- Email: [email protected]
Mixed-Pattern Immune Deposit Glomerulonephritis in a Child With Inherited Deficiency of the Third Component of Complement
CASE REPORTS
Mixed-Pattern Immune Deposit Glomerulonephritis in a Child With Inherited Deficiency of the Third Component of Complement Michael S. Borzy, MD, and Donald Houghton, MD • The renal histopathology of a 7-year-old Laotian male with inherited deficiency of the third component of complement, recurrent infections, and persistent hematuria and proteinuria is described. The histologic changes are predominantly those of mesanglopathic disease with isolated changes resembling type I membranoproliferative glomerulonephritis and transmembranous glomerulonephritis. IgG, IgA, IgM, C4, and fibrinogen, but not C3, were detected by immunofluorescence in mesangial zones and in segments of capillary walls. A normal distribution of C3b receptors was present along all capillary walls. This report provides additional support for the association of congenital C3 deficiency and immune deposit glomerulonephritis. INDEX WORDS: C3 deficiency; mesangiopathic glomerulonephritis; immune deposits.
T
HE THIRD component of complement (C3) plays a major role in host defense. It is a pivotal component of both the classical and alternative pathways of complement activation, and its major cleavage product, C3b, is an important serum opsonin. 1 In addition, C3 is involved in the solubilization of immune complexes 2 and the modulation of the lymphocyte response to antigenic stimulation.3,4 Fourteen patients with congenital C3 deficiency (C3D) have been reported,5-13 12 of whom have had increased susceptibility to bacterial infections. Cutaneous vasculitis documented by skin biopsy has been reported in four patients with C3D,5.8.13 but immune deposit glomerulonephritis has been described previously in only one patient. 14 This report of a second patient with C3D who has both recurrent infections and immune deposit glomerulonephritis provides further descriptive information concerning the renal histopathology of this newly recognized association of C3D and glomerulonephritis.
From the Departmellt of Pediatrics. Doernbecher Memorial Hospitalfor Children . Oregon Health Sciences University, and the Department of Pathology; Oregon Health Sciences University. Supported in part by grants from the Oregon Chapter of the American Cancer Society and the Medical Research Foundation of Oregon. Address reprint requests to Michael S. Borzy. MD. Division of Pediatric Immunology/Rheumatology. Departmellf of Pediatrics. Doernbecher Memorial Hospital for Children. Oregon Health Sciences University. 3181 SW Sam Jackson Park Rd, Portland, OR 97201 . © 1985 by The National Kidney Foundation. Inc. 0272-6386/85/010054-06$3.00/0
54
MATERIALS AND METHODS The clinical and laboratory features of the patient . a 7-yearold Laotian male with homozygous C3D (I % of normal serum C3 hemolytic activity and undetectable antigenic C3 by radial immunodiffusion and Laurell rocket electroimmunoassay using 1% agarose containing anti-C3 antibody 'S,'6) will be reported in detail elsewhere (Borzy MS , Gewurz A, Wolff L, et al: Inherited deficiency of the third component of complement in a child with recurrent infections and glomerulonephritis, in preparation). Briefly, he has experienced two episodes of Streptococcus pneumoniae meningitis and three bouts of lobar pneumonia . Microscopic hematuria and trace proteinuria were first detected at 20 months of age during his second hospital admission for meningitis. Persistent microscopic hematuria and trace proteinuria without cellular casts have been noted on urinalyses over the subsequent 5-year period; creatinine clearance, BP, and serum albumin and cholesterol have been normal. Twentyfour-hour urinary protein was less than 150 mg/m2 on two occasions. He has no clinical features of autoimmune disease, and neither autoantibodies (rheumatoid factor; antinuclear, antidsDNA, and anti-Sm antibodies) nor circulating immune complexes using the Clq binding assay and the Raji cell radioimmunoassay were detected. Family studies showed subnormal levels of serum C3 hemolytic activity in both parents (father = 49 % of normal ; mother = 59%) and two (sister = 37% of normal ; sister = 62 %) of three siblings suggesting an autosomal codominant inheritance of the gene for C3D in this family. Renal tissue obtained by open biopsy was divided for light, electron, and immunofluorescence microscopy. Tissue for light microscopy was fixed in 2 % phosphate-buffered glutaraldehyde, embedded in methacrylate, and sectioned at I /Lm. Tissue sections were stained with hematoxy lin-eosin and Jones methenamine silver stains. Tissue for electron microscopy was fixed in glutaraldehyde, post fixed in cacodylate-buffered osmium tetroxide, embedded in araldite, sectioned, and stained with lead citrate and uranyl acetate _ It was examined in a Philips EM-2oo electron microscope (Philips Electronics Instrument Co, Eindhoven, The Netherlands .)_ Tissue for immunofluorescence was frozen at - 60 °C in frozen-section embedding medium, and sectioned in a refrigerated microtome. Frozen sections were directly stained with fluoresceinated antisera to IgG , IgM , IgA , C3 , C4, fibrinogen , and albumin
American Journal of Kidney Diseases, Vol V, No 1, January 1985
C3 DEFICIENCY WITH GLOMERULONEPHRITIS (Meloy Laboratories . Springfield . Va). In addition, glomeru lar C3b receptors were detected indirectly using rabbit anti-human C3b receptor antibody. kindly provided by Dr Douglas Fearon , Harvard Medical School, Boston, and fluoresceinated sheep anti-rabbit immunoglobulin (Cappel Laboratories, Cochranville, Pa). Control studies for direct flu orescent tests included concurrent staining of known positive and negative control tissues with all reagents. Study tissue failed to stai n with fluorescei nated rabbit nonimmune serum. Control studies with the C3b receptor antiserum included direct tissue application of secondary antiserum (fluoresceinated sheep anti-rabbit immunoglobulin) and stain ing with secondary antiserum after initial application of rabbit nonimmune serum . Renal tissue from two other cases , one without demonstrable immune deposits (lipoid nephrosis) and the other with immune deposits (active diffuse proliferative lupus nephritis), were also stained for C3 receptors.
