DETECTION OF IMMUNOGLOBULINS AND OTHER SERUM PROTEINS IN THE DERMAL AND GLOMERULAR CAPILLARY WALLS FROM PATIENTS WITH DIABETES MELLITUS

Deposition of immunoglobulins or acute phase reactant (APR) proteins in the dermal and glomerular capillary walls from patients with diabetes mellitus was examined to determine whether development of microangiopathy in such patients was related to exudation and/or entrapment of these proteins. Skin and renal biopsy specimens were obtained from patients with diabetic nephropathy and diabetes mellitus without nephropathy. These biopsy samples were stained with FITC-labeled anti-human IgG, IgA, IgM, C3, APR proteins, and β-Hpoprotein antisera. Linear depositions of IgA, IgG and/or APR proteins were observed in the dermal and/or glomerular capillary walls from some patients with diabetic nephropathy or diabetes mellitus without nephropathy. It is indicated that deposition of such immunoglobulins in the dermal and glomerular capillary walls might be due to exudation and/or entrapment of these substances in patients with diabetes mellitus.     

Immunofluorescence staining in unfixed or fixed renal biopsy specimens from patients with diabetic nephropathy

Immunofluorescence staining in unfixed or fixed renal biopsy specimens were evaluated in nine patients with diabetic nephropathy in order to elucidate if immunofluorescence staining is applicable in fixed renal tissues in such patients. Renal biopsy specimens were embedded in gelatin or paraffin matrix. Renal biopsy specimens embedded in paraffin matrix were digested with 0.05% protease. Immunofluorescent studies of kidney tissues were performed by staining with FITC-labeled heavy chain specific anti-human IgG, IgA, IgM, acute phase reactant (APR) proteins such as alpha 1-anti-trypsin (alpha 1-AT), haptoglobin (Hpt) and beta-lipoprotein (beta-Lp) antisera, and then examined with a fluorescent microscope. Linear and nodular deposition of IgG, IgA, IgM, alpha 1-AT, Hpt, and beta-Lp were observed in the glomerular capillary walls of the renal specimens embedded in paraffin matrix. The staining patterns in specimens embedded in paraffin matrix was similar to that embedded in gelatin matrix. There was no significant difference in the intensity or distribution of IgG, IgM, alpha 1-AT, and beta-Lp deposition among the two different conditions of immunofluorescence in patients with diabetic nephropathy. It was suggested that immunofluorescence staining in renal biopsy specimens embedded in paraffin matrix after digestion with protease is useful for the evaluation of IgG, IgM, APR proteins, and beta-Lp in glomeruli from patients with diabetic nephropathy.     

IMMUNOFLUORESCENCE STAINING IN UNFIXED OR FIXED RENAL BIOPSY SPECIMENS FROM PATIENTS WITH DIABETIC NEPHROPATHY

Immunofluorescence staining in unfixed or fixed renal biopsy specimens were evaluated in nine patients with diabetic nephropathy in order to elucidate if immunofluorescence staining is applicable in fixed renal tissues in such patients. Renal biopsy specimens were embedded in gelatin or paraffin matrix. Renal biopsy specimens embedded in paraffin matrix were digested with 0.05% protease. Immunofluorescent studies of kidney tissues were performed by staining with FITC-labeled heavy chain specific anti-human IgG, IgA, IgM, acute phase reactant (APR) proteins such as α-1-anti-trypsin (αl-AT), haptoglobin (Hpt) and β-lipoprotein (β -Lp) antisera, and then examined with a fluorescent microscope. Linear and nodular deposition of IgG, IgA, IgM, α 1-AT, Hpt, and β -Lp were observed in the glomerular capillary walls of the renal specimens embedded in paraffin matrix. The staining patterns in specimens embedded in paraffin matrix was similar to that embedded in gelatin matrix. There was no significant difference in the intensity or distribution of IgG, IgM, αl-AT, and β-Lp deposition among the two different conditions of immunofluorescence in patients with diabetic nephropathy. It was suggested that immunofluorescence staining in renal biopsy specimens embedded in paraffin matrix after digestion with protease is useful for the evaluation of IgG, IgM, APR proteins, and β -Lp in glomeruli from patiens with diabetic nephropathy.     

EFFECT OF IMMUNOGLOBULIN DEPOSITION ON THE AMOUNT OF GLOMERULAR SIALIC ACIDS IN PATIENTS WITH MEMBRANOUS NEPHROPATHY

A study of double immunofluorescence staining of immunoglobulins and sialic acids in the glomeruli from patients with membranous nephropathy was described. Renal biopsy specimens from patients with membranous nephropathy were stained with rhodamine-labeled limulus polyphemus (LPA) and tricum vulgaris (WGA). The binding of LPA or WGA was hardly observed in the glomerular capillary walls from patients with membranous nephropathy although the deposition of immunoglobulin was marked. Both binding of LPA and deposition of IgG in the glomerular capillary walls were observed in only one out of nine patients with membranous nephropathy (11%). In that case, binding of LPA was observed in the inside of glomerular capillary walls although the deposition of IgG was observed on the outside of the walls. It was suggested that decrease of sialic acids in the glomerular capillary walls might be due to deposition of IgG in some patients with membranous nephropathy.     

