Nephritis-Associated Plasmin Receptor and Acute Poststreptococcal Glomerulonephritis: Characterization of the Antigen and Associated Immune Response
Nobuyuki Yoshizawa*,
Kazuo Yamakami*,
Masayuki Fujino*,
Takashi Oda*,
Kikuko Tamura,
Koichi Matsumoto,
Tetsuzo Sugisaki and
Michael D.P. Boyle¶
Departments of *Public Health and Pediatrics, National Defense Medical College, Saitama, Second Department of Internal Medicine, Nihon University School of Medicine, and Department of Nephrology, Showa University School of Medicine, Tokyo, Japan; and ¶Department of Biology, Juniata College, Huntington, Pennsylvania.
Correspondence to Dr. Nobuyuki Yoshizawa, Department of Public Health, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Phone: 81-4-2995-1575; Fax: 81-4-2996-5196; E-mail: yoshizawa-npr{at}umin.ac.jp
ABSTRACT. The role of nephritis-associated antigen as a virulencefactor for acute poststreptococcal glomerulonephritis (APSGN)remains to be fully clarified. Nephritis-associated plasminreceptor (NAPlr) was previously isolated from group A streptococcus(GAS) and shown to bind plasmin(ogen). The nucleotide sequenceof the naplr gene from GAS isolates obtained from patients withAPSGN was determined. The sequence of the putative open readingframe (1011 bp) showed 99.8% identity among isolated strains.Homology screen revealed an exact match with streptococcal glyceraldehyde-3-phosphatedehydrogenase (GAPDH). NAPlr exhibited GAPDH activity in zymography,and it activated the complement pathway in vitro. In APSGN kidneybiopsy specimens, NAPlr was observed mainly in the early stageof the disease (1 to 14 d after onset) but was not colocalizedwith either C3 or IgG as assessed by double immunofluorescencestaining. Sera of patients with APSGN, patients with GAS infectionwithout renal involvement, nonrenal pediatric patients, andhealthy adults as controls were assayed for anti-NAPlr antibodytiters. Anti-NAPlr antibodies were present most frequently inAPSGN sera, and antibody titers were also significantly higherthan in patients with GAS infection alone or in other controlpatients. Moreover, antibody titers remained elevated duringthe entire 10-yr follow-up period.
Group A streptococcus (GAS) causes various levels of infectionranging from mild pharyngitis to severe streptococcal toxicshock syndrome. One sequela of GAS infection is acute poststreptococcalglomerulonephritis (APSGN), which is associated with long-termrenal dysfunction in some patients (1). However, only certainstrains appear to cause APSGN (2), and only these strains producenephritis-associated antigens (3).
A number of streptococcal proteins, including nephritis strain-associatedprotein, streptococcal pyrogenic exotoxin B (SPEB), preabsorbingantigen, and NAPlr, are involved in the pathogenesis of APSGN(4–7). Nephritogenic antigens are expressed by so-callednephritis-associated serotypes, accumulate in the glomeruliof patients with APSGN, and induce high antibody titers in thesepatients. NAPlr is such a nephritogenic antigen; it is expressedby streptococcus strains historically associated with APSGN,it is highly antigenic, and it is localized in affected glomeruli(7). However, we cannot exclude the possibility that NAPlr isidentical to previously described nephritogenic antigens becausesome other streptococcal proteins also exhibit plasmin(ogen)-bindingactivity (4,8,9). Only a partial amino acid sequence has beenavailable for NAPlr (7); however, the protein may be homologousto streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(10,11) and may show GAPDH activity. Thus, additional analysisof NAPlr is needed.
APSGN-related antibodies against putative nephritogenic antigenshave been identified (12–14). High levels of anti-SPEBantibody were present in patients with APSGN (6), and subsetsof APSGN kidney specimens were positive for anti-SPEB antibody.In addition, increased levels of anti-zymogen antibody appearto be a marker of APSGN (15,16). Glomeruli from patients withearly APSGN can be stained with IgG obtained from the sera ofconvalescing patients (17). Reactivity is typically observedon the endothelial side of the glomerular basement membrane(GBM) and in the mesangial matrix (18). The streptococcal antigen,such as preabsorbing antigen, has also been detected in theglomeruli of patients in relatively early stages of APSGN (5).