RESULTS
Tissue cores examined by light microscopy contained more than 30 glomeruli per level. In sections stained with hematoxylin-eosin, renal histology was essentially normal. Specifically, there was no glomerular hypercellularity or sclerosis,
55
and the tubulointerstitial compartment and vasculature were normal. In silver-stained sections , many glomeruli had normal or near-normal appearances , but accretion of mesangial matix and glomerular basement membrane irregularities were evident in about half (Fig I). The affected basement membrane segments were thickened with subepithelial and subendothelial irregularities suggesting the presence of immune deposits. Splitting and duplication of glomerular basement membranes occurred only rarely. The glomerular basement membrane changes were localized to a few capillaries in most involved glomerular tufts. Eight glomeruli were examined by electron microscopy. All of them contained voluminous electron-dense deposits throughout the mesangial axis; most peripheral capillary walls were entirely normal. In some tufts, however, a few capillaries contained large subendothelial electron-dense deposits accompanied by interposed mesangial cell cytoplasmic processes and associated with the formation of multiple thin layers of new subendothelial
Fig 1. This glomerulus stained with Jones methenamine silver demonstrates generalized glomerular basement membrane changes. Many segments are irregularly thickened and scalloped. The subepithelial irregularities and projections in some capillary segments resemble those of membranous glomerulonephritis. These kinds of changes were found in only a few of the tufts in the biopsy (x 570).
56
basal lamina (Fig 2A). In other loops, isolated subepithelial electron-dense deposits (Fig 2B), intramembranous deposits, capillary collapse, and associated glomerular basement membrane thickening were prominent features. Direct immunofluorescence studies documented the presence ofIgG, IgA, IgM, and C4 in all glomeruli. IgM (Fig 3A) was deposited uniformly in mesangial zones with extension to local segments of glomerular capillary walls. IgG, IgA, and C4 were distributed in a segmental pattern; in some
BORZY AND HOUGHTON
instances, deposits outlined individual capillary walls (Fig 3B). Stains for C3 and albumin were negative. C3b receptors were present uniformly along all capillary walls in the study case and in the lipoid nephrosis case. In the lupus nephritis case, however, capillary wall staining was discontinuous, a phenomenon previously described by Kazatchkine et al. 17 Staining for C3b receptors was unaffected by prior application of nonfluoresceinated anti-human immunoglobulin.
Fig 2. Electron micrographs of glomerular capillary loops. (A) Voh.lminous electron-dense deposits occupy and expand mesangial and subendothelial zones. These deposits and the associated mesangial interposition, foot process collapse, and formation of new layers of subendothelial basal lamina are characteristic features of membranoproliferative glomerulonephritis. Adjacent capillary walls and most of those in other glomeruli demonstrate only mild nonspecific changes. Mesangial electron deposits were present diffusely (x 6,050). (6) A partially collapsed capillary in the same glomerulus demonstrates mesangial interposition, partial foot process collapse, glomerular basement membrane thickening and duplication, intramembranous deposits, and several subepithelial electrondense deposits (arrows) ( x 9,900).
C3 DEFICIENCY WITH GLOMERULONEPHRITIS
Fig 3. Direct immunofluorescent staining for (A) IgM and (8) C4. Deposits of IgM have a generalized mesangial and local capillary wall distribution. Voluminous deposits of C4 outline a few, apparently thickened, segments of capillary wall. Deposits of IgG and IgA were present in the same pattern as seen for C4. C3 was not detectable.