Effect of immunoglobulin deposition on the amount of glomerular sialic acids in patients with membranous nephropathy

A study of double immunofluorescence staining of immunoglobulins and sialic acids in the glomeruli from patients with membranous nephropathy was described. Renal biopsy specimens from patients with membranous nephropathy were stained with rhodamine-labeled limulus polyphemus (LPA) and tricum vulgaris (WGA). The binding of LPA or WGA was hardly observed in the glomerular capillary walls from patients with membranous nephropathy although the deposition of immunoglobulin was marked. Both binding of LPA and deposition of IgG in the glomerular capillary walls were observed in only one out of nine patients with membranous nephropathy (11%). In that case, binding of LPA was observed in the inside of glomerular capillary walls although the deposition of IgG was observed on the outside of the walls. It was suggested that decrease of sialic acids in the glomerular capillary walls might be due to deposition of IgG in some patients with membranous nephropathy.     

Computer imaging analysis of glomerular extracellular components in patients with IgA nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC-1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non-collagenous (NC-1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC-labeled anti-mouse Ig antisera. Marked staining of the 7S or NC-1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy.     

Computer Imaging Analysis of Glomerular Extracellular Components in Patients with IgA Nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC 1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non collagenous (NC 1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC labeled anti mouse Ig antisera. Marked staining of the 7S or NC 1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy. Acta Pathol Jpn 39: 296 305, 1989.     

Double immunofluorescence studies of immunoglobulins, complement components and their control proteins in patients with IgA nephropathy

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and beta 1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. beta 1H globulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.     

IgA nephropathy associated with chronic hepatitis B virus infection in adults: the pathogenetic role of HBsAG

Five adult cases of IgA nephropathy associated with chronic hepatitis B virus infection were studied. Serum HBsAg and anti-HBc were present in five patients and HBeAg in four patients. Glomerular changes were typical of primary IgA nephropathy in four patients, and a mixed picture of IgA and membranous nephropathy was demonstrated in one patient. Immunofluorescence microscopy using polyclonal and monoclonal antibodies against HBsAg, HBcAg, and HBeAg revealed mesangial deposits of HBsAg in renal biopsies from four patients. One renal biopsy showed only mesangial and capillary HBcAg by polyclonal antiserum, and virus-like particles were demonstrated in the intramembranous electron-dense deposits on ultrastructural examination. Mesangial HBeAg was not detected in the renal biopsies from these patients with IgA nephropathy. As for the single patient with a mixed picture of IgA and membranous nephropathy, granular deposits of HBeAg with a distribution similar to IgG were detected in the glomerular capillary walls in addition to the mesangial deposition of HBsAg. These findings suggest that HBsAg rather than HBeAg may play a role of the pathogenesis in some of the adult patients with IgA nephropathy associated with chronic hepatitis B virus infection.     

Immunoelectron microscopy of IgA nephropathy

The distribution of IgA, IgG, IgM, C3, and albumin in kidney biopsy specimens from 11 children and adults with recurrent gross and microscopic hematuria and IgA nephropathy and 7 control specimens were evaluated by the direct peroxidase-labeled antibody method and electron microscopy. Granular masses of reaction product (RP), representing IgA, IgG, IgM, and C3, were observed within the mesangial matrix of glomeruli from all patients with IgA nephropathy. Occasional smaller masses of IgA-RP and C3-RP were noted along the peripheral glomerular capillary loops, the tubular basement membranes, and within the interstitial matrix of some patients. Large amounts of IgA-RP and C3-RP were present within the walls of small renal arterioles of several patients. These observations support the concept that immune-complex deposits are involved in the pathogenesis of IgA nephropathy and suggest that vascular deposits may have a more important role in the progression of the disease in some patients.     

DOUBLE IMMUNOFLUORESCENCE STUDIES OF IMMUNOGLOBULINS, COMPLEMENT COMPONENTS AND THEIR CONTROL PROTEINS IN PATIENTS WITH IgA NEPHROPATHY

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and β1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. β1H gobulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.     

