The Immunohistopathology of Glomerular Antigens II. The Glomerular Basement Membrane, Actomyosin, and Fibroblast Surface Antigens in Normal, Diseased. and Transplanted Human Kidneys Jon 1. Scheinman, MD, Alfred J. Fish, MD, Arthur J. Matas, MD, and Alfred F. Michael, MD
Immunofluorescent studies have demonstrated that actomyosin (AMY) is present in the mesangium in a restrictive pattern, whereas fibroblast surface antigen (FSA) has a more extensive mesangial distribution. Antibody to glomerular basement membrane (GBM) is localized to the GBM. One hundred ninety-six tissue samples, including 17 from normal subjects, 94 from patients with primary renal diseases, and 85 from transplanted kidneys. were examined for changes in distribution of AMY, FSA, and GBMI antigens. The distribution of A-MY and FSA in the mesangium is markedly increased in width in patients with diabetic nephropathy, and the GBM is thickened. AMY and FSA are mildly increased in patients with glomerulonephritis and GBM is normal. Patients with membranoproliferative glomerulonephritis (MPGN) show a loss of all glomerular antigens. Increased mesangial A-MY was found in 12 of 37 transplanted kidneys in diabetic patients vs 4 of 33 nondiabetics 0 to 4 years after transplantation (P < 0.025). This difference is more notable at 2 to 4 vears, with 5 of 9 diabetics showing increased AMY vs 0 of 6 nondiabetic patients (P < 0.0005). In MPGN, 5 of 8 tissues showed decreased mesangial A-MY vs 3 of 36 diabetic patients (P < 0.0005) and 1 of 32 patients with other glomerulopathies (P < 0.0005). GBM thickening is not associated with specific pretransplant diseases. In transplanted kidneys, the pattern of FSA is dissociated from that of AMY: over half of all patients have increased FSA, even when .AM*Y is normal. Whether this unique finding in transplanted kidneys reflects an increase in the synthesis of FSA by mesangial cells or the failure to clear this (circulating) antigen from the mesangial matrix is unknown. (Am J Pathol 90:71-8, 1978)
PREVIOUS STUDIES HAV-E DEMONSTRATED a marked and consistent
increase in the distribution of mesangial actomvosin (ANIY) antigen 1 in diabetic nephropathy and its loss in membranoproliferative glomerulonephritis, with more subtle alterations in other glomerulopathies. Glomerular basement membrane (GB\I) antigen was localized to the thickened basement membranes in patients with diabetic nephropathv and From the Departments of Pediatrics and Surgery. Univ ersity of Nlinnesota Medical School. Mlinneapolis. \linnesota Supported by a Grant-In-Aid from the American Heart Association and the American Diabetes Association of Minnesota. by grants from the American Diabetes Association and the Minnesota Xrthritis Foundation. and by Grants HL 06314 and XI-01704-14 from the US Public Health Ser-ice Dr Scheinman is the recipient of a Research Career Development Avvard from the NIAMD =K04A\100126)i Accepted for publication August 31. 1977. Xddress reprint requests to Dr Jon I Scheinman. Department of Pediatrics. Box 491 Mayo \lemorial Building. University of Minnesota \Medical School. \inneapolis. NN 5.5455. 71
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membranous nephropathy. In this study we explore, in addition, the immunohistopathology of the fibroblast surface antigen (FSA) or fibronectin2 in primary renal diseases and further examine all these antigens in transplanted human kidneys. Materials and Methods Antsm Antim-aid Antihuman uterine smooth muscle actomyosin (anti-AMY) was made in rabbits immunized with purified AMY and characterized as previously described.' Myosin was isolated by the method of Pollard, Thomas, and Niederman,' was relatively homogeneous on acrylamide gel electrophoresis 4 in sodium dodecyl sulfate (Figure IA), and had ATPase activity (measured in 0.6 M KC1, 8 mM MgCGl, 15 mM Tris at room temperature) of 3 AM P/mg/30 min, similar to human uterine AMY.1 Inorganic phosphorus was determined colorimetrically.' Foot pad immunization of 3 rabbits with 1 mg of mvosin in complete Freund's adjuvant was repeated subcutaneously after 3 weeks, and bleeds taken 2 to 4 weeks later were used for immunodiffusion and immunofluorescence. Both arterial walls and mesangium were stained in the human kidney, but only the arterial wall was stained in rat tissue. Antimyosin reacted against AMY and myosin on immunodiffusion 1 with lines of identity (Figure IB); the tissue-staining reactivities of anti-AMY and antimyosin were identical and both were completely inhibited by absorption with mvosin. Anti-AMY was also inhibited by absorption with a sonicated, Iyophilized preparation of whole human glomeruli. Rabbit antihuman glomerular basement membrane (GBM) antiserums have been previously characterized.1 Antifibroblast surface antigen (anti-FSA) was made in rabbits immunized with lvophilized human foreskin fibroblast cultures incorporated in complete Freund's adjuvant as previously described." Bleeds obtained 2 to 3 weeks after a single booster injection were used. This antiserum reacted with the mesangium of human, rat, and chicken kidnev, as observed by indirect immunofluorescent techniques. Antiserums obtained later in the course of immunization also showed reactivitv with the GBM. The in vivo localization of anti-FSA in the primate mesangium and resultant nephrotoxicity were previously reported.' Anti-FSA reacted only undiluted, on immunodiffusion, with normal human serum, a reaction which disappeared after passage over a human serum, Sepharose 4B (Biorad, Oxnard, Calif) immunosorbent column,7 without change in glomerular mesangial staining. Absorption with human and bovine plasminogen-rich fibrinogen (generously provided by Dr. Kenneth Miller, Departments of Pediatrics and Laboratorv Medicine), dialyzed and lyophilized fetal calf serum (Rehatuin F.S., Reheis, Phoenix, Ariz), human A and B red blood cell antigens, human skin collagen,l purified AMY and chicken gizzard tropomysin (generously provided by Dr. Judith Schollmeyer, Department of Laboratorv Medicine and Pathology) also did not abolish the staining. Anti-FSA had no cytotoxic reactivity with a panel of lymphocytes bearing known HLA specificities (kindly performed by Dr. Edmund Yunis, Department of Laboratory Medicine and Pathology). Absorption of anti-FSA with cold-insoluble globulin (CIG), isolated from cryoprecipitated human plasma (provided by Dr. Kenneth Miller) bv the fractionation steps of Mosseson and Umfleet,' completely abolished the immunofluorescent localization of anti-FSA on normal kidney sections and on monolayers of cultured human fibroblasts and glomerular cells.' This preparation of CIG also completely absorbed the staining of kidney sections and cultured cell monolayers by anti-CIG (generously provided by Dr. Lan Bo Chen, Cold Spring Harbor Laboratory).
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-St Direct and indirect immunofluorescent stainings were performed on frozen tissue sections of human kidney as described previously.' The IgG fractions of rabbit antiserums were used directly after labeling with fluorescein isothiocyanate (FITC) 10 or indirectlv in combination with FITC-labeled goat antirabbit IgG.11 Tissue sections were examined using a Zeiss fluorescence microscope with FITC interference filter and OG-4 barrier filter; more precise localization was obtained with a Zeiss Universal microscope with epifluorescence filters (FITC exciter filter 2XKP500, 1X455; dichroic mirror reflector IX510; and barrier filter IXLP528), allowing direct comparisons with phase or Nomarski optics. Pat One hundred ninety-six biopsy specimens and nephrectomy or autopsy (less than 4 hours post mortem) tissue samples (from the Departments of Pediatrics, Surgery, and Medicine) were studied. Seventeen normal kidney tissue specimens were transplant donor biopsy or normal tissue specimens obtained during the surgical removal of kidneys only partially involved by tumors. Specimens were from patients with primary renal diseases. including 25 with diabetic nephropathy, 19 with membranoproliferative glomerulonephritis (MPGN),- and 50 with other glomerulopathies, including 19 with focal (anaphylactoid purpura or IgA nephropathy) or diffuse proliferative glomerulonephritis, 16 with membranous nephropathy, including 6 with systemic lupus erythematosus, 2 with malignant hypertensive nephropathy, and 13 with idiopathic nephrotic syndrome (8 with focal sclerosis). Transplant kidney tissue was obtained for diagnostic purposes to evaluate the presence of homograft rejection or recurrence of disease. The diseases of the patients from whom these transplant tissues were obtainedf included 44 tissue specimens from 42 patients with diabetic nephropathy and from 9 with MPGN;t 19 biopsy specimens from 14 patients with other glomerulopathies (7 with undefined end-stage glomerulonephritis, 2 with systemic lupus erythematosus [membranous], and 1 each with idiopathic membranous nephropathy, Goodpasture's disease, nephrotic syndrome with focal sclerosis, congenital nephrotic syndrome, and scleroderma [hypertension]); and 13 biopsy specimens from 12 patients with nonglomerular diseases (5 with hypoplasia or dysplasia, 3 with polycystic disease, and 1 each with congenital ureteral obstruction, medullary cystic disease, chronic interstitial nephritis, and cystinosis).