In the study presented here, we determined the amino acid sequenceof NAPlr purified from GAS strains in patients with APSGN. Wealso examined GAPDH activity, complement activation, and immuneresponses to NAPlr in patients with APSGN.
GAS and Preparation of NAPlr
Strains of group A -hemolytic streptococci belonging to T types1, 4, and 12 and M types 12 and 49 were isolated from the pharynxof five patients with APSGN. Growth conditions and purificationof NAPlr were as described previously (7). We had prepared NAPlrin the presence of protease inhibitors and had confirmed theabsence of proteolytic activity in the purified fraction (7).
DNA Sequencing
Genomic DNA of GAS T types 1, 4, and 12 and M types 12 and 49were purified with SepaGene (Sanko Junyaku, Tokyo, Japan) andused as templates to amplify a fragment of naplr by PCR. Thefollowing primers were used: forward primers, 5'-AAGTTAAAGAAGGTGGAT-3',5'-AGCTGCTTCAAACGATAG-3', and 5'-TATATTTGGTGGGTTTTG-3'; reverseprimers, 5'-CAGCTTCTTTCTTCTAG-3', 5'-GAATGCATCGTGAAGAGC-3',and. 5'-CCCCTTCCATCTTAGCCTTTTTGTA-3' at a concentration of 1µM each. Primers were designed from the results of partialamino acid sequencing of purified NAPlr (7) and preliminaryDNA sequence analysis. The PCR temperature profile was carriedout as follows: consisting of an initial denaturation stop of95°C for 5 min, followed by 99 cycles of a denaturationstep of 95°C for 30 s, a primer annealing step at 55°Cfor 20 s, and an extension step at 60°C for 4 min. The amplifiedDNA fragments were sequenced with a BigDye Terminator ReadyReaction Kit and an ABI PRISM 377 XL DNA sequencer (AppliedBiosystems, Foster City, CA). The nucleotide and deduced aminoacid sequences were analyzed with the Query GenBank Database(NCBI, http://www.ncbi.nlm.nih.gov/GenBank/index.html) and GENETYX-MACsoftware (Software Development, Tokyo, Japan).
GAPDH Activity Assay
Similarity of NAPlr to GAPDH was assessed by Western blot analysis(7). After SDS-PAGE and transfer of purified NAPlr to PVDF membranes(Millipore, Billerica, MA), proteins were reacted with mouseanti-Bacillus GAPDH antibody (1:1000 in PBS containing 0.1%Tween 20; Chemicon, Temecula, CA) followed by incubation withan horseradish peroxidase–labeled goat anti-mouse IgG(1:2000 in PBS containing Tween 20; BioSource, Camarillo, CA).An ECL kit (Amersham Biosciences, Piscataway, NJ) was used tovisualize immunecomplexes. Bacillus GAPDH (Sigma, St. Louis,MO) was included in each assay as a control.
For zymographic analysis of GAPDH activity, purified NAPlr (1µg protein) was separated by electrophoresis on 8% polyacrylamidegels in Tris-glycine (pH 8.9) at 4°C. After electrophoresis,gels were gently washed with 0.1 M Na2HPO4 (pH 8.5) for 10 min,then with 0.05 M Na2HPO4 (pH 8.5) for 10 min. Gels were incubatedfor 20 to 30 min at room temperature in substrate buffer containing2.5 mM glyceraldehyde-3-phosphate (Sigma), 0.5 mM NAD+, 300µg/ml nitroblue tetrazolium, and 20 µg/ml phenazinemethosulfate (Wako Pure Chemical Industries, Osaka, Japan) in50 mM Na2HPO4 (pH 8.5). GAPDH activity was detected as a blueband. Bacillus GAPDH (Sigma) was also included in the assayas a positive control.