Glomerular deposition of IgA in experimental hepatic cirrhosis

Wistar rats rendered cirrhotic with carbon tetrachloride excreted significant proteinuria and hematuria. Serum levels of IgA and IgG were significantly elevated in cirrhotic animals. They showed mild mesangial proliferation and immunofluorescent studies revealed deposits of IgA and IgG predominantly in mesangial areas and along capillary walls. These findings were very similar to those seen in patients with hepatic cirrhosis or IgA nephropathy. The deposits of IgA were also found in hepatic tissue from cirrhotic animals. The intensity and distribution of glomerular IgA deposits were not diminished after treatment with acid buffer. These results suggest that glomerular IgA are IgA polymers and decreased hepatic clearance of hepatic IgA polymers may be responsible for the glomerular deposition of IgA.     

Overlapping IgA and membranous nephropathy

Four patients with overlapping IgA nephropathy and membranous nephropathy recently have been reported from two different medical centers in Japan. The authors describe two patients from the United States with the concurrence of IgA and membranous nephropathy and a third patient who may have the same overlapping glomerular lesions. All three patients had hematuria and nephrotic range proteinuria. Renal biopsy specimens from two of the patients revealed focal proliferative glomerular lesions, large mesangial and numerous subepithelial electron-dense deposits, exclusively mesangial intense immunostaining for IgA, and extensive granular capillary wall immunostaining for IgG. The third patient is a sibling of one of the other patients and was shown to have focal proliferative glomerular lesions, mesangial immunostaining for IgA, and numerous subepithelial electron-dense deposits. Pathogenic possibilities are discussed.     

Immunohistochemical study of the membrane attack complex of complement in IgA nephropathy

Summary The localization of the membrane attack complex of complement (MAC) was examined in the normal human kidneys and in biopsy specimens from patients with primary IgA nephropathy by immunofluorescent and immunoelectron microscopies. Immunofluorescent staining for MAC was significantly more intense than in the normal kidneys, and was observed in the mesangium and occasionally along the glomerular capillary walls of 22 of 30 patients with IgA nephropathy. By dualstaining, the MAC deposits were generally concordant with the deposits of IgA, C3, C5 and C9, or of IgG, when present. C1_q or C4 was infrequently observed in the glomeruli. Immunoelectron microscopy revealed various staining patterns of glomerular MAC deposition; homogeneous fine-granular staining beneath the glomerular basement membrane (GBM) in the paramesangial zone, patchy staining within the mesangial electron dense deposits (EDD), and ring-shaped or ribbon-like staining, associated with the striated membrane structures (SMS), in the matrix of the mesangium, GBM and tubular basement membrane (TBM). This study suggests that the terminal complement system is activated, mainly by an alternative complement pathway mechanism, in the mesangium of IgA nephropathy, and is associated with the paramesangial lesion and EDD. MAC deposition in glomerular SMS may also result from in situ activation rather than trapping from the circulation. There was little correlation between glomerular MAC deposition and proteinuria or renal histology of patients with IgA nephropathy.     

Evaluation of the staining findings of immunofluorescence in unfixed or fixed renal biopsy specimens from patients with IgA nephropathy and membranous nephropathy

A study on the evaluation of staining findings of immunofluorescence in unfixed or fixed renal biopsy specimens is described. Renal biopsy specimens obtained from ten patients with IgA nephropathy and membranous nephropathy were embedded in gelatin or paraffin matrix. Renal biopsy specimens embedded in paraffin matrix were digested with 0.05% protease. The specimens were stained with FITC-conjugated anti-human IgA, IgG, IgM or C3 antisera at 4 degrees C overnight. IgA, IgG or IgM were markedly observed in glomeruli using unfixed materials embedded in gelatin matrix or 10% neutral buffered formalin fixed materials embedded in paraffin matrix from patients with IgA nephropathy and membranous nephropathy. There was no significant difference in the intensity or distribution of IgA, IgG or IgM deposition among the two different conditions of immunofluorescence in patients with such diseases. Although the deposition of IgA using unfixed materials embedded in gelatin matrix was prominently coarse granular or lumpy in glomeruli from patients with IgA nephropathy, that of IgA using 10% formalin fixed materials embedded in paraffin matrix was fine granular and/or interrupted linear in glomeruli. It was suggested that the immunofluorescence in renal biopsy specimens embedded in paraffin matrix after digestion with protease is useful for the evaluation of immunoglobulins in glomeruli from patients with IgA nephropathy or membranous nephropathy.     