Interpretation of Results The immunofluorescent patterns of tissues from normal patients and from patients with renal diseases were evaluated as previously described.1 Transplant tissues were evaluated blindly by one author (JIS). Selected tissues, representing a wide range of immunofluorescent alterations, were independently evaluated (as unknowns) by other investigators (AFM and AJF). For anti-AMY and anti-FSA, the width and extent of the stained mesangium were evaluated independently, as normal, decreased, moderately increased, or markedly increased. Width was evaluated in the proximal mesangium between adjacent * Fourteen of 19 MPGN patients were studied by electron microscopy; only 1 patient had GBM1 dense deposits." An attempt was made to select comparable biopsy specimens with respect to clinical state, prior renal disease, and duration of the graft. The diagnosis of transplant rejection was made on the basis of clinical course and routine histology by one author (AJM), without knowledge of immunohistopathologic findings. Patients with acute rejection had decreases in renal function within 1 month of biopsy, while those with chronic rejection had decreased renal function, not significantly different from the previous month, arising from episodes of acute rejection. One of 5 available for evaluation by electron microscopy had GBM dense deposits.1"
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capillary loops. Extent was a measure of the degree of staining extending to the periphery of the glomerulus. For anti-GBM, thickening of the stained GBMI was evaluated; tissues in X hich the glomerular capillary wall was markedly thickened but not fullY stained by the antibodv were not graded as abnormal in this s'stem.
Results Normal Kidney Tissues
As reported previously." anti-GBM (Figure 2A) stains the GBM within the glomerulus. Anti-AMY (Figure 2B) and antimvosin fix to the mesangium in a relatively restrictive pattern, while anti-FSA (Figure 2C) stains the mesangium more widelv and extensively. Bowvman's capsule and capillary and tubular basement membranes are stained strongly by antiGBM: interstitial tissues and arteriolar muscularis are not stained by antiGBM. Anti-AMY stains both large vessel and peritubular capillary endothelia and strands of material in the interstitium (possibly part of the capillar w-all). The muscularis of arteries and arterioles is strongly stained. No striated structures in the arterial wall have been observed. Anti-FSA staining of Bowman's capsule and tubular basement membranes is minimal and cannot be clearly distinguished because of the contiguous staining of adjacent interstitium. Arterial and arteriolar inner media are not stained, although the outer media and adventitia are stained. Venule and arteriole endothelia are only minimally and irregularly- stained. Anti-CIG stains the human glomerular mesangium brilliantly in a pattern identical to anti-FSA (Figures 2D and E). Interstitial tissue is also stained, with an accentuation of peritubular capillary walls. Endothelial staining of arterioles and veins is present but scattered (Figure 2F) rather than continuous (as seen with anti-AMY). Glomerular and tubular basement membranes are not stained. The staining by both anti-CIG and antiFSA on normal human kidney is completely inhibited by absorption with CIG. Primary Renal Diseases
Among the patients with glomerulopathies (Table 1), an increased extent of anti-AMY staining is seen in patients with diffuse proliferative glomerulonephritis (including lupus glomerulonephritis), membranous nephropathv, and hypertensive nephropathv. Anti-FSA staining patterns are similarly altered, although increased extent of mesangial staining for this antigen, normall- more extensive than AMY, is less dramatic. Patients with focal proliferative glomerulonephritis, including SLE with mesangial expansion, have a moderate focal increase in mesangial width stained by
GLOMERULAR ANTIGEN IMMUNOHISTOPATHOLOGY
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anti-AMY and anti-FSA. Areas of advanced focal sclerosis in patients with nephrotic syndrome do not stain with anti-AMY or anti-FSA; however, early focal sclerotic lesions in 2 patients are associated with localized increases in mesangial staining. Only patients with membranous nephropathy show thickened anti-GBM staining of the GBM. MPGN is accompanied by a loss of staining of the GBM by anti-GBM; anti-AMY does not stain the expanded mesangium. Anti-FSA staining of glomeruli from 15 of 17 patients with MPGN shows a similar marked decrease in mesangial staining. Tissue specimens from patients with MPGN with GBM dense deposits show the same loss of staining by anti-GBM and anti-FSA. In patients with diabetic nephropathy there is a marked increase in glomerular mesangial AMY; anti-FSA staining of the mesangium is markedly increased (Figure 3A). The same pattem of distribution is seen with anti-CIG on the tissue of a patient with diabetic nephropathy (Figure 3B). Staining by both anti-FSA and anti-CIG antiserums is abolished by absorption with CIG. Marked GBM thickening, which often stains as a bilaminar structure with anti-GBM, is seen in patients with diabetic nephropathy. Although a diffuse increase in all antigens is observed in the expanded interstitium in patients with advanced renal diseases, all glomerular antigens, including FSA, are absent from sclerotic glomeruli (in end-stage kidneys). Severely thickened arteriolar media and intima, seen especially in patients with diabetic nephropathy and hypertensive diseases, show neither staining with anti-GBM and anti-FSA nor increased staining with anti-AMY, the only antigen normally found in these loci. Trated Kidney Tssu
As seen in Table 1 and Text-figure IA, increased width of distribution of mesangial AMY antigen is not a regular feature of kidneys of recipients with previous glomerulopathies and nonglomerular diseases. However, 4 of 7 biopsy specimens obtained 3 or more years after transplantation in the glomerulopathy group have increased anti-AMY staining with normal width (Figure 4A and B), the typical finding in patients with primarn glomerulopathies. A significant proportion (5 of 8) of transplant recipients with prior MPGN have decreased anti-AMY mesangial staining (Figure 5), as found in patients with primary MPGN, compared with diabetics ( P < 0.0005) and patients with other glomerulopathies (P < 0.0005). The other evidence of recurrent MPGN in these patients included hypocomplementemia in 5 of 8; increased capillary loop thickening in the 7 examined by light microscopy, with 3 of 7 having basement membrane splitting; and typical lobular changes in 4 of 7. One of 5 tissue specimens
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related to time after transplantation. The time scale is compressed after 5 vears. N = normal, = markedly increased width. Closed decreased staining, t = increased mesangial width, and triangles (B and D) represent tissues from transplanted diabetic patients. Open circles (A and C) represent tissues from transplanted patients with previous nonglomerular diseases and miscellaneous glomerulopathies. Cloed circles (A and C) represent membranoproliferative glomerulonephritis (MPGN). Note the decreased AMY in MPGN in A and the lack of time-related increase in AMY in other tissues as is seen in B. Note the greater number of tissues with increased FSA (C) than AMY (A), unrelated to time, and the absence of decreased FSA in MPGN. Tissue samples obtained from diabetics at 4 'ears mav show a greater proportion with increased FSA (D) compared with tissue samples obtained from nondiabetics after 4 years (C).
evaluated by electron microscopy exhibited typical dense deposits, markedly increased anti-AMY and anti-GBM staining, and markedly increased anti-FSA. Immunofluorescent microscopy for immunoglobulins and complement components showed heavy CS mesangial and peripheral lobular staining as well as properdin in all but 2 of 7 tissues examined. Approximately one third of transplant tissues in the diabetic group have
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increased width of anti-AMY staining (Figure 6). There is a progressive increase with time in the proportion of tissues with moderate as well as marked increased in width of anti-AMY staining (Text-figure 1B), significantly different from nondiabetic tissue from 1 to 4 years (P < 0.025) and especially after 2 years (P < 0.0005). The distribution of FSA in normal subjects and in patients with primarv renal disease is more extensive than that of anti-AMY, except in patients with diabetic nephropathy, in whom both antigens are maximally increased. In none of the nontransplant groups is AMY normal and FSA increased. In contrast, it is evident from Table 1 and Text-figures IC and D that over half of tissues in all groups of transplant recipients have increased mesangial FSA (Figure 7), even in the presence of normal AMY. For example, in kidneys transplanted to patients with MPGN there is increased mesangial FSA (6 of 8) but decreased AMY (5 of 8). Between 1 and 4 years after transplantation, more diabetic (19 of 29) than nondiabetic tissues (6 of 14) exhibit increased FSA (P < 0.05). No association can be observed between clinical rejection and the selective increase in FSA in transplanted kidneys. When transplant tissues from patients with glomerulopathies and nonglomerular disease are combined, 4 of 7 biopsy specimens from patients without rejection and 15 of 25 from patients with rejection have increased FSA. In the diabetic group of recipients, 20 of 32 without any evidence of rejection and 8 of 12 with acute or chronic rejection have increases in FSA. However, most diabetics from whom tissue specimens were obtained in the first year had rejection, whereas few of the patients from whom tissue specimens were obtained later had experienced rejection. In view of the remarkable differences between anti-AMY patterns and anti-FSA in transplanted tissues, and their similarities in nontransplanted kidneys, further examination of the identity of FSA is warranted. Direct comparisons of anti-FSA and anti-CIG localization have been made on two normal tissue samples and three transplant biopsy specimens including two with moderately increased mesangial FSA and one with marked increase in FSA. The mesangial staining patterns of anti-FSA (Figure 8A) and anti-CIG (Figure 8B) are similar; however, with anti-CIG there is some increased extraglomerular endothelial staining, which is not found with anti-FSA. Increased GBM thickness observed with anti-GBM is found after transplantation, somewhat more prominently in later biopsy specimens (Figure 9). Decreased anti-GBM staining was seen in 3 of 8 biopsy specimens in the transplanted MPGN group, as found in primary MPGN. Increased anti-GBM staining was not more prominent in the diabetic than in the
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glomerulopathy group of transplant recipients, of whom only 2 had a pretransplant diagnosis of membranous nephropathy (the only type of disease in the primary glomerulopathies accompanied by increased GBM thickness stained by anti-GBM). Discussion