DISCUSSION
The histologic changes reflect more than one pattern of glomerular injury. All glomeruli contained mesangial immunoglobulin and electrondense deposits, though few, if any, exhibited mesangial hypercell ularity. Such a pattern is commonly seen in mild mesangial deposit (mesangiopathic) glomerulonephritides, such as IgA nephropathy, in which IgA predominates in the deposits. As many as half of the glomeruli also had subendothelial deposits, which while common in mesangiopathic glomerulonephritis, were exceptionally voluminous and accompanied by capillary wall reaction more typical of type I membranopro" !iferative glomerulonephritis. It might be argued that these capillary wall changes were part of the mesangiopathic process, or that the combination of findings were part of very early type I membrano-
57
proliferative glomerulonephritis. However, subepithelial deposits that were also present are not usually so prominent in either membranoproliferative glomerulonephritis or mesangiopathic glomerulonephritis. The presence of these latter deposits suggest that at least a second antigenantibody system existed, one which led to transmembranous deposition or formation of immune complexes. Such a mixed-pattern glomerulonephritis is not unique to this setting. A similar combination is sometimes described as type III membranoproliferative glomerulonephritis,18 or as "glomerulonephritis with deposits at multiple sites" 19 when it occurs as a primary disorder. The pattern may also be seen in the glomerular disease of systemic lupus erythematosus. 20 Berger et al 14 described a child with congenital C3D and type I membranoproliferative glomerulonephritis with diffuse glomerular involvement. They also found scattered subepithelial electrondense deposits. The patterns of glomerular involvement in their patient and ours are similar, differing primarily in extent and severity of injury. In another family with hereditary C3D, Pussel et al \0 found clinical evidence (microscopic hematuria and/or proteinuria) of glomerular disease in three members with homozygous C3D; however, renal tissue was not examined in any case. One female sibling in this family who was heterozygous for C3D was found to have microscopic hematuria and proteinuria with two episodes of nephrotic syndrome, and renal biopsy tissue showed features of membranoproliferative glomerulonephritis by light microscopy with absent immunoglobulin, but positive C3, staining on immunofluorescence. The association of hypocomplementemia and the primary membranoproliferative glomerulonephritides is well known. 21 Glomerulonephritis has also been reported in other settings of acquired 22 and inherited 2324 C3 depletion. Furthermore , immune complex disease , including glomerulonephritis and systemic lupus erythematosus or lupus-like syndromes, occur with congenital absence or states of depletion of other early components (CI, C4, C2) of complementl .2S.26 Because glomerular disease is not always found in patients with C3D, it might be inferred that the relationship between glomerulonephritis and C3D is an indirect or circumstantial one. However, the frequency of the association is unknown because sig-
58
SORZY AND HOUGHTON
nificant glomerular disease may be clinically inapparent. 27 It has been proposed that immune complex disease and complement deficiency are associated through linkage of genes controlling immune responsiveness and complement production 26.28 that are located within the major histocompatibility complex (MHC) on chromosome 6. Genes for C2 , C4, and factor B are located within the MHC,29 but the gene for C3 is located on chromosome 19,30 making linkage with MHC immune response genes unlikely in C3D . In addition, Peters and Williams 31 have proposed that the glomerulonephritis associated with complement deficiency results from the repeated infections and the lack of immune complex solubilization caused by complement deficiency. Thus, the increased antigen exposure and the heightened likelihood of forming pathogenic immune complexes might lead to the activation , sequentially or concurrently, of multiple immune complex systems resulting in the development of mixed or varied patterns of glomerular injury. Antibiotic prophylaxis against bacterial infection failed to prevent immune complex depo-
sit ion and the development and progression of glomerulonephritis in this patient and the patient reported by Berger et al. 14 Although recurrent or chronic infection with organisms unaffected by antibiotics remains a possibility, this child has not had symptoms of chronic infection. The finding of a normal distribution of C3b receptors in the patient's glomeruli confirms a similar observation 14 and suggests that the cell surface expression of receptor is independent of its ligand . Although the role of C3b receptors on glomerular podocytes in the pathogenesis of the renal disease in C3D is unclear, it is interesting to speculate that the C3b receptor may possess cross-reactive binding affinity with an undefined product(s) of a noncomplement mediator system and that this interaction may play a part in the observed immune deposit disease. ACKNOWLEDGMENT The authors thank Anita Gewurz , MD , for complement determinations, Linda Germain and Don Anderson for expert technical assistance, and Norma Fritz for expert secretarial assistance.
