam, are possibly involved in the process of glomerulosclerosis: (2) both IV col and 200-KD Lam, in particular 200-KD Lam, are greatly involved in the process of new basement membrane-like materials formation in MN.     

Effects of endothelin receptor antagonists on the progression of diabetic nephropathy

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage renal disease in European countries and is associated with an enhanced renal synthesis of endothelin (ET)-1. ETs are - beside their potent vasoconstrictor properties - very potent profibrotic acting paracrine hormones especially in the kidney. METHODS: We analyzed in rats with streptozotocin-induced diabetes the effects of an ETA-type (ETA) receptor antagonist (LU 135252) in comparison to a combined ETA/ETB receptor antagonist (LU 224332) on the expression of interstitial and glomerular collagen type I, III and IV as well as on fibronectin and laminin by quantitative immunohistochemistry using a computer-aided image analysis system. Global glomerular matrix deposition was analyzed after PAS staining. In addition to the morphometric examination of the kidneys, we also investigated GFR, urinary albumin and total protein excretion. The diabetic rats were treated for 36 weeks. RESULTS: Treatment with either LU 135252 or LU 224332 normalized the amount of PAS-positive material within the glomeruli. The expression of glomerular fibronectin and type IV collagen was increased 36 weeks after induction of diabetes. The overexpression of these two matrix proteins within the glomeruli of diabetic rats was completely abolished by both ET receptor antagonists, whereas protein excretion was only reduced by about 50% as compared to diabetic rats without treatment. CONCLUSION: The present study indicates that ETA receptor antagonists as well as combined ETA/ETB receptor antagonists reduce proteinuria and completely normalize the renal matrix protein expression in hyperglycemic rats with streptozotocin-induced diabetes. The antifibrotic effect seems to be mediated via the ETA receptor. ET receptor antagonists might be a new approach in the treatment of diabetic nephropathy.     

1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study

Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.     

Collagen distribution in human membranous glomerulonephritis

In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (1/2, 3, 4, 5, 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high-resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the 3, 4, and 5 chains of type IV collagen. In contrast, the 1/2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type VI collagen play an important role in the thickening of the GBM in human MGN.     

Histochemical and immunohistochemical studies of diseased human glomeruli

In order to study the localization of Lentil lectin (LCH)-binding glycoresidues in glomeruli from patients with a variety of glomerulopathies, and to elucidate the relationship between LCH-binding sugars and the components of the extracellular matrix, laminin and type IV collagen, investigations of formalin-fixed, paraffin-embedded kidney tissues digested with trypsin were carried out by the direct and indirect immunofluorescence microscopy techniques. The glomerular basement membrane (GBM) and the mesangium reacted well with LCH, whereas areas with sclerotic lesions exhibited a decreased reactivity. The pattern of LCH binding to the GBM in various glomerulopathies was similar to that of laminin but different from that of type IV collagen. The pattern of localization of LCH-reacting sites and of laminin in the GBM included the double linear lines in diabetic nephropathy, inner linear line with outer projections (spikes) in membranous nephropathy, and reduplicated basement membrane in membranoproliferative glomerulonephritis. The results obtained by enzyme-linked immunoadsorbent assay showed that LCH had a stronger reactivity for laminin than for type IV collagen or fibronectin. These findings suggest that LCH is more reactive with laminin than with other components of the glomerular extracellular matrix.     

Urine and serum levels of the carboxyterminal domain (NCl) of collagen IV in membranous glomerulonephritis and diabetic nephropathy

Serum and urinary concentrations of NCl, the non collagenous globular domain of collagen IV, were used as markers for turnover of basement membranes. NCl levels were studied in membranous glomerulonephritis and diabetic nephropathy. Thirteen patients with membranous glomerulonephritis and 8 insulin-dependent diabetic patients with diabetic nephropathy were compared to 16 apparently healthy control subjects. The patients with membranous glomerulonephritis had lower levels of NCl in serum and urine compared to the control subjects. In comparison, the patients with diabetic nephropathy had similar levels of NCl in serum and urine as the control subjects. Furthermore, among patients with membranous glomerulonephritis, those with hypertension had higher serum levels of NCl than those without, which may indicate that hemodynamic factors influence the basement membrane collagen metabolism. It is suggested that there are differences in basement membrane turnover in membranous glomerulonephritis and diabetic nephropathy although there are similarities in glomerular histopathological features. Other possible mechanism are discussed. Further studies are needed to confirm the suggested mechanism.