In the present study and our previous work I we have sought alterations in the immunohistopathology of normal glomerular antigens. The contractile protein, smooth muscle actomyosin (AMY), is normally localized by immunofluorescence to a restricted distribution in the glomertular mesangium."14 In human diabetic nephropathy it is markedly increased, filling the expanded mesangium. MPGN, having an expanded mesangial area, shows a decrease or disappearance of AMY. In contrast, other glomerulonephritic diseases, with less pronounced mesangial involvement, demonstrate extension of AMY to peripheral parts of the glomerulus, rather than the widened mesangium observed in diabetic nephropathy. In human renal homotransplantation we find evidence of the development of similar changes in the transplanted kidneys of patients whose own kidneys were previously lost due to those primary diseases. The fibroblast surface antigen (FSA), recently termed "fibronectin,"15 is a high-molecular-weight glycoprotein believed to be involved in the surface regulation of cellular function.16 We describe its distinctive distribution in the human glomerulus as mesangial but clearly more extensive than the distribution of AMY. In primary renal diseases its alterations are similar to those of AMY. However, the transplanted kidney reveals a marked disparity: FSA is strikingly increased in all groups of patients, more in diabetics over 1 year after transplantation. The proportion of nondiabetic patients with increased FSA did not progress with time after transplantation. The lack of nondiabetic tissue samples obtained at different times after transplantation from patients without rejection precluded determining the relationship between increased FSA mesangial width and clinical rejection. The glomerular distribution of GBM antigen in patients with primary renal diseases demonstrates thickening of the GBM in patients with diabetic nephropathy and membranous nephropathy. After transplantation, milder degrees of GBM thickening are seen, unrelated to the previous disease in the original kidney but apparently related to time after transplantation. In trying to explain these interesting immunohistopathologic alterations in glomerular antigens, the specificity and functional significance of the antigens must be explored. While some investigators have doubted the presence of myosin in the glomerulus,17 the observation of glomerular contractility in vitro 18 sug-
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gests that some form of contractile system is present. The basic functional components of all muscle contractile systems are myosin and actin. The present study verifies that anti-AMY has specificity for the myosin component. Autoantibodies 19 and heterologous antiserums 20 to actin have been seen to fix to glomeruli, although localization within the mesangium has not been convincingly demonstrated. '21 Tropomysin would be expected to accompany actin and myosin as a regulator of a contractile function and has been reported, by immunofluorescent localization, to have a distribution similar to actomysin in the mesangium.n However, our antichicken gizzard tropomyosin and anti-a-actinin ' stain human vascular media brilliantly, without striated structures," and only weakly and diffusely in the human and avian glomerulus, less than in interstitial tissue.2' What then is the function of glomerular myosin? It may have a role different from that in the arteriolar muscularis in regulating glomerular circulation and ultrafiltration. Recent micropuncture studies suggest that within the glomerulus there is a unique reactivity to angiotensin II and its analogues, differing from that of the arteriolar resistance vessels.2' The different species specificity of anti-AMY reactions with arterial media and glomerular mesangium reported here supports a tissue specialization. In patients with diabetic nephropathy the markedly thickened arteriolar media often has diminished anti-AMY staining, while the mesangium and vascular intima have increased staining. The increased AMY antigens of patients with early primary diabetic nephropathy might represent a generalized increase in the contractile function of mesangial cells in the metabolic environment of diabetes mellitus. The process by which accentuated mesangial smooth muscle function might lead to the loss of renal function in diabetes is unclear. At later stages of the disease the massive increase in AMY and FSA in the mesangium may reflect a failure of the mesangium to remove cellular degradation products, as suggested by studies of the processing of macromolecular aggregates in experimental diabetes in rats.X An increased width of mesangial anti-AMY staining develops within a few years in diabetic transplant recipients, which is greater than that observed in nondiabetic hosts. The development of distinctive lesions of diabetic nephropathy in the nondiabetic kidney transplanted into the human diabetic host is supported by other evidence from this laboratory: arteriolar hyalinosis 26 and renal extracellular basement membrane immunofluorescence for IgG and albumin 2" were recently reported in renal homografts in human diabetic recipients. Observations on renal transplantation in experimental diabetes in rats '* provides further support for
Vol. 90, No. 1 January 1978
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the hypothesis that the renal lesions develop as a consequence of the metabolic alterations. Decreased mesangial AMY is found in the original and transplanted kidneys of patients with MPGN, which supports the morphologic evidence of recurrence of that lesion.12 On the basis of glomerular and vascular cell culture studies,2' we presume that AMY is normally present within mesangial cells, while FSA is a cell surface antigen ' in the mesangial matrix. Anti-FSA elicited to an undefined mixture of antigens from human skin fibroblasts 6 has a similar tissue distribution to that of the FSA (or "fibronectin") previously described 2; the cross reaction with CIG and absorption with this antigen I provide identification. The limitations of light microscopy preclude our certainty of the mesangial cell origin of FSA in glomeruli of normal, diseased, and transplanted kidneys. We cannot exclude the possibility that FSA is also present in the glomerular endothelial cell. High-resolution light microscopy, using phase and Nomarski optics, compared with immunofluorescent techniques, shows that FSA (and CIG) remains beneath the basement membrane of glomerular capillary loops but seldom surrounds cross-sectional loops. In this area it resembles endothelial staining for factor VIII antigen.' Glomerular smooth muscle (possibly mesangial) cells in culture do not show the contact inhibition of skin fibroblasts. They further differ from fibroblasts by having less surface FSA; however, they have more surface FSA than do (umbilical vein) endothelial cells.' Contact inhibition of cultured fibroblasts is associated with retention of surface FSA.1' The proliferation of the mesangial cells of normal kidneys may then be limited bv normal FSA. Binding of FSA by its antibody was seen experimentally bv in vivo injection into primates,6 resulting in mesangial cell proliferation and proteinuria, perhaps a consequence of the binding of FSA. In patients with primarv renal diseases, FSA is found in the mesangium in a pattern similar to that of AMY; the dissociation between the findings for anti-AMY and anti-FSA staining of the glomerulus of nondiabetic transplanted kidneys is remarkable: mesangial anti-FSA staining can be markedly increased, even when anti-AMY staining is normal. An example is the decrease in anti-AMY staining in recurrent MPGN, while anti-FSA staining is normal or increased. The presence of significant levels of FSA in plasma6 and the known channeling of circulating macromolecular aggregates through the mesangium 31 suggest that the increased FSA in the mesangium of transplanted kidneys could represent an altered balance between the total synthesis and release from extrarenal sites and normal clearance of this antigen through the mesangium. Its relationship to the rejection process has not been determined.
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Heterologous antiserum to GB M is directed against numerous antigenic determinants. The most prominent are probably the noncollagen components 32 in view of the relatively poor antigenicity of collagen. Our antiGBM reacts by immunoprecipitation wvith collagenase-digested (and presumably noncollagep) GBM. In patients with renal diseases involving severe GBM thickening, one can detect a bilaminar staining of the GBMI with anti-GBM antiserums,' suggesting that antigenically "normal" GBMI may be deposited from both the endothelial and epithelial sides of the GBM. Bilaminar staining is found not only in tissue samples from patients with diabetes mellitus and membranous nephropathv but also in some long-term transplant tissues. In transplantation, thickened GBM staining is neither associated with proteinuria or clinical rejection nor found distinctively in patients whose pretransplant disease was diabetic nephropathv or glomerulonephritis; it is thus related to some undefined phenomenon, as previously observed by conventional histologic techniques.3 The observations of this study may provide both defined markers of the glomerular mesangial cell and new parameters of alteration in this cell system associated wvith glomerular injurv. In many glomerular disorders (diabetes mellitus, MPGN, proliferative glomerulopathies, and transplantation nephropathy) there exists considerable overlap in the histologic findings of mesangial cell proliferation and increased mesangial matrix. In this study distinctive changes in AMY and FSA glomerular mesangial antigenic markers wvere observed; more precise, specific pathologic observations can be similarly made for each disorder and may provide nex insights into the pathogenesis of these glomerular lesions. References 1. Scheinman JI. Fish AJ. Michael AF: The imrnunohistopathology of glomerular antigens: The glomerular basement membrane. collagen, and actomyosin antigens in normal and diseased kidneys. J Clin Invest 34:1144-1154. 1974 2. Linder E. V'aheri A. Ruoslahti E. Wartiovaara J: Distribution of fibroblast surface antigen in the developing chick embryo. J Exp Med 142:41-49, 1973 3. Pollard TD, Thomas SM. Niederman R: Human platelet myosin. I. Purification by a rapid method applicable to other non-muscle cells. Anal Biochem 60:258-266.1974 4. W7eber K. Osborn NI: The reliability of molecular weight determinations by dodecyl sulfate-polvacylamide gel electrophoresis. J Biol Chem 244:4406-4412, 1969 3. Chen PS Jr. Toribara TY. Warner H: Microdetermination of phosphorus. Anal Chem 28:1 736-1738. 1956 6. Fish AJ, NMichael AF. Xernier RL. Brow-n DM: Human glomerular cells in tissue culture. Lab Invest 33:3.30-341, 197 19 7. Parikh I, March S. Cuatrecasas P: Topics in the methodology of substitution reactions with agarose. Methods Enzymol :34:77-102. 1974 8. Nfosesson MNXV. Umfleet RA: The cold-insoluble globulin of human plasma. I.