REFERENCES I. Agnello V: Complement deficiency states. Medicine 57:1-23 , 1978 2. Czop J, Nussenzweig V: Studies on the mechanism of solubili zation of immune precipitates by serum. J Exp Med 143 :615-630, 1976 3. Perl mann H, Perlmann P, Schreiber RD, et aJ: Interaction of target cell-bound C3bi and C3b with human lymphocyte receptors: Enhancement of antibody mediated cellular toxicity. J Exp Med 153:1592-1603, 1981 4. Koopman WJ, Sandberg AL , Wahl SM. et al: Interaction of soluble C3 fragments with guinea pig lymphocytes: Comparison of effects of C3a, C3b, C3c and C3d on Iymphokine production and lymphocyte proliferation. J ImmunoII17:331 336, 1976 5. Alper CA, Colten HR , Rosen FS, et al: Homozygous deficiency of C3 in a patient with repeated infections. Lancet 2:1179-1181,1972 6. Ballow M , Shira JE, Harden L, et al: Complete absence of the third component of complement in man. J Clin Invest 56:703-710, 1975 7. Grace HJ, Brereton-Stiles GG, Vos GH, et al: A family with partial and total deficiency of complement C3. South Afr Med J 50:139-140, 1976 8. Osofsky SG , Thompson BH, Lint TF, et al: Hereditary deficiency of the third component of complement in a child with fever, skin rash , and arthralgias: Response to transfusion of whole blood. J Pediatr 90:180-186, 1977 9. Davis AE, Davis JS, Rabson AR , et al: Homozygous C3 deficiency: Detection of C3 by radioimmunoassay. Clin Immunol Immunopathol 8:543- 550, 1977
10. Pussell BA, Bourke E, Nayef M, et al: Complement deficiency and nephritis. Lancet 1:675-677, 1980 II. Sano Y, Nishimukai H, Kitamura H , et al: Hereditary deficiency of the third component of complement in two sisters with system ic lupus erythematosus-like symptoms. Arthritis Rheum 24: 1255-1260, 1981 12. Kue-Hsuing H, Ching- Yuang L, Ting-Chien L: Complete absence of the third component of complement in a patient with repeated infections. Clin Immunol ImmunopathoI20:305312, 1981 \3. Roord JJ , Daha M, Kuis W, et al: Inherited deficiency of the third component of comple ment associated with recurrent pyogenic infections, circulating immune complexes, and vasculitis in a Dutch family. Pediatrics 71:81-87 , 1983 14. Berger M, Balow JE. Werlson CB, et al: Circulating immune complexes and glomerulonephritis in a patient with congenital absence of the third component of complement. N Engl J Med 308 :1009-1012 , 1983 15 . Lim D, Gewurz A, Lin! TF, et al : Absence of the sixth component of complement in a patient with repeated episodes of meningitis. J Pediatr 89:42-47, 1976 16. Laurell CB: Quantitative estimation of proteins by electrophoresi s in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 17 . Kazatchkine MD, Fearon DT, Appay MD, et al: Immunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases. J Clin Invest 69:900-9 12, 1982 18. Bu rkholder PM, Hyman LR, Krueger RP: Characterization of mixed membranous a nd proliferati ve g lomeru-
C3 DEFICIENCY WITH GLOMERULONEPHRITIS lonephritis: Recognition of three varieties, in Kincaid-Smith P, Mather TH, Becker EL (eds): Glomerulonephritis 1. New York, Wiley and Sons, 1973, pp 557-559 19. Cameron lS: Pathogenesis and treatment of membranous nephropathy. Kidney Int 15: 88-103, 1979 20. Heptinstall RH: Membranoproliferative glomerulonephritis, in Heptinstall RH (ed): Pathology of the Kidney. Boston, Little, Brown, 1983, pp 479-518 21. Hill GS: Systemic lupus erythematosus and mixed connective tissue disease, in Heptinstall RH (ed): Pathology of the Kidney. Boston, Little, Brown, 1983, pp 839-906 22. Peters DK, Williams DG, Charlesworth lA, et al: Mesangiocapillary nephritis, partial lipodystrophy, and hypocomplementemia. Lancet 2:535-538, 1973 23. McLean RH, Weinstein A, Damjanov I, et al: Hypomorphic variant of C3, arthritis, and chronic glomerulonephritis. J Pediatr 93:937-943, 1978 24. Marder HK, Coleman TH, Forristal J, et al: An inherited defect in the C3 convertase, C3b, Bb, associated with glomerulonephritis. Kidney Int 23:749-758, 1983 25. Thompson RA, Haeney M, Reid KBM, et al: A genetic defect of the Clq subcomponent of complement associated with
59 childhood (immune complex) nephritis. N Engl J Med 303:2224 , 1980 26. Kim Y, Friend PS, Dresner IG, et al: Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulonephritis. Am J Med 62:765-771, 1977 27. Bennett WM, Bardana EJ, Wuepper K, et al: Partial lipodystrophy, C3 nephritis factor and clinically inapparent mesangiocapillary glomerulonephritis. Am 1 Med 62:757-760, 1977 28. Glass D, Raum D, Gibson D, et al: Inherited deficiency of the second component of complement. J Clin Invest 58:853861, 1976 29. Lachmann Pl, Hobart MJ: Complement genetics in relation to HLA. Br Med Bull 34:247-252, 1978 30. Whitehead AS, Solomon E, Chambers Sp, et al: Assignment of the structural gene for the third component of human complement to chromosome 19. Proc Natl Acad Sci USA 79:5021-5025, 1982 31. Peters DK, Williams DG: Complement and mesangiocapillary glomerulonephritis: Role of complement deficiency in the pathogenesis of nephritis. Nephron 13: 189-197, 1974
Mixed-Pattern Immune Deposit Glomerulonephritis in a Child With Inherited Deficiency of the Third Component of Complement Michael S. Borzy, MD, and Donald Houghton, MD • The renal histopathology of a 7-year-old Laotian male with inherited deficiency of the third component of complement, recurrent infections, and persistent hematuria and proteinuria is described. The histologic changes are predominantly those of mesanglopathic disease with isolated changes resembling type I membranoproliferative glomerulonephritis and transmembranous glomerulonephritis. IgG, IgA, IgM, C4, and fibrinogen, but not C3, were detected by immunofluorescence in mesangial zones and in segments of capillary walls. A normal distribution of C3b receptors was present along all capillary walls. This report provides additional support for the association of congenital C3 deficiency and immune deposit glomerulonephritis. INDEX WORDS: C3 deficiency; mesangiopathic glomerulonephritis; immune deposits.
