Journal of Clinical Investigation Vol. 45, No. 2, 1966 Acute Poststreptococcal Glomerulonephritis: Immune Deposit Disease * ALFRED F. MICHAEL, JR.,t KEITH N. DRUMMOND,t ROBERT A. GOOD,§ AND ROBERT L. VERNIER || WITH THE TECHNICAL ASSISTANCE OF AGNES M. OPSTAD AND JOYCE E. LOUNBERG (From the Pediatric Research Laboratories of the Variety Club Heart Hospital and the Department of Pediatrics, University of Minnesota, Minneapolis, Minn.) The possible role of immunologic mechanisms in the kidney in acute glomerulonephritis have also acute poststreptococcal glomerulonephritis was revealed the presence of discrete electron dense suggested in 1908 by Schick (2), who compared masses adjacent to the epithelial surface of the the delay in appearance of serum sickness after glomerular basement membrane (11-18). injection of heterologous serum to the latent pe- The purpose of this paper is to describe immuno- riod between scarlet fever and onset of acute glo- fluorescent and electron microscopic observations merulonephritis. Evidence in support of this con- of the kidney in 16 children with acute poststrepto- cept is the depression of serum complement during coccal glomerulonephritis. This study demon- the early stages of the disease (3) and glomerular strates 1) the presence of discrete deposits of yG- localization of immunoglobulin. Immunofluores- and fl3c-globulins along the glomerular basement cent studies have revealed either no glomerular membrane and its epithelial surface that are similar deposition of a-globulin (4) or a diffuse involve- in size and location to the dense masses seen by ment of the capillary wall (5-9). Seegal, Andres, both thin section microscopy and electron micros- Hsu, and Zabriskie (10) demonstrated the pres- copy; 2) the characteristic and unique specificity of ence of 7 S y-globulin, /31c-globulin, and strepto- this lesion in acute poststreptococcal glomerulo- coccal antigen in the glomeruli of most patients nephritis, its difference from other glomerular dis- with this disease. By studies employing ferritin- eases, and the similarity to experimental antigen- labeled antibodies, these proteins could be demon- antibody complex nephritis; and 3) the resolution strated in the mesangium, between the endothelial of these deposits during recovery from the disease cells, and within and adjacent to the basement and their absence from the glomeruli 10 years af- membrane. Electron microscopic observations of ter epidemic acute poststreptococcal glomerulo- nephritis. * Submitted for publication May 3, 1965; accepted No- vember 4, 1965. Methods Aided by grants from the U. S. Public Health Service (AI-02168 and HE-05662), the American Heart Associ- Patients. The major portion of the study concerned ation, and the Cardiovascular Clinical Research Center sixteen children with acute poststreptococcal glomerulo- (HE-06314). nephritis having the characteristic clinical course and re- Presented in part at the American Society for Clinical nal histology as well as prior P-hemolytic streptococcal Investigation, Fifty-sixth Annual Meeting, May 4, 1964, infections and elevated or rising antistreptococcal anti- Atlantic City, N. J. (1). body titers. A total of 22 renal biopsies were performed t Established Investigator, American Heart Associa- on these patients by the method previously described tion. (19) and at the times indicated in Table I. As part of t Present address: Dept. of Pediatrics, McGill Uni- a separate follow-up study (20) 10 years after well- versity, Montreal Children's Hospital, Montreal, Canada. documented acute poststreptococcal glomerulonephritis § American Legion Memorial Heart Research Profes- (Red Lake, Minnesota, epidemic of 1953), additional re- sor of Pediatrics and Microbiology. nal biopsies were performed on 14 children and studied Address requests for reprints to Dr. Robert A. Good, by fluorescent microscopy. Dept. of Pediatrics, University of Minnesota Medical Electron and fluorescent microscopic observations of School, Minneapolis, Minn. 55455. the kidney were also made in one patient with lupus 11 Established Investigator, American Heart Association. nephritis and one with Goodpasture's disease. Present address: Dept. of Pediatrics, Center for Health Histologic methods. Part of each biopsy was placed Sciences, University of California, Los Angeles, Calif. in 10% buffered formalin (pH 7.35) for standard paraffin 237 238 MICHAEL, DRUMMOND, GOOD, AND VERNIER embedding and sectioning and subsequently stained with man fibrinogen was prepared from human Fraction I 5 hematoxylin-eosin, azocarmine, and periodic-acid Schiff. according to the method of Laki (30, 31). 