Complement Activation by NAPlr
To analyze complement activation by NAPlr, we incubated normalhuman serum (50 µl) for 1 h at 37°C with NAPlr (10µg/50 µl in physiologic saline). In some samples,10 µl of 0.1 M EGTA and/or 0.1 M EDTA was added beforeincubation to differentiate between the two complement activationpathways. The reactions were separated by electrophoresis on1.1% agarose gels in veronal buffer (pH 8.6) with an ionic strengthof 0.05 (Wako). Conversion of C3 was examined with anti-humanC3 antibody (ICN, Costa Mesa, CA). Zymosan (Sigma) was usedas a control for complement activation.
The product of NAPlr cleavage of C3, iC3b, was assayed by ELISA.For sample preparation, 50 µl of human serum, diluted1:40 with physiologic saline, was incubated with 50 µlof NAPlr (ranging from 0.01 µg to 6.25 µg) at 37°Cfor 1 h. The level of iC3b in each sample was measured witha commercial iC3b EIA kit (Quidel, San Diego, CA) accordingto the manufacturer’s instructions.
Patients and Control Subjects
Sera from 50 patients with APSGN (27 men and 23 women), diagnosedby renal biopsy, (Table 1) were tested for levels of anti-NAPlrantibody. Serum samples were obtained at the time of biopsy(1 to 90 d after disease onset); anti-NAPlr antibody levelswere determined and taken as the initial antibody titers. Sampleswere collected over a 10-yr follow-up period and used to monitorantibody levels in each patient. The diagnosis of APSGN wasconfirmed by the presence of proteinuria, hematuria, hypocomplementemia,history of antecedent streptococcal infection with titers ofanti-streptolysin O (ASO) and/or anti-streptokinase (ASK), andrenal biopsy. Fifty age-matched patients with GAS upper respiratorytract infection without detectable renal involvement (26 menand 24 women) (Table 2) were included as subjects. GAS upperrespiratory tract infection was diagnosed on the basis of clinicalsign with significant elevation of ASO and/or ASK titers. Thecontrol groups included 100 nonrenal pediatric patients and100 healthy adults. Pediatric patients were categorized by ageinto two groups: pediatric I (age 0.2 to 10 yr, n = 50, 27 boys,23 girls) and pediatric II (age 11 to 20 yr, n = 50, 23 boysand 27 girls). Healthy adults were also categorized by age intotwo groups: adult I (age 25 to 35 yr, n = 50, 25 men and 25women, age matched with patients with APSGN), and adult II (age52 to 59 yr, n = 50, 25 men and 25 women). These subjects showedno signs of recent streptococcal infection. Informed consentwas obtained from all subjects in each group.
Table 2. Clinical and laboratory features of patients with group A streptococcal infection without renal involvement, children, and normal adults
Measurement of Serum Anti-NAPlr Antibody
Serum anti-NAPlr antibody was measured by Western blot analysisas described previously (7). Affinity-purified NAPlr (7) wasseparated by SDS-PAGE (10% polyacrylamide gel) and the proteinswere transferred to PVDF membranes (Millipore) at 0.8 mA/cm2for 50 min in a semidry transfer cell (Bio-Rad Laboratories,Hercules, CA). Membranes were blocked with 5% nonfat milk in10 mM Tris-HCl (pH 7.2) containing 0.15 M NaCl and 0.1% Tween20 (TBS-Tween) for 1 h. The membranes were incubated with eachserum sample (1:500 to 1:2000 in nonfat milk/TBS-Tween) for1 h. Membranes were washed with TBS-Tween and then incubatedwith horseradish peroxidase–conjugated anti-human IgGantibody (1:2000 in nonfat milk/TBS; American Qualex, San Clemente,CA) for 1 h. Immune complexes were visualized by developmentwith ECL (Amersham). Pooled sera from convalescing patientswere included as positive controls, and pooled sera from age-matchedhealthy donors were used as negative controls. NAPlr bands werequantified with a Densitometry System and Imaging Software (ATTO,Tokyo, Japan). The level of anti-NAPlr antibody was determinedrelative to the density of the positive control band (titer:1000 units) and that of the age-matched healthy control band(titer: 60 to 140 units).