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Purification, primary characterization, and relationship to fibrinogen and other coldinsoluble fraction components. J Biol Chem 245:5728-5736, 1970 Scheinman JI, Fish AJ: Glomerular antigens of glomerular and vascular cells in culture. Fed Proc 36:1055, 1977 (Abstr) Cebra JJ, Goldstein G: Chromatographic purification of tetramethylrhodamineimmune globulin conjugates and their use in the cellular localization of rabbit -globulin polypeptide chains. J Immunol 95:230-245, 1965 Michael AF Jr, Drummond KN, Good RA, Vernier RL: Acute poststreptococcal glomerulonephritis: Immune deposit disease. J Clin Invest 45:237-248, 1966 McLean RH, Geiger H, Burke B, Simmons R, Najarian J, Vernier RL, Michael AF: Recurrence of membranoproliferative glomerulonephritis following a kidnev transplantation: Serum complement component studies. Am J Med 60:60-72, 1976 Kim Y, Vernier RL: Personal communication Becker CG: Demonstration of actomyosin in mesangial cells of the renal glomerulus. Am J Pathol 66:97-110, 1972 Kuusela P, Ruoslahti E, Engwall E, Vaheri A: Immunological interspecies crossreactions of fibroblast surface antigen (fibronectin). Immunochemistrv 13:639-642, 1976 Yamada KM, Yamada SS, Pastan I: Cell surface protein partiallv restores morphology, adhesiveness, and contact inhibition of movement to transformed fibroblasts. Proc Natl Acad Sci USA 73:1217-1221, 1976 Rukosuev VS, Nanaev AK: Histogenesis of mesangial cells of the renal glomeruli. Bull Exp Biol Med 79:115-117, 1975 Bernik MB: Contractile activity of human glomeruli in culture. Nephron 6:1-10, 1969 Whittingham S, Mackav IR, Irwin J: Autoimmune hepatitis. Immunofluorescence reactions with cytoplasm of smooth muscle and renal glomerular cells. Lancet 1:1333-1335, 1966 Miller F, Lazarides E; Elias J: Application of immunologic probes for contractile proteins to tissue sections. Clin Immunol Immunopathol 5:416-428, 1976 Accinni L, Natali PG, Vassallo L, Hsu KS, DeMartino C: Immunoelectron microscopic evidence of contractile proteins in the cellular and acellular components of mouse kidney glomeruli. Cell Tissue Res 162:297-312, 1975 Becker CG, Hardy AM, Dubin T: Contractile and relaxing proteins of smooth muscle, endothelial cells and platelets. Platelets, Thrombosis and Inhibitors. Edited bv P Didisheim. New York, FK Schattaur Verlay, 1974, pp 25-34 Schollmeyer JV, Furcht LT, Goll DE, Robson RM, Steomer MH: Localization of a-actinin and tropomvosin in smooth muscle and normal and transformed cells. Cell Motility. Cold Spring Harbor Symposium on Cellular Proliferation, 1976, pp
361-387 24. Scheinman JI, Schollmever JV: Unpublished observations 25. Blantz RC, Konnen KS, Tucker BJ: Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat. J Clin Invest 57:419434, 1976 26. Mauer SM, Steffes MW, Michael AF, Brown DM: Studies of diabetic nephropathv in animals and man. Diabetes 25:850-857, 1976 27. Mauer SM, Miller K, Goetz FC, Barbosa J, Simmons RL, Najarian JS, Michael AF: Immunopathology of renal extracellular membranes in kidneys transplanted into patients with diabetes mellitus. Diabetes 25:709-712, 1976 28. Scheinman JI, Fish AJ, Brown DM, Michael AF: Human glomerular smooth muscle (mesangial) cells in culture. Lab Invest 34:150-158, 1976 29. Ruoslahti E, Vaheri A: Interaction of soluble fibroblast surface antigen with fibrinogen and fibrin: Identitv with cold-insoluble globulin of human plasma. J Exp Med 141:497-501, 1975
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:30. Hover LW. de Los Santos RP. Hover JR: Antihemophilic factor antigen: Localization in endothelial cells bv immunofluorescent microscopy. J Clin Invest 52:273 7-2744. 197.3 31. Elema JD. Hover JR. Vernier RL: The glomerular mesangium: Uptake and transport of intravenously injected colloidal carbon in rats. Kidney Int 9:395-406. 1976 32. Marquardt H. Wilson CB. Dixon FJ: Human glomerular basement membrane. Selective solubilization with chaotropes and chemical and immunologic characterization of its components. Biochemistry 12:3260-3266. 1973 33. Fish AJ. Herdman RC. Kelly WN'D. Good RA: Glomerular changes in w-ell-functioning human renal homografts. Transplantation 3: 13.38-1343. 1967
Acknowledgments The overall technical assistance of Kathryn LaCroix and Kathleen Carmodv: of Cr-stal Blocher. Kim Pinkham. and Lore Lang in immunohistology; and of Marshall Hoff in illustrations and photography and the secretarial assistance of Nancy Kirschling are gratefully acknowledged. WN e also wish to thank Drs. S. Michael Mauer. John Najarian. and Richard Simmons for supplying tissues: Dr. Lan Bo Chen for supplying antiserum to cold-insoluble globulin: and Dr. Judith Schollmeyer for supplying antiserums to chicken gizzard a-actinin and tropomyosin. We especially thank Drs. Y. Kim and R. L. Vernier for the prepublication communication of their electron microscopic findings in MIPGN- and in recurrent MIPGN- in the transplanted kidney.
Figure LA-Polyacrylamide eketrophoresis gels (7.5%) loaded with 75 gg protein, stained with Coomassie blue. Human uterine myosin, left gel, shows myosin heavy chain (M), myosin rod (R), and small bands of unidentified peptides (X). Light chains, not seen in this reproduction, were faintly visualized at (L). In the gel on the right, human uterine actin (A) is run as a marker. B-Ouchterlony immunodiffusion in 1% Noble agar, 0.5 M KCI, diffused for 3 days at 4 C, stained with Buffalo black. Actomyosin, approximately 5 mg/ml in 50% glycerol, 0.5 M KCI, in center well. Antiserums, clockwise: 1, 2, and 3 are undiluted antiserums from 3 rabbits
immunized with myosin. 4 is anti-AMY IgG, 10 mg/ml, sharing the line of identity with the antimyosins.
Fgure 2-Immunofluorescent localization of glomerular antigens in the normal glomerulus. A-Anti-GBM localization to the GBM. (x 590) B-Antiactomyosin (AMY) staining of the mesangium in a restrictive pattern. The adjacent wall of the arteriole (A) is strongly stained. (x C-Antifibroblast surface antigen (FSA) staining of the mesangium is more extensive 590) than anti-AMY (B). (x 510) D-Anti-CIG staining of the mesangium, in the same extensive as pattern anti-FSA (Bt Arrows are directed to unstained peripheral capillary loops. (Epifluorescence, x 350) E-Phase micrograph of the same field as D. (x 350) F-Anti-CIG staining of normal kidney shows mild reactivity of endothelium (E) of arteriole and peritubular capillary (C). Bowman's capsule (BC), between arrows, is unstained. A light interstitial staining cannot be specifically localized. (Epifluorescence, x 700)
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Fgu 3-Immunofluorescent localization of anti-FSA and anti-CIG in a patient with diabetic nephropathy. AAnti-FSA staininq of expanded mesangium (M). (x 590) B-Anti-CIG stain of a portion of a glomerulus from the CIG stain of a portion of a glomerulus from the same tissue as in A, with unstained mesangium (arrows). The muscularis of the arteriole (above A) is not stained, and only scattered endothelial staining is visible. (x 1800) Fge 4-Increased extent (not width) of anti-AMY staining in 2-year transplant biopsy specimen of a patient with previous (and recurrent) membranous nephropathy. A-Arrows point to the (unstained) outer aspect of peripheral capillary loops, showing that staining for AMY remains interior to the GBM. Note lack of continuity in staining of B-Phase contrast view of the same field as in A, showing the locatwo capillary loops. (Epifluorescence, x 750) tion of basement membranes (arrows).
Fqu 5-Decreased anti-AMY staining of 8-year transplant biopsy of a patient with MPGN. Decreased glomerular staining contrasts with preserved staining of the wall of an adjacent arteriole (A). (x 590) Fe 6Markedly increased anti-AMY staining of mesangial width in 4-year transplant biopsy of a diabetic patient. (x 380) Fe 7-Three-year transplant biopsy of a diabetic patient with normal renal function and no evidence of rejection. Anti-FSA stains a markedly widened mesangium. Note (arrow) the light staining of arterial endothelium. (x 380).