4) Human Light microscopy was carried out on 21 of the 22 biopsies. fibrin was prepared from human fibrinogen; 25.0 mg of A total of 12 to 30 glomeruli were evaluated in 17 of the fibrinogen was diluted in isotonic saline to a final con- 21 biopsies; in each of the remaining specimens, five to centration of 5.0 mg per ml, and then 10.0 ml of 0.075 M six glomeruli were present. sodium chloride in phosphate buffer (pH 6.1) and 0.5 ml Another portion of the biopsy specimen was immedi- of bovine thrombin were added. After standing for 1 ately cut into cubes (0.5 mme) under a drop of buffered hour, the clots were washed with distilled water, lyophi- (1%o) osmic acid solution, dehydrated by standard tech- lized, and suspended in particulate form in 0.15 M saline. niques, and embedded in Vestopal-W1 polyester resin. Antisera were also prepared against 5) human albumin Frac- Sections 0.5 to 1.0 j in thickness cut from these blocks (Cohn Fraction V),6 6) rabbit y-globulin (Cohn were stained by either the Wright-Giemsa (21) or basic tion II), and 7) heat-killed and ground-cell suspension fuchsin method (22) and examined under oil immersion of nephritogenic, type 1, group A, fi-hemolytic strepto- light microscopy. Very thin sections for electron micros- coccus. 8) Extracellular products of the same strepto- copy were stained with either uranyl acetate (23) or lead coccus were prepared according to the method of Wan- citrate (24) and examined with either an RCA EMU-3D namaker (32) and concentrated by ammonium sulfate or a Phillips EMU-200 electron microscope at original precipitation or by pervaporation. magnifications of 2,500 to 75,000 times and enlarged Preparation of antibody. Antibody to these human anti- photographically as desired. gens was prepared in rabbits. Immunizations were car- Immunofluorescent methods (25-27) were carried out ried out with alum-precipitated human 'yG-globulin and on renal biopsy tissue that had been frozen in isopentane albumin, fibrin and Pic-globulin incorporated in complete in liquid nitrogen, fixed to a small cellulose sponge,2 and Freund's adjuvant,8 and a saline solution of fibrinogen. sectioned in a Lipshaw cryostat. Fluorescent staining of Alum-precipitated rabbit y-globulin was injected into unfixed tissue was carried out according to the method goats. The specificity of the antisera was shown by im- of Ortega and Mellors (28). In some instances an indi- munoelectrophoresis of the immunizing antigen and whole rect technique was also employed. Inhibition of fluores- serum developed against the antisera. Antisera showing cence was carried out by pretreating the section once or more than one major precipitin band were not used. twice with unlabeled antiserum. A horse anti-'yM-globulin serum,9 employed in these The sections were viewed in a Zeiss microscope with studies, formed one major line on immunoelectrophoresis an HB-200 light source or a mercury lamp (GE-AHG) against normal serum; an additional faint line was also mounted in a Scopicon water-cooled unit. The ultra- seen in the a2-globulin .region. violet activating and heat absorption filters were UG-1 Antisera to the various streptococcal preparations were and KG-1, respectively. The barrier filters consisted of obtained by immunizing rabbits three times weekly for OG-4 and a 2A Wratten (Kodak). Photographs were a total of 2 months with 0.5 ml of antigen in solution or taken with an Exacta camera using Kodak Tri-X or suspension. Antigen preparations were injected intra- high speed Ektachrome film. The intensity and amount venously during the first month and subcutaneously dur- of fluorescence were arbitrarily graded as negative, trace, ing the second month of immunization. The development 1+, 2 +, and 3 +. Six to 18 glomeruli were available of antibodies to multiple antigens in these crude vaccine for evaluation from each of the 22 biopsies. materials was demonstrated by the appearance of a num- Preparation of antisera. Antisera were prepared against ber of lines of precipitation in a double-diffusion agar the following antigens: 1) Human -yG-globulin was iso- system. lated by DEAE Sephadex A-50 chromatography; 3 im- The ammonium sulfate-precipitated globulins were di- munoelectrophoresis of this antigen against antihuman alyzed and then conjugated with fluorescein-isothiocya- serum revealed a single precipitin line. 2) Human Pic- nate 10 (33, 34) with 0.015 g per g of protein. The la- globulin, a part of the third component of complement, beled antiserum was then passed through a column of was prepared from 400 ml of fresh human serum by the Sephadex G-25 to remove the free fluorescein (35, 36). column chromatographic method of Muller-Eberhard, Antisera were stored as small portions at - 220 C until Nilsson, and Aronsson (29); the 8,c-globulin isolated used. Immediately before use, the antisera were ab- formed one precipitin line with both antihuman serum and with known rabbit anti-Pic-globulin serum.4 3) Hu- 5 Provided by the American Red Cross and Dr. Rich- of 1 ard von Korff, Department Biochemistry, University Martin Jaeger Co., Geneva, Switzerland. of Minnesota, Minneapolis, Minn. 2 no. 1, obtained from Histomed, Paterson, Onkosponge 6 Cutter's normal human serum albumin, Cutter Labora- N. J. tories, Berkeley, Calif. 8 Some of the -yG-globulin used was obtained from Im- munology, Inc., Lombard, Ill. 7 Rabbit y-globulin (Fraction II), Nutritional Bio- 4 During the initial part of this investigation the rab- chemical Corp., Cleveland, Ohio.