EVALUATION OF THE STAINING FINDINGS OF IMMUNOFLUORESCENCE IN UNFIXED OR FIXED RENAL BIOPSY SPECIMENS FROM PATIENTS WITH IGA NEPHROPATHY and MEMBRANOUS NEPHROPATHY

A study on the evaluation of staining findings of immunofluorescence in unfixed or fixed renal biopsy specimens is described. Renal biopsy specimens obtained from ten patients with IgA nephropathy and membranous nephropathy were embedded in gelatin or paraffin matrix. Renal biopsy specimens embedded in paraffin matrix were digested with 0.05% protease. The specimens were stained with FITC-conjugated anti-human IgA, IgG, IgM or C3 antisera at 4°C overnight. IgA, IgG or IgM were markedly observed in glomeruli using unfixed materials embedded in gelatin matrix or 10% neutral buffered formalin fixed materials embedded in paraffin matrix from patients with IgA nephropathy and membranous nephropathy. There was no significant difference in the intensity or distribution of IgA, IgG or IgM deposition among the two different conditions of immunofluorescence in patients with such diseases. Although the deposition of IgA using unfixed materials embedded in gelatin matrix was prominently coarse granular or lumpy in glomeruli from patients with IgA nephropathy, that of IgA using 10% formalin fixed materials embedded in paraffin matrix was fine granular and/or interrupted linear in glomeruli. It was suggested that the immunofluorescence in renal biopsy specimens embedded in paraffin matrix after digestion with protease is useful for the evaluation of immunoglobulins in glomeruli from patients with IgA nephropathy or membranous nephropathy. ACTA PATHOL. JPN. 35 : 315–321, 1985.     

GLOMERULAR DEPOSITION OF IgA IN EXPERIMENTAL HEPATIC CIRRHOSIS

Wistar rats rendered cirrhotic with carbon tetrachloride excreted significant proteinuria and hematuria. Serum levels of IgA and IgG were significantly elevated in cirrhotic animals. They showed mild mesangial proliferation and immunofiuorescent studies revealed deposits of IgA and IgG predominantly in mesangial areas and along capillary walls. These findings were very similar to those seen in patients with hepatic cirrhosis or IgA nephropathy. The deposits of IgA were also found in hepatic tissue from cirrhotic animals. The intensity and distribution of glomerular IgA deposits were not diminished after treatment with acid buffer. These results suggest that glomerular IgA are IgA polymers and decreased hepatic clearance of hepatic IgA polymers may be responsible for the glomerular deposition of IgA. ACTA PATHOL. JPN. 35: 561–567, 1985.     

Studies of kidney and muscle biopsy specimens from identical twins discordant for type I diabetes mellitus

To distinguish metabolic from genetic factors in the development of microangiopathy in diabetes, we evaluated biopsy specimens of kidney and quadriceps muscle from seven pairs of identical twins who were discordant for Type I (insulin-dependent) diabetes mellitus. Two of the diabetic patients had clinical diabetic nephropathy, including hypertension, marked albuminuria, and a substantially reduced creatinine clearance; the other five had normal renal function and only minor clinical indications of complications. All the twins of the diabetic patients had normal glomerular basement membrane widths and normal fractional volumes of the glomerular mesangium. Values for glomerular basement membrane width, tubular basement membrane width, and mesangial volume in each diabetic twin exceeded the values in the respective sibling (P less than or equal to 0.0035), even if the value in the diabetic twin lay within established normal ranges. Values for muscle capillary basement membrane width in the diabetic twins did not differ from those in their siblings (P = 0.5). Our observations suggest that the metabolic abnormalities of diabetes are necessary, if not sufficient, for the development of glomerular abnormalities. We also conclude that in diabetic patients, alterations in muscle capillary basement membrane width do not necessarily accompany pathologic lesions in the kidney.     

Patho- and morphogenesis of diabetes mellitus and early diabetic nephropathy

Heterogeneity of diabetes mellitus, as the specialists see it nowadays, is determined by the presence of histocompatibility antigens and the onset of immune autoaggression against beta-cells of islet of Langerhans which leads to formation of circulating immune complexes and complexes in situ. This may give rise to diabetic nephropathy. In florid diabetes mellitus there also appeared disturbances induced by metabolic derangement, relevant antigens being then exogenic insulin and low-molecular lipoproteins entailing drastic changes of the basal membrane of glomerular capillaries, plasma saturation of the vascular walls and eventual hyalinosis and capillary obliteration.     

Glomerular membranopathy in adolescents with insulin-dependent diabetes

Kidney biopsy specimens from 15 patients ranging in age from 11 to 19 years with two- to 14-year histories of insulin-dependent diabetes mellitus were evaluated electron microscopically. Although the mean duration of disease was only eight years, the glomerular basement membrane (GBM) in these patients showed a variety of alterations typical of insulin-dependent diabetes mellitus. Saccular glomerular microaneurysms, previously little recognized, were seen in six of the specimens. These lesions, always associated with breaks in the paramesangial BM, were morphologically distinct from the ectatic capillary loops and glomerular capillary aneurysms described previously in diabetic glomerulopathy. All of the patients with such aneurysms also had other severe GBM alterations. Lytic or mechanical injury to the structurally and biochemically altered diabetic GBM may be responsible for the formation of microaneurysms.