Fgwe 8A-Mildly
increased mesangial anti-FSA staining. Staining of adjacent sections of a 2-year nondiabetic transplant biopsy specimen. The arterial media is unstained, but adventitium is stained (A). Tubule B-Anti-CIG staining (T) is seen in A and B. (x 590)
is the same as on adjacent section. Note intimal staining of the arteriole at arrow, the same vessel seen Fiu 9-Antiat the top of the field in Figure 9. GBM staining of markedly thickened GBM (between arrows) in 10-year transplant biopsy specimen from a patient with primary renal hypoplasia. Note bilaminar pattern, with accentuation of the inner aspect. (x 1260)
Immunofluorescent studies have demonstrated that actomyosin (AMY) is present in the mesangium in a restrictive pattern, whereas fibroblast surface antigen (FSA) has a more extensive mesangial distribution. Antibody to glomerular basement membrane (GBM) is localized to the GBM. One hundred ninety-six tissue samples, including 17 from normal subjects, 94 from patients with primary renal diseases, and 85 from transplanted kidneys. were examined for changes in distribution of AMY, FSA, and GBMI antigens. The distribution of A-MY and FSA in the mesangium is markedly increased in width in patients with diabetic nephropathy, and the GBM is thickened. AMY and FSA are mildly increased in patients with glomerulonephritis and GBM is normal. Patients with membranoproliferative glomerulonephritis (MPGN) show a loss of all glomerular antigens. Increased mesangial A-MY was found in 12 of 37 transplanted kidneys in diabetic patients vs 4 of 33 nondiabetics 0 to 4 years after transplantation (P < 0.025). This difference is more notable at 2 to 4 vears, with 5 of 9 diabetics showing increased AMY vs 0 of 6 nondiabetic patients (P < 0.0005). In MPGN, 5 of 8 tissues showed decreased mesangial A-MY vs 3 of 36 diabetic patients (P < 0.0005) and 1 of 32 patients with other glomerulopathies (P < 0.0005). GBM thickening is not associated with specific pretransplant diseases. In transplanted kidneys, the pattern of FSA is dissociated from that of AMY: over half of all patients have increased FSA, even when .AM*Y is normal. Whether this unique finding in transplanted kidneys reflects an increase in the synthesis of FSA by mesangial cells or the failure to clear this (circulating) antigen from the mesangial matrix is unknown. (Am J Pathol 90:71-8, 1978)
PREVIOUS STUDIES HAV-E DEMONSTRATED a marked and consistent
increase in the distribution of mesangial actomvosin (ANIY) antigen 1 in diabetic nephropathy and its loss in membranoproliferative glomerulonephritis, with more subtle alterations in other glomerulopathies. Glomerular basement membrane (GB\I) antigen was localized to the thickened basement membranes in patients with diabetic nephropathv and From the Departments of Pediatrics and Surgery. Univ ersity of Nlinnesota Medical School. Mlinneapolis. \linnesota Supported by a Grant-In-Aid from the American Heart Association and the American Diabetes Association of Minnesota. by grants from the American Diabetes Association and the Minnesota Xrthritis Foundation. and by Grants HL 06314 and XI-01704-14 from the US Public Health Ser-ice Dr Scheinman is the recipient of a Research Career Development Avvard from the NIAMD =K04A\100126)i Accepted for publication August 31. 1977. Xddress reprint requests to Dr Jon I Scheinman. Department of Pediatrics. Box 491 Mayo \lemorial Building. University of Minnesota \Medical School. \inneapolis. NN 5.5455. 71
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membranous nephropathy. In this study we explore, in addition, the immunohistopathology of the fibroblast surface antigen (FSA) or fibronectin2 in primary renal diseases and further examine all these antigens in transplanted human kidneys. Materials and Methods Antsm Antim-aid Antihuman uterine smooth muscle actomyosin (anti-AMY) was made in rabbits immunized with purified AMY and characterized as previously described.' Myosin was isolated by the method of Pollard, Thomas, and Niederman,' was relatively homogeneous on acrylamide gel electrophoresis 4 in sodium dodecyl sulfate (Figure IA), and had ATPase activity (measured in 0.6 M KC1, 8 mM MgCGl, 15 mM Tris at room temperature) of 3 AM P/mg/30 min, similar to human uterine AMY.1 Inorganic phosphorus was determined colorimetrically.' Foot pad immunization of 3 rabbits with 1 mg of mvosin in complete Freund's adjuvant was repeated subcutaneously after 3 weeks, and bleeds taken 2 to 4 weeks later were used for immunodiffusion and immunofluorescence. Both arterial walls and mesangium were stained in the human kidney, but only the arterial wall was stained in rat tissue. Antimyosin reacted against AMY and myosin on immunodiffusion 1 with lines of identity (Figure IB); the tissue-staining reactivities of anti-AMY and antimyosin were identical and both were completely inhibited by absorption with mvosin. Anti-AMY was also inhibited by absorption with a sonicated, Iyophilized preparation of whole human glomeruli. Rabbit antihuman glomerular basement membrane (GBM) antiserums have been previously characterized.1 Antifibroblast surface antigen (anti-FSA) was made in rabbits immunized with lvophilized human foreskin fibroblast cultures incorporated in complete Freund's adjuvant as previously described." Bleeds obtained 2 to 3 weeks after a single booster injection were used. This antiserum reacted with the mesangium of human, rat, and chicken kidnev, as observed by indirect immunofluorescent techniques. Antiserums obtained later in the course of immunization also showed reactivitv with the GBM. The in vivo localization of anti-FSA in the primate mesangium and resultant nephrotoxicity were previously reported.' Anti-FSA reacted only undiluted, on immunodiffusion, with normal human serum, a reaction which disappeared after passage over a human serum, Sepharose 4B (Biorad, Oxnard, Calif) immunosorbent column,7 without change in glomerular mesangial staining. Absorption with human and bovine plasminogen-rich fibrinogen (generously provided by Dr. Kenneth Miller, Departments of Pediatrics and Laboratorv Medicine), dialyzed and lyophilized fetal calf serum (Rehatuin F.S., Reheis, Phoenix, Ariz), human A and B red blood cell antigens, human skin collagen,l purified AMY and chicken gizzard tropomysin (generously provided by Dr. Judith Schollmeyer, Department of Laboratorv Medicine and Pathology) also did not abolish the staining. Anti-FSA had no cytotoxic reactivity with a panel of lymphocytes bearing known HLA specificities (kindly performed by Dr. Edmund Yunis, Department of Laboratory Medicine and Pathology). Absorption of anti-FSA with cold-insoluble globulin (CIG), isolated from cryoprecipitated human plasma (provided by Dr. Kenneth Miller) bv the fractionation steps of Mosseson and Umfleet,' completely abolished the immunofluorescent localization of anti-FSA on normal kidney sections and on monolayers of cultured human fibroblasts and glomerular cells.' This preparation of CIG also completely absorbed the staining of kidney sections and cultured cell monolayers by anti-CIG (generously provided by Dr. Lan Bo Chen, Cold Spring Harbor Laboratory).
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-St Direct and indirect immunofluorescent stainings were performed on frozen tissue sections of human kidney as described previously.' The IgG fractions of rabbit antiserums were used directly after labeling with fluorescein isothiocyanate (FITC) 10 or indirectlv in combination with FITC-labeled goat antirabbit IgG.11 Tissue sections were examined using a Zeiss fluorescence microscope with FITC interference filter and OG-4 barrier filter; more precise localization was obtained with a Zeiss Universal microscope with epifluorescence filters (FITC exciter filter 2XKP500, 1X455; dichroic mirror reflector IX510; and barrier filter IXLP528), allowing direct comparisons with phase or Nomarski optics. Pat One hundred ninety-six biopsy specimens and nephrectomy or autopsy (less than 4 hours post mortem) tissue samples (from the Departments of Pediatrics, Surgery, and Medicine) were studied. Seventeen normal kidney tissue specimens were transplant donor biopsy or normal tissue specimens obtained during the surgical removal of kidneys only partially involved by tumors. Specimens were from patients with primary renal diseases. including 25 with diabetic nephropathy, 19 with membranoproliferative glomerulonephritis (MPGN),- and 50 with other glomerulopathies, including 19 with focal (anaphylactoid purpura or IgA nephropathy) or diffuse proliferative glomerulonephritis, 16 with membranous nephropathy, including 6 with systemic lupus erythematosus, 2 with malignant hypertensive nephropathy, and 13 with idiopathic nephrotic syndrome (8 with focal sclerosis). Transplant kidney tissue was obtained for diagnostic purposes to evaluate the presence of homograft rejection or recurrence of disease. The diseases of the patients from whom these transplant tissues were obtainedf included 44 tissue specimens from 42 patients with diabetic nephropathy and from 9 with MPGN;t 19 biopsy specimens from 14 patients with other glomerulopathies (7 with undefined end-stage glomerulonephritis, 2 with systemic lupus erythematosus [membranous], and 1 each with idiopathic membranous nephropathy, Goodpasture's disease, nephrotic syndrome with focal sclerosis, congenital nephrotic syndrome, and scleroderma [hypertension]); and 13 biopsy specimens from 12 patients with nonglomerular diseases (5 with hypoplasia or dysplasia, 3 with polycystic disease, and 1 each with congenital ureteral obstruction, medullary cystic disease, chronic interstitial nephritis, and cystinosis).