T
HE THIRD component of complement (C3) plays a major role in host defense. It is a pivotal component of both the classical and alternative pathways of complement activation, and its major cleavage product, C3b, is an important serum opsonin. 1 In addition, C3 is involved in the solubilization of immune complexes 2 and the modulation of the lymphocyte response to antigenic stimulation.3,4 Fourteen patients with congenital C3 deficiency (C3D) have been reported,5-13 12 of whom have had increased susceptibility to bacterial infections. Cutaneous vasculitis documented by skin biopsy has been reported in four patients with C3D,5.8.13 but immune deposit glomerulonephritis has been described previously in only one patient. 14 This report of a second patient with C3D who has both recurrent infections and immune deposit glomerulonephritis provides further descriptive information concerning the renal histopathology of this newly recognized association of C3D and glomerulonephritis.
From the Departmellt of Pediatrics. Doernbecher Memorial Hospitalfor Children . Oregon Health Sciences University, and the Department of Pathology; Oregon Health Sciences University. Supported in part by grants from the Oregon Chapter of the American Cancer Society and the Medical Research Foundation of Oregon. Address reprint requests to Michael S. Borzy. MD. Division of Pediatric Immunology/Rheumatology. Departmellf of Pediatrics. Doernbecher Memorial Hospital for Children. Oregon Health Sciences University. 3181 SW Sam Jackson Park Rd, Portland, OR 97201 . © 1985 by The National Kidney Foundation. Inc. 0272-6386/85/010054-06$3.00/0
54
MATERIALS AND METHODS The clinical and laboratory features of the patient . a 7-yearold Laotian male with homozygous C3D (I % of normal serum C3 hemolytic activity and undetectable antigenic C3 by radial immunodiffusion and Laurell rocket electroimmunoassay using 1% agarose containing anti-C3 antibody 'S,'6) will be reported in detail elsewhere (Borzy MS , Gewurz A, Wolff L, et al: Inherited deficiency of the third component of complement in a child with recurrent infections and glomerulonephritis, in preparation). Briefly, he has experienced two episodes of Streptococcus pneumoniae meningitis and three bouts of lobar pneumonia . Microscopic hematuria and trace proteinuria were first detected at 20 months of age during his second hospital admission for meningitis. Persistent microscopic hematuria and trace proteinuria without cellular casts have been noted on urinalyses over the subsequent 5-year period; creatinine clearance, BP, and serum albumin and cholesterol have been normal. Twentyfour-hour urinary protein was less than 150 mg/m2 on two occasions. He has no clinical features of autoimmune disease, and neither autoantibodies (rheumatoid factor; antinuclear, antidsDNA, and anti-Sm antibodies) nor circulating immune complexes using the Clq binding assay and the Raji cell radioimmunoassay were detected. Family studies showed subnormal levels of serum C3 hemolytic activity in both parents (father = 49 % of normal ; mother = 59%) and two (sister = 37% of normal ; sister = 62 %) of three siblings suggesting an autosomal codominant inheritance of the gene for C3D in this family. Renal tissue obtained by open biopsy was divided for light, electron, and immunofluorescence microscopy. Tissue for light microscopy was fixed in 2 % phosphate-buffered glutaraldehyde, embedded in methacrylate, and sectioned at I /Lm. Tissue sections were stained with hematoxy lin-eosin and Jones methenamine silver stains. Tissue for electron microscopy was fixed in glutaraldehyde, post fixed in cacodylate-buffered osmium tetroxide, embedded in araldite, sectioned, and stained with lead citrate and uranyl acetate _ It was examined in a Philips EM-2oo electron microscope (Philips Electronics Instrument Co, Eindhoven, The Netherlands .)_ Tissue for immunofluorescence was frozen at - 60 °C in frozen-section embedding medium, and sectioned in a refrigerated microtome. Frozen sections were directly stained with fluoresceinated antisera to IgG , IgM , IgA , C3 , C4, fibrinogen , and albumin
American Journal of Kidney Diseases, Vol V, No 1, January 1985
C3 DEFICIENCY WITH GLOMERULONEPHRITIS (Meloy Laboratories . Springfield . Va). In addition, glomeru lar C3b receptors were detected indirectly using rabbit anti-human C3b receptor antibody. kindly provided by Dr Douglas Fearon , Harvard Medical School, Boston, and fluoresceinated sheep anti-rabbit immunoglobulin (Cappel Laboratories, Cochranville, Pa). Control studies for direct flu orescent tests included concurrent staining of known positive and negative control tissues with all reagents. Study tissue failed to stai n with fluorescei nated rabbit nonimmune serum. Control studies with the C3b receptor antiserum included direct tissue application of secondary antiserum (fluoresceinated sheep anti-rabbit immunoglobulin) and stain ing with secondary antiserum after initial application of rabbit nonimmune serum . Renal tissue from two other cases , one without demonstrable immune deposits (lipoid nephrosis) and the other with immune deposits (active diffuse proliferative lupus nephritis), were also stained for C3 receptors.