Interpretation of Results The immunofluorescent patterns of tissues from normal patients and from patients with renal diseases were evaluated as previously described.1 Transplant tissues were evaluated blindly by one author (JIS). Selected tissues, representing a wide range of immunofluorescent alterations, were independently evaluated (as unknowns) by other investigators (AFM and AJF). For anti-AMY and anti-FSA, the width and extent of the stained mesangium were evaluated independently, as normal, decreased, moderately increased, or markedly increased. Width was evaluated in the proximal mesangium between adjacent * Fourteen of 19 MPGN patients were studied by electron microscopy; only 1 patient had GBM1 dense deposits." An attempt was made to select comparable biopsy specimens with respect to clinical state, prior renal disease, and duration of the graft. The diagnosis of transplant rejection was made on the basis of clinical course and routine histology by one author (AJM), without knowledge of immunohistopathologic findings. Patients with acute rejection had decreases in renal function within 1 month of biopsy, while those with chronic rejection had decreased renal function, not significantly different from the previous month, arising from episodes of acute rejection. One of 5 available for evaluation by electron microscopy had GBM dense deposits.1"
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capillary loops. Extent was a measure of the degree of staining extending to the periphery of the glomerulus. For anti-GBM, thickening of the stained GBMI was evaluated; tissues in X hich the glomerular capillary wall was markedly thickened but not fullY stained by the antibodv were not graded as abnormal in this s'stem.
Results Normal Kidney Tissues
As reported previously." anti-GBM (Figure 2A) stains the GBM within the glomerulus. Anti-AMY (Figure 2B) and antimvosin fix to the mesangium in a relatively restrictive pattern, while anti-FSA (Figure 2C) stains the mesangium more widelv and extensively. Bowvman's capsule and capillary and tubular basement membranes are stained strongly by antiGBM: interstitial tissues and arteriolar muscularis are not stained by antiGBM. Anti-AMY stains both large vessel and peritubular capillary endothelia and strands of material in the interstitium (possibly part of the capillar w-all). The muscularis of arteries and arterioles is strongly stained. No striated structures in the arterial wall have been observed. Anti-FSA staining of Bowman's capsule and tubular basement membranes is minimal and cannot be clearly distinguished because of the contiguous staining of adjacent interstitium. Arterial and arteriolar inner media are not stained, although the outer media and adventitia are stained. Venule and arteriole endothelia are only minimally and irregularly- stained. Anti-CIG stains the human glomerular mesangium brilliantly in a pattern identical to anti-FSA (Figures 2D and E). Interstitial tissue is also stained, with an accentuation of peritubular capillary walls. Endothelial staining of arterioles and veins is present but scattered (Figure 2F) rather than continuous (as seen with anti-AMY). Glomerular and tubular basement membranes are not stained. The staining by both anti-CIG and antiFSA on normal human kidney is completely inhibited by absorption with CIG. Primary Renal Diseases
Among the patients with glomerulopathies (Table 1), an increased extent of anti-AMY staining is seen in patients with diffuse proliferative glomerulonephritis (including lupus glomerulonephritis), membranous nephropathv, and hypertensive nephropathv. Anti-FSA staining patterns are similarly altered, although increased extent of mesangial staining for this antigen, normall- more extensive than AMY, is less dramatic. Patients with focal proliferative glomerulonephritis, including SLE with mesangial expansion, have a moderate focal increase in mesangial width stained by
GLOMERULAR ANTIGEN IMMUNOHISTOPATHOLOGY
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anti-AMY and anti-FSA. Areas of advanced focal sclerosis in patients with nephrotic syndrome do not stain with anti-AMY or anti-FSA; however, early focal sclerotic lesions in 2 patients are associated with localized increases in mesangial staining. Only patients with membranous nephropathy show thickened anti-GBM staining of the GBM. MPGN is accompanied by a loss of staining of the GBM by anti-GBM; anti-AMY does not stain the expanded mesangium. Anti-FSA staining of glomeruli from 15 of 17 patients with MPGN shows a similar marked decrease in mesangial staining. Tissue specimens from patients with MPGN with GBM dense deposits show the same loss of staining by anti-GBM and anti-FSA. In patients with diabetic nephropathy there is a marked increase in glomerular mesangial AMY; anti-FSA staining of the mesangium is markedly increased (Figure 3A). The same pattem of distribution is seen with anti-CIG on the tissue of a patient with diabetic nephropathy (Figure 3B). Staining by both anti-FSA and anti-CIG antiserums is abolished by absorption with CIG. Marked GBM thickening, which often stains as a bilaminar structure with anti-GBM, is seen in patients with diabetic nephropathy. Although a diffuse increase in all antigens is observed in the expanded interstitium in patients with advanced renal diseases, all glomerular antigens, including FSA, are absent from sclerotic glomeruli (in end-stage kidneys). Severely thickened arteriolar media and intima, seen especially in patients with diabetic nephropathy and hypertensive diseases, show neither staining with anti-GBM and anti-FSA nor increased staining with anti-AMY, the only antigen normally found in these loci. Trated Kidney Tssu
As seen in Table 1 and Text-figure IA, increased width of distribution of mesangial AMY antigen is not a regular feature of kidneys of recipients with previous glomerulopathies and nonglomerular diseases. However, 4 of 7 biopsy specimens obtained 3 or more years after transplantation in the glomerulopathy group have increased anti-AMY staining with normal width (Figure 4A and B), the typical finding in patients with primarn glomerulopathies. A significant proportion (5 of 8) of transplant recipients with prior MPGN have decreased anti-AMY mesangial staining (Figure 5), as found in patients with primary MPGN, compared with diabetics ( P < 0.0005) and patients with other glomerulopathies (P < 0.0005). The other evidence of recurrent MPGN in these patients included hypocomplementemia in 5 of 8; increased capillary loop thickening in the 7 examined by light microscopy, with 3 of 7 having basement membrane splitting; and typical lobular changes in 4 of 7. One of 5 tissue specimens
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related to time after transplantation. The time scale is compressed after 5 vears. N = normal, = markedly increased width. Closed decreased staining, t = increased mesangial width, and triangles (B and D) represent tissues from transplanted diabetic patients. Open circles (A and C) represent tissues from transplanted patients with previous nonglomerular diseases and miscellaneous glomerulopathies. Cloed circles (A and C) represent membranoproliferative glomerulonephritis (MPGN). Note the decreased AMY in MPGN in A and the lack of time-related increase in AMY in other tissues as is seen in B. Note the greater number of tissues with increased FSA (C) than AMY (A), unrelated to time, and the absence of decreased FSA in MPGN. Tissue samples obtained from diabetics at 4 'ears mav show a greater proportion with increased FSA (D) compared with tissue samples obtained from nondiabetics after 4 years (C).
evaluated by electron microscopy exhibited typical dense deposits, markedly increased anti-AMY and anti-GBM staining, and markedly increased anti-FSA. Immunofluorescent microscopy for immunoglobulins and complement components showed heavy CS mesangial and peripheral lobular staining as well as properdin in all but 2 of 7 tissues examined. Approximately one third of transplant tissues in the diabetic group have
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increased width of anti-AMY staining (Figure 6). There is a progressive increase with time in the proportion of tissues with moderate as well as marked increased in width of anti-AMY staining (Text-figure 1B), significantly different from nondiabetic tissue from 1 to 4 years (P < 0.025) and especially after 2 years (P < 0.0005). The distribution of FSA in normal subjects and in patients with primarv renal disease is more extensive than that of anti-AMY, except in patients with diabetic nephropathy, in whom both antigens are maximally increased. In none of the nontransplant groups is AMY normal and FSA increased. In contrast, it is evident from Table 1 and Text-figures IC and D that over half of tissues in all groups of transplant recipients have increased mesangial FSA (Figure 7), even in the presence of normal AMY. For example, in kidneys transplanted to patients with MPGN there is increased mesangial FSA (6 of 8) but decreased AMY (5 of 8). Between 1 and 4 years after transplantation, more diabetic (19 of 29) than nondiabetic tissues (6 of 14) exhibit increased FSA (P < 0.05). No association can be observed between clinical rejection and the selective increase in FSA in transplanted kidneys. When transplant tissues from patients with glomerulopathies and nonglomerular disease are combined, 4 of 7 biopsy specimens from patients without rejection and 15 of 25 from patients with rejection have increased FSA. In the diabetic group of recipients, 20 of 32 without any evidence of rejection and 8 of 12 with acute or chronic rejection have increases in FSA. However, most diabetics from whom tissue specimens were obtained in the first year had rejection, whereas few of the patients from whom tissue specimens were obtained later had experienced rejection. In view of the remarkable differences between anti-AMY patterns and anti-FSA in transplanted tissues, and their similarities in nontransplanted kidneys, further examination of the identity of FSA is warranted. Direct comparisons of anti-FSA and anti-CIG localization have been made on two normal tissue samples and three transplant biopsy specimens including two with moderately increased mesangial FSA and one with marked increase in FSA. The mesangial staining patterns of anti-FSA (Figure 8A) and anti-CIG (Figure 8B) are similar; however, with anti-CIG there is some increased extraglomerular endothelial staining, which is not found with anti-FSA. Increased GBM thickness observed with anti-GBM is found after transplantation, somewhat more prominently in later biopsy specimens (Figure 9). Decreased anti-GBM staining was seen in 3 of 8 biopsy specimens in the transplanted MPGN group, as found in primary MPGN. Increased anti-GBM staining was not more prominent in the diabetic than in the
Vol. 90, No..l January 1978
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glomerulopathy group of transplant recipients, of whom only 2 had a pretransplant diagnosis of membranous nephropathy (the only type of disease in the primary glomerulopathies accompanied by increased GBM thickness stained by anti-GBM). Discussion
In the present study and our previous work I we have sought alterations in the immunohistopathology of normal glomerular antigens. The contractile protein, smooth muscle actomyosin (AMY), is normally localized by immunofluorescence to a restricted distribution in the glomertular mesangium."14 In human diabetic nephropathy it is markedly increased, filling the expanded mesangium. MPGN, having an expanded mesangial area, shows a decrease or disappearance of AMY. In contrast, other glomerulonephritic diseases, with less pronounced mesangial involvement, demonstrate extension of AMY to peripheral parts of the glomerulus, rather than the widened mesangium observed in diabetic nephropathy. In human renal homotransplantation we find evidence of the development of similar changes in the transplanted kidneys of patients whose own kidneys were previously lost due to those primary diseases. The fibroblast surface antigen (FSA), recently termed "fibronectin,"15 is a high-molecular-weight glycoprotein believed to be involved in the surface regulation of cellular function.16 We describe its distinctive distribution in the human glomerulus as mesangial but clearly more extensive than the distribution of AMY. In primary renal diseases its alterations are similar to those of AMY. However, the transplanted kidney reveals a marked disparity: FSA is strikingly increased in all groups of patients, more in diabetics over 1 year after transplantation. The proportion of nondiabetic patients with increased FSA did not progress with time after transplantation. The lack of nondiabetic tissue samples obtained at different times after transplantation from patients without rejection precluded determining the relationship between increased FSA mesangial width and clinical rejection. The glomerular distribution of GBM antigen in patients with primary renal diseases demonstrates thickening of the GBM in patients with diabetic nephropathy and membranous nephropathy. After transplantation, milder degrees of GBM thickening are seen, unrelated to the previous disease in the original kidney but apparently related to time after transplantation. In trying to explain these interesting immunohistopathologic alterations in glomerular antigens, the specificity and functional significance of the antigens must be explored. While some investigators have doubted the presence of myosin in the glomerulus,17 the observation of glomerular contractility in vitro 18 sug-
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gests that some form of contractile system is present. The basic functional components of all muscle contractile systems are myosin and actin. The present study verifies that anti-AMY has specificity for the myosin component. Autoantibodies 19 and heterologous antiserums 20 to actin have been seen to fix to glomeruli, although localization within the mesangium has not been convincingly demonstrated. '21 Tropomysin would be expected to accompany actin and myosin as a regulator of a contractile function and has been reported, by immunofluorescent localization, to have a distribution similar to actomysin in the mesangium.n However, our antichicken gizzard tropomyosin and anti-a-actinin ' stain human vascular media brilliantly, without striated structures," and only weakly and diffusely in the human and avian glomerulus, less than in interstitial tissue.2' What then is the function of glomerular myosin? It may have a role different from that in the arteriolar muscularis in regulating glomerular circulation and ultrafiltration. Recent micropuncture studies suggest that within the glomerulus there is a unique reactivity to angiotensin II and its analogues, differing from that of the arteriolar resistance vessels.2' The different species specificity of anti-AMY reactions with arterial media and glomerular mesangium reported here supports a tissue specialization. In patients with diabetic nephropathy the markedly thickened arteriolar media often has diminished anti-AMY staining, while the mesangium and vascular intima have increased staining. The increased AMY antigens of patients with early primary diabetic nephropathy might represent a generalized increase in the contractile function of mesangial cells in the metabolic environment of diabetes mellitus. The process by which accentuated mesangial smooth muscle function might lead to the loss of renal function in diabetes is unclear. At later stages of the disease the massive increase in AMY and FSA in the mesangium may reflect a failure of the mesangium to remove cellular degradation products, as suggested by studies of the processing of macromolecular aggregates in experimental diabetes in rats.X An increased width of mesangial anti-AMY staining develops within a few years in diabetic transplant recipients, which is greater than that observed in nondiabetic hosts. The development of distinctive lesions of diabetic nephropathy in the nondiabetic kidney transplanted into the human diabetic host is supported by other evidence from this laboratory: arteriolar hyalinosis 26 and renal extracellular basement membrane immunofluorescence for IgG and albumin 2" were recently reported in renal homografts in human diabetic recipients. Observations on renal transplantation in experimental diabetes in rats '* provides further support for
Vol. 90, No. 1 January 1978
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the hypothesis that the renal lesions develop as a consequence of the metabolic alterations. Decreased mesangial AMY is found in the original and transplanted kidneys of patients with MPGN, which supports the morphologic evidence of recurrence of that lesion.12 On the basis of glomerular and vascular cell culture studies,2' we presume that AMY is normally present within mesangial cells, while FSA is a cell surface antigen ' in the mesangial matrix. Anti-FSA elicited to an undefined mixture of antigens from human skin fibroblasts 6 has a similar tissue distribution to that of the FSA (or "fibronectin") previously described 2; the cross reaction with CIG and absorption with this antigen I provide identification. The limitations of light microscopy preclude our certainty of the mesangial cell origin of FSA in glomeruli of normal, diseased, and transplanted kidneys. We cannot exclude the possibility that FSA is also present in the glomerular endothelial cell. High-resolution light microscopy, using phase and Nomarski optics, compared with immunofluorescent techniques, shows that FSA (and CIG) remains beneath the basement membrane of glomerular capillary loops but seldom surrounds cross-sectional loops. In this area it resembles endothelial staining for factor VIII antigen.' Glomerular smooth muscle (possibly mesangial) cells in culture do not show the contact inhibition of skin fibroblasts. They further differ from fibroblasts by having less surface FSA; however, they have more surface FSA than do (umbilical vein) endothelial cells.' Contact inhibition of cultured fibroblasts is associated with retention of surface FSA.1' The proliferation of the mesangial cells of normal kidneys may then be limited bv normal FSA. Binding of FSA by its antibody was seen experimentally bv in vivo injection into primates,6 resulting in mesangial cell proliferation and proteinuria, perhaps a consequence of the binding of FSA. In patients with primarv renal diseases, FSA is found in the mesangium in a pattern similar to that of AMY; the dissociation between the findings for anti-AMY and anti-FSA staining of the glomerulus of nondiabetic transplanted kidneys is remarkable: mesangial anti-FSA staining can be markedly increased, even when anti-AMY staining is normal. An example is the decrease in anti-AMY staining in recurrent MPGN, while anti-FSA staining is normal or increased. The presence of significant levels of FSA in plasma6 and the known channeling of circulating macromolecular aggregates through the mesangium 31 suggest that the increased FSA in the mesangium of transplanted kidneys could represent an altered balance between the total synthesis and release from extrarenal sites and normal clearance of this antigen through the mesangium. Its relationship to the rejection process has not been determined.
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Heterologous antiserum to GB M is directed against numerous antigenic determinants. The most prominent are probably the noncollagen components 32 in view of the relatively poor antigenicity of collagen. Our antiGBM reacts by immunoprecipitation wvith collagenase-digested (and presumably noncollagep) GBM. In patients with renal diseases involving severe GBM thickening, one can detect a bilaminar staining of the GBMI with anti-GBM antiserums,' suggesting that antigenically "normal" GBMI may be deposited from both the endothelial and epithelial sides of the GBM. Bilaminar staining is found not only in tissue samples from patients with diabetes mellitus and membranous nephropathv but also in some long-term transplant tissues. In transplantation, thickened GBM staining is neither associated with proteinuria or clinical rejection nor found distinctively in patients whose pretransplant disease was diabetic nephropathv or glomerulonephritis; it is thus related to some undefined phenomenon, as previously observed by conventional histologic techniques.3 The observations of this study may provide both defined markers of the glomerular mesangial cell and new parameters of alteration in this cell system associated wvith glomerular injurv. In many glomerular disorders (diabetes mellitus, MPGN, proliferative glomerulopathies, and transplantation nephropathy) there exists considerable overlap in the histologic findings of mesangial cell proliferation and increased mesangial matrix. In this study distinctive changes in AMY and FSA glomerular mesangial antigenic markers wvere observed; more precise, specific pathologic observations can be similarly made for each disorder and may provide nex insights into the pathogenesis of these glomerular lesions. References 1. Scheinman JI. Fish AJ. Michael AF: The imrnunohistopathology of glomerular antigens: The glomerular basement membrane. collagen, and actomyosin antigens in normal and diseased kidneys. J Clin Invest 34:1144-1154. 1974 2. Linder E. V'aheri A. Ruoslahti E. Wartiovaara J: Distribution of fibroblast surface antigen in the developing chick embryo. J Exp Med 142:41-49, 1973 3. Pollard TD, Thomas SM. Niederman R: Human platelet myosin. I. Purification by a rapid method applicable to other non-muscle cells. Anal Biochem 60:258-266.1974 4. W7eber K. Osborn NI: The reliability of molecular weight determinations by dodecyl sulfate-polvacylamide gel electrophoresis. J Biol Chem 244:4406-4412, 1969 3. Chen PS Jr. Toribara TY. Warner H: Microdetermination of phosphorus. Anal Chem 28:1 736-1738. 1956 6. Fish AJ, NMichael AF. Xernier RL. Brow-n DM: Human glomerular cells in tissue culture. Lab Invest 33:3.30-341, 197 19 7. Parikh I, March S. Cuatrecasas P: Topics in the methodology of substitution reactions with agarose. Methods Enzymol :34:77-102. 1974 8. Nfosesson MNXV. Umfleet RA: The cold-insoluble globulin of human plasma. I.
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10. 11. 12.
13. 14. 15.