RESULTS
Tissue cores examined by light microscopy contained more than 30 glomeruli per level. In sections stained with hematoxylin-eosin, renal histology was essentially normal. Specifically, there was no glomerular hypercellularity or sclerosis,
55
and the tubulointerstitial compartment and vasculature were normal. In silver-stained sections , many glomeruli had normal or near-normal appearances , but accretion of mesangial matix and glomerular basement membrane irregularities were evident in about half (Fig I). The affected basement membrane segments were thickened with subepithelial and subendothelial irregularities suggesting the presence of immune deposits. Splitting and duplication of glomerular basement membranes occurred only rarely. The glomerular basement membrane changes were localized to a few capillaries in most involved glomerular tufts. Eight glomeruli were examined by electron microscopy. All of them contained voluminous electron-dense deposits throughout the mesangial axis; most peripheral capillary walls were entirely normal. In some tufts, however, a few capillaries contained large subendothelial electron-dense deposits accompanied by interposed mesangial cell cytoplasmic processes and associated with the formation of multiple thin layers of new subendothelial
Fig 1. This glomerulus stained with Jones methenamine silver demonstrates generalized glomerular basement membrane changes. Many segments are irregularly thickened and scalloped. The subepithelial irregularities and projections in some capillary segments resemble those of membranous glomerulonephritis. These kinds of changes were found in only a few of the tufts in the biopsy (x 570).
56
basal lamina (Fig 2A). In other loops, isolated subepithelial electron-dense deposits (Fig 2B), intramembranous deposits, capillary collapse, and associated glomerular basement membrane thickening were prominent features. Direct immunofluorescence studies documented the presence ofIgG, IgA, IgM, and C4 in all glomeruli. IgM (Fig 3A) was deposited uniformly in mesangial zones with extension to local segments of glomerular capillary walls. IgG, IgA, and C4 were distributed in a segmental pattern; in some
BORZY AND HOUGHTON
instances, deposits outlined individual capillary walls (Fig 3B). Stains for C3 and albumin were negative. C3b receptors were present uniformly along all capillary walls in the study case and in the lipoid nephrosis case. In the lupus nephritis case, however, capillary wall staining was discontinuous, a phenomenon previously described by Kazatchkine et al. 17 Staining for C3b receptors was unaffected by prior application of nonfluoresceinated anti-human immunoglobulin.
Fig 2. Electron micrographs of glomerular capillary loops. (A) Voh.lminous electron-dense deposits occupy and expand mesangial and subendothelial zones. These deposits and the associated mesangial interposition, foot process collapse, and formation of new layers of subendothelial basal lamina are characteristic features of membranoproliferative glomerulonephritis. Adjacent capillary walls and most of those in other glomeruli demonstrate only mild nonspecific changes. Mesangial electron deposits were present diffusely (x 6,050). (6) A partially collapsed capillary in the same glomerulus demonstrates mesangial interposition, partial foot process collapse, glomerular basement membrane thickening and duplication, intramembranous deposits, and several subepithelial electrondense deposits (arrows) ( x 9,900).
C3 DEFICIENCY WITH GLOMERULONEPHRITIS
Fig 3. Direct immunofluorescent staining for (A) IgM and (8) C4. Deposits of IgM have a generalized mesangial and local capillary wall distribution. Voluminous deposits of C4 outline a few, apparently thickened, segments of capillary wall. Deposits of IgG and IgA were present in the same pattern as seen for C4. C3 was not detectable.
DISCUSSION
The histologic changes reflect more than one pattern of glomerular injury. All glomeruli contained mesangial immunoglobulin and electrondense deposits, though few, if any, exhibited mesangial hypercell ularity. Such a pattern is commonly seen in mild mesangial deposit (mesangiopathic) glomerulonephritides, such as IgA nephropathy, in which IgA predominates in the deposits. As many as half of the glomeruli also had subendothelial deposits, which while common in mesangiopathic glomerulonephritis, were exceptionally voluminous and accompanied by capillary wall reaction more typical of type I membranopro" !iferative glomerulonephritis. It might be argued that these capillary wall changes were part of the mesangiopathic process, or that the combination of findings were part of very early type I membrano-
57
proliferative glomerulonephritis. However, subepithelial deposits that were also present are not usually so prominent in either membranoproliferative glomerulonephritis or mesangiopathic glomerulonephritis. The presence of these latter deposits suggest that at least a second antigenantibody system existed, one which led to transmembranous deposition or formation of immune complexes. Such a mixed-pattern glomerulonephritis is not unique to this setting. A similar combination is sometimes described as type III membranoproliferative glomerulonephritis,18 or as "glomerulonephritis with deposits at multiple sites" 19 when it occurs as a primary disorder. The pattern may also be seen in the glomerular disease of systemic lupus erythematosus. 20 Berger et al 14 described a child with congenital C3D and type I membranoproliferative glomerulonephritis with diffuse glomerular involvement. They also found scattered subepithelial electrondense deposits. The patterns of glomerular involvement in their patient and ours are similar, differing primarily in extent and severity of injury. In another family with hereditary C3D, Pussel et al \0 found clinical evidence (microscopic hematuria and/or proteinuria) of glomerular disease in three members with homozygous C3D; however, renal tissue was not examined in any case. One female sibling in this family who was heterozygous for C3D was found to have microscopic hematuria and proteinuria with two episodes of nephrotic syndrome, and renal biopsy tissue showed features of membranoproliferative glomerulonephritis by light microscopy with absent immunoglobulin, but positive C3, staining on immunofluorescence. The association of hypocomplementemia and the primary membranoproliferative glomerulonephritides is well known. 21 Glomerulonephritis has also been reported in other settings of acquired 22 and inherited 2324 C3 depletion. Furthermore , immune complex disease , including glomerulonephritis and systemic lupus erythematosus or lupus-like syndromes, occur with congenital absence or states of depletion of other early components (CI, C4, C2) of complementl .2S.26 Because glomerular disease is not always found in patients with C3D, it might be inferred that the relationship between glomerulonephritis and C3D is an indirect or circumstantial one. However, the frequency of the association is unknown because sig-
58
SORZY AND HOUGHTON
nificant glomerular disease may be clinically inapparent. 27 It has been proposed that immune complex disease and complement deficiency are associated through linkage of genes controlling immune responsiveness and complement production 26.28 that are located within the major histocompatibility complex (MHC) on chromosome 6. Genes for C2 , C4, and factor B are located within the MHC,29 but the gene for C3 is located on chromosome 19,30 making linkage with MHC immune response genes unlikely in C3D . In addition, Peters and Williams 31 have proposed that the glomerulonephritis associated with complement deficiency results from the repeated infections and the lack of immune complex solubilization caused by complement deficiency. Thus, the increased antigen exposure and the heightened likelihood of forming pathogenic immune complexes might lead to the activation , sequentially or concurrently, of multiple immune complex systems resulting in the development of mixed or varied patterns of glomerular injury. Antibiotic prophylaxis against bacterial infection failed to prevent immune complex depo-
sit ion and the development and progression of glomerulonephritis in this patient and the patient reported by Berger et al. 14 Although recurrent or chronic infection with organisms unaffected by antibiotics remains a possibility, this child has not had symptoms of chronic infection. The finding of a normal distribution of C3b receptors in the patient's glomeruli confirms a similar observation 14 and suggests that the cell surface expression of receptor is independent of its ligand . Although the role of C3b receptors on glomerular podocytes in the pathogenesis of the renal disease in C3D is unclear, it is interesting to speculate that the C3b receptor may possess cross-reactive binding affinity with an undefined product(s) of a noncomplement mediator system and that this interaction may play a part in the observed immune deposit disease. ACKNOWLEDGMENT The authors thank Anita Gewurz , MD , for complement determinations, Linda Germain and Don Anderson for expert technical assistance, and Norma Fritz for expert secretarial assistance.