16. 17.
18. 19.
20. 21. 22. 23.
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Purification, primary characterization, and relationship to fibrinogen and other coldinsoluble fraction components. J Biol Chem 245:5728-5736, 1970 Scheinman JI, Fish AJ: Glomerular antigens of glomerular and vascular cells in culture. Fed Proc 36:1055, 1977 (Abstr) Cebra JJ, Goldstein G: Chromatographic purification of tetramethylrhodamineimmune globulin conjugates and their use in the cellular localization of rabbit -globulin polypeptide chains. J Immunol 95:230-245, 1965 Michael AF Jr, Drummond KN, Good RA, Vernier RL: Acute poststreptococcal glomerulonephritis: Immune deposit disease. J Clin Invest 45:237-248, 1966 McLean RH, Geiger H, Burke B, Simmons R, Najarian J, Vernier RL, Michael AF: Recurrence of membranoproliferative glomerulonephritis following a kidnev transplantation: Serum complement component studies. Am J Med 60:60-72, 1976 Kim Y, Vernier RL: Personal communication Becker CG: Demonstration of actomyosin in mesangial cells of the renal glomerulus. Am J Pathol 66:97-110, 1972 Kuusela P, Ruoslahti E, Engwall E, Vaheri A: Immunological interspecies crossreactions of fibroblast surface antigen (fibronectin). Immunochemistrv 13:639-642, 1976 Yamada KM, Yamada SS, Pastan I: Cell surface protein partiallv restores morphology, adhesiveness, and contact inhibition of movement to transformed fibroblasts. Proc Natl Acad Sci USA 73:1217-1221, 1976 Rukosuev VS, Nanaev AK: Histogenesis of mesangial cells of the renal glomeruli. Bull Exp Biol Med 79:115-117, 1975 Bernik MB: Contractile activity of human glomeruli in culture. Nephron 6:1-10, 1969 Whittingham S, Mackav IR, Irwin J: Autoimmune hepatitis. Immunofluorescence reactions with cytoplasm of smooth muscle and renal glomerular cells. Lancet 1:1333-1335, 1966 Miller F, Lazarides E; Elias J: Application of immunologic probes for contractile proteins to tissue sections. Clin Immunol Immunopathol 5:416-428, 1976 Accinni L, Natali PG, Vassallo L, Hsu KS, DeMartino C: Immunoelectron microscopic evidence of contractile proteins in the cellular and acellular components of mouse kidney glomeruli. Cell Tissue Res 162:297-312, 1975 Becker CG, Hardy AM, Dubin T: Contractile and relaxing proteins of smooth muscle, endothelial cells and platelets. Platelets, Thrombosis and Inhibitors. Edited bv P Didisheim. New York, FK Schattaur Verlay, 1974, pp 25-34 Schollmeyer JV, Furcht LT, Goll DE, Robson RM, Steomer MH: Localization of a-actinin and tropomvosin in smooth muscle and normal and transformed cells. Cell Motility. Cold Spring Harbor Symposium on Cellular Proliferation, 1976, pp
361-387 24. Scheinman JI, Schollmever JV: Unpublished observations 25. Blantz RC, Konnen KS, Tucker BJ: Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat. J Clin Invest 57:419434, 1976 26. Mauer SM, Steffes MW, Michael AF, Brown DM: Studies of diabetic nephropathv in animals and man. Diabetes 25:850-857, 1976 27. Mauer SM, Miller K, Goetz FC, Barbosa J, Simmons RL, Najarian JS, Michael AF: Immunopathology of renal extracellular membranes in kidneys transplanted into patients with diabetes mellitus. Diabetes 25:709-712, 1976 28. Scheinman JI, Fish AJ, Brown DM, Michael AF: Human glomerular smooth muscle (mesangial) cells in culture. Lab Invest 34:150-158, 1976 29. Ruoslahti E, Vaheri A: Interaction of soluble fibroblast surface antigen with fibrinogen and fibrin: Identitv with cold-insoluble globulin of human plasma. J Exp Med 141:497-501, 1975
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:30. Hover LW. de Los Santos RP. Hover JR: Antihemophilic factor antigen: Localization in endothelial cells bv immunofluorescent microscopy. J Clin Invest 52:273 7-2744. 197.3 31. Elema JD. Hover JR. Vernier RL: The glomerular mesangium: Uptake and transport of intravenously injected colloidal carbon in rats. Kidney Int 9:395-406. 1976 32. Marquardt H. Wilson CB. Dixon FJ: Human glomerular basement membrane. Selective solubilization with chaotropes and chemical and immunologic characterization of its components. Biochemistry 12:3260-3266. 1973 33. Fish AJ. Herdman RC. Kelly WN'D. Good RA: Glomerular changes in w-ell-functioning human renal homografts. Transplantation 3: 13.38-1343. 1967
Acknowledgments The overall technical assistance of Kathryn LaCroix and Kathleen Carmodv: of Cr-stal Blocher. Kim Pinkham. and Lore Lang in immunohistology; and of Marshall Hoff in illustrations and photography and the secretarial assistance of Nancy Kirschling are gratefully acknowledged. WN e also wish to thank Drs. S. Michael Mauer. John Najarian. and Richard Simmons for supplying tissues: Dr. Lan Bo Chen for supplying antiserum to cold-insoluble globulin: and Dr. Judith Schollmeyer for supplying antiserums to chicken gizzard a-actinin and tropomyosin. We especially thank Drs. Y. Kim and R. L. Vernier for the prepublication communication of their electron microscopic findings in MIPGN- and in recurrent MIPGN- in the transplanted kidney.
Figure LA-Polyacrylamide eketrophoresis gels (7.5%) loaded with 75 gg protein, stained with Coomassie blue. Human uterine myosin, left gel, shows myosin heavy chain (M), myosin rod (R), and small bands of unidentified peptides (X). Light chains, not seen in this reproduction, were faintly visualized at (L). In the gel on the right, human uterine actin (A) is run as a marker. B-Ouchterlony immunodiffusion in 1% Noble agar, 0.5 M KCI, diffused for 3 days at 4 C, stained with Buffalo black. Actomyosin, approximately 5 mg/ml in 50% glycerol, 0.5 M KCI, in center well. Antiserums, clockwise: 1, 2, and 3 are undiluted antiserums from 3 rabbits
immunized with myosin. 4 is anti-AMY IgG, 10 mg/ml, sharing the line of identity with the antimyosins.
Fgure 2-Immunofluorescent localization of glomerular antigens in the normal glomerulus. A-Anti-GBM localization to the GBM. (x 590) B-Antiactomyosin (AMY) staining of the mesangium in a restrictive pattern. The adjacent wall of the arteriole (A) is strongly stained. (x C-Antifibroblast surface antigen (FSA) staining of the mesangium is more extensive 590) than anti-AMY (B). (x 510) D-Anti-CIG staining of the mesangium, in the same extensive as pattern anti-FSA (Bt Arrows are directed to unstained peripheral capillary loops. (Epifluorescence, x 350) E-Phase micrograph of the same field as D. (x 350) F-Anti-CIG staining of normal kidney shows mild reactivity of endothelium (E) of arteriole and peritubular capillary (C). Bowman's capsule (BC), between arrows, is unstained. A light interstitial staining cannot be specifically localized. (Epifluorescence, x 700)
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Fgu 3-Immunofluorescent localization of anti-FSA and anti-CIG in a patient with diabetic nephropathy. AAnti-FSA staininq of expanded mesangium (M). (x 590) B-Anti-CIG stain of a portion of a glomerulus from the CIG stain of a portion of a glomerulus from the same tissue as in A, with unstained mesangium (arrows). The muscularis of the arteriole (above A) is not stained, and only scattered endothelial staining is visible. (x 1800) Fge 4-Increased extent (not width) of anti-AMY staining in 2-year transplant biopsy specimen of a patient with previous (and recurrent) membranous nephropathy. A-Arrows point to the (unstained) outer aspect of peripheral capillary loops, showing that staining for AMY remains interior to the GBM. Note lack of continuity in staining of B-Phase contrast view of the same field as in A, showing the locatwo capillary loops. (Epifluorescence, x 750) tion of basement membranes (arrows).
Fqu 5-Decreased anti-AMY staining of 8-year transplant biopsy of a patient with MPGN. Decreased glomerular staining contrasts with preserved staining of the wall of an adjacent arteriole (A). (x 590) Fe 6Markedly increased anti-AMY staining of mesangial width in 4-year transplant biopsy of a diabetic patient. (x 380) Fe 7-Three-year transplant biopsy of a diabetic patient with normal renal function and no evidence of rejection. Anti-FSA stains a markedly widened mesangium. Note (arrow) the light staining of arterial endothelium. (x 380).
Fgwe 8A-Mildly
increased mesangial anti-FSA staining. Staining of adjacent sections of a 2-year nondiabetic transplant biopsy specimen. The arterial media is unstained, but adventitium is stained (A). Tubule B-Anti-CIG staining (T) is seen in A and B. (x 590)
is the same as on adjacent section. Note intimal staining of the arteriole at arrow, the same vessel seen Fiu 9-Antiat the top of the field in Figure 9. GBM staining of markedly thickened GBM (between arrows) in 10-year transplant biopsy specimen from a patient with primary renal hypoplasia. Note bilaminar pattern, with accentuation of the inner aspect. (x 1260)