REFERENCES I. Agnello V: Complement deficiency states. Medicine 57:1-23 , 1978 2. Czop J, Nussenzweig V: Studies on the mechanism of solubili zation of immune precipitates by serum. J Exp Med 143 :615-630, 1976 3. Perl mann H, Perlmann P, Schreiber RD, et aJ: Interaction of target cell-bound C3bi and C3b with human lymphocyte receptors: Enhancement of antibody mediated cellular toxicity. J Exp Med 153:1592-1603, 1981 4. Koopman WJ, Sandberg AL , Wahl SM. et al: Interaction of soluble C3 fragments with guinea pig lymphocytes: Comparison of effects of C3a, C3b, C3c and C3d on Iymphokine production and lymphocyte proliferation. J ImmunoII17:331 336, 1976 5. Alper CA, Colten HR , Rosen FS, et al: Homozygous deficiency of C3 in a patient with repeated infections. Lancet 2:1179-1181,1972 6. Ballow M , Shira JE, Harden L, et al: Complete absence of the third component of complement in man. J Clin Invest 56:703-710, 1975 7. Grace HJ, Brereton-Stiles GG, Vos GH, et al: A family with partial and total deficiency of complement C3. South Afr Med J 50:139-140, 1976 8. Osofsky SG , Thompson BH, Lint TF, et al: Hereditary deficiency of the third component of complement in a child with fever, skin rash , and arthralgias: Response to transfusion of whole blood. J Pediatr 90:180-186, 1977 9. Davis AE, Davis JS, Rabson AR , et al: Homozygous C3 deficiency: Detection of C3 by radioimmunoassay. Clin Immunol Immunopathol 8:543- 550, 1977
10. Pussell BA, Bourke E, Nayef M, et al: Complement deficiency and nephritis. Lancet 1:675-677, 1980 II. Sano Y, Nishimukai H, Kitamura H , et al: Hereditary deficiency of the third component of complement in two sisters with system ic lupus erythematosus-like symptoms. Arthritis Rheum 24: 1255-1260, 1981 12. Kue-Hsuing H, Ching- Yuang L, Ting-Chien L: Complete absence of the third component of complement in a patient with repeated infections. Clin Immunol ImmunopathoI20:305312, 1981 \3. Roord JJ , Daha M, Kuis W, et al: Inherited deficiency of the third component of comple ment associated with recurrent pyogenic infections, circulating immune complexes, and vasculitis in a Dutch family. Pediatrics 71:81-87 , 1983 14. Berger M, Balow JE. Werlson CB, et al: Circulating immune complexes and glomerulonephritis in a patient with congenital absence of the third component of complement. N Engl J Med 308 :1009-1012 , 1983 15 . Lim D, Gewurz A, Lin! TF, et al : Absence of the sixth component of complement in a patient with repeated episodes of meningitis. J Pediatr 89:42-47, 1976 16. Laurell CB: Quantitative estimation of proteins by electrophoresi s in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 17 . Kazatchkine MD, Fearon DT, Appay MD, et al: Immunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases. J Clin Invest 69:900-9 12, 1982 18. Bu rkholder PM, Hyman LR, Krueger RP: Characterization of mixed membranous a nd proliferati ve g lomeru-
C3 DEFICIENCY WITH GLOMERULONEPHRITIS lonephritis: Recognition of three varieties, in Kincaid-Smith P, Mather TH, Becker EL (eds): Glomerulonephritis 1. New York, Wiley and Sons, 1973, pp 557-559 19. Cameron lS: Pathogenesis and treatment of membranous nephropathy. Kidney Int 15: 88-103, 1979 20. Heptinstall RH: Membranoproliferative glomerulonephritis, in Heptinstall RH (ed): Pathology of the Kidney. Boston, Little, Brown, 1983, pp 479-518 21. Hill GS: Systemic lupus erythematosus and mixed connective tissue disease, in Heptinstall RH (ed): Pathology of the Kidney. Boston, Little, Brown, 1983, pp 839-906 22. Peters DK, Williams DG, Charlesworth lA, et al: Mesangiocapillary nephritis, partial lipodystrophy, and hypocomplementemia. Lancet 2:535-538, 1973 23. McLean RH, Weinstein A, Damjanov I, et al: Hypomorphic variant of C3, arthritis, and chronic glomerulonephritis. J Pediatr 93:937-943, 1978 24. Marder HK, Coleman TH, Forristal J, et al: An inherited defect in the C3 convertase, C3b, Bb, associated with glomerulonephritis. Kidney Int 23:749-758, 1983 25. Thompson RA, Haeney M, Reid KBM, et al: A genetic defect of the Clq subcomponent of complement associated with
59 childhood (immune complex) nephritis. N Engl J Med 303:2224 , 1980 26. Kim Y, Friend PS, Dresner IG, et al: Inherited deficiency of the second component of complement (C2) with membranoproliferative glomerulonephritis. Am J Med 62:765-771, 1977 27. Bennett WM, Bardana EJ, Wuepper K, et al: Partial lipodystrophy, C3 nephritis factor and clinically inapparent mesangiocapillary glomerulonephritis. Am 1 Med 62:757-760, 1977 28. Glass D, Raum D, Gibson D, et al: Inherited deficiency of the second component of complement. J Clin Invest 58:853861, 1976 29. Lachmann Pl, Hobart MJ: Complement genetics in relation to HLA. Br Med Bull 34:247-252, 1978 30. Whitehead AS, Solomon E, Chambers Sp, et al: Assignment of the structural gene for the third component of human complement to chromosome 19. Proc Natl Acad Sci USA 79:5021-5025, 1982 31. Peters DK, Williams DG: Complement and mesangiocapillary glomerulonephritis: Role of complement deficiency in the pathogenesis of nephritis. Nephron 13: 189-197, 1974