Immunofluorescence Microscopy
Direct and indirect immunofluorescence microscopy, FITC-conjugatedrabbit anti-NAPlr antibody, and monoclonal antibody to recombinantPlr were as described by Yamakami et al. (7). Briefly, directimmunofluorescence was used for the detection of NAPlr, complementcomponents (C3, C1q, C4, P), immunoglobulins (IgG, IgA, IgM),fibrinogen, and plasminogen (ICN, Irvine, CA). Indirect immunofluorescencewas used to detect other complement components (C5, C9, S, MAC)(ICN). As a negative control, sections were pretreated witheither unlabeled rabbit anti-NAPlr antibody or serum from aconvalescing patient with APSGN. NAPlr-C3 and NAPlr-IgG colocationassays were performed with double staining for NAPlr and C3or IgG in renal sections from several NAPlr-positive patients.To examine colocalization of NAPlr and C3, we labeled anti-NAPlrantibody (1 mg protein) with Alexa Fluor 594 (Molecular Probes,Eugene, OR), according to the manufacturer’s instructionsand applied the labeled antibody with FITC-labeled anti-C3 antibody(ICN). For NAPlr-IgG colocalization experiments, Alexa Fluor594-labeled goat anti-human IgG antibody (Molecular Probes)and FITC-labeled anti-NAPlr antibody were applied simultaneouslyto the sections.
Statistical Analyses
Statistical analyses of ASO titers and anti-NAPlr antibody titersin the present report were performed by unpaired t test. Two-tailedP values of less than 0.05 were considered statistically significant.
naplr Gene Sequences
The full-length nucleotide sequence of the naplr gene of T type12 is shown in Figure 1. Among sequences from the five-nephritogenicstrains analyzed, only two nucleotides in the open-reading frame(ORF) differed; however, the predicted NAPlr amino acid sequencewas identical among strains. The predicted naplr ORF is 1011bp long, and the putative promoter contains a conserved TATAbox at –10 and a CAT box (TTGCAT) at –35. In addition,a potential ribosome binding site (TAAGGAGG) is located ninenucleotides upstream from the predicted ATG start codon. Guanineat position 1066 of naplr was substituted for thymine in comparisonto the nucleotide sequence of the plr gene encoding plasminreceptor (Plr), which is identified as GAPDH of GAS strain 64/14(10; GenBank Database accession number M95569). Thus, NAPlrand Plr showed 99.8% identity at nucleotide and 99.7% identityat amino acid levels.
Figure 1. Nucleotide sequence and predicted amino acid sequence of the naplr gene in group A streptococci T type 12. Putative conserved promoter sequences (–35 and –10) and ribosome-binding site sequence (RBS) are indicated by bars. The predicted transcription start site, +1, is denoted by an asterisk. The predicted ATG start codon and TAA stop codon are indicated by bars. Positions of the PCR primers and their orientations are indicated by arrows.
The naplr ORF encodes a 336 amino acid polypeptide with a predictedisoelectric point of 5.2 and a predicted molecular mass of 35.8kD. The predicted molecular mass was lower than that determinedby SDS-PAGE (43 kD), which may reflect the amino acid compositionsof NAPlr. The N-terminal amino acid sequence of purified NAPlrcontained the following five residues: VVKVG. The N-terminalamino acid sequence of native NAPlr was homologous to the deducedN-terminal sequence of NAPlr, with the exception of an additionalN-terminal methionine.
Functional Analysis of NAPlr
On the basis of the naplr nucleotide sequence, which was homologousto the GAPDH sequence (11), NAPlr was tested for reactivitywith anti-GAPDH antibody and for GAPDH activity. Western blotanalysis revealed that NAPlr reacted with anti-Bacillus GAPDHantibody (Figure 2, left). In addition, zymographic analysisshowed that both purified NAPlr and crude extract each containedactivity in single bands that had identical migration profiles(Figure 2, right).
Figure 2. Antigenic and functional similarities between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and NAPlr. The Western blot profile of RCS and NAPlr shows that both proteins reacted with anti-Bacillus GAPDH antibody (left). The zymogram profile indicates that both RCS and NAPlr have GAPDH activity (right). The positions of protein standards are shown (kD) on the left of panel. RCS, ruptured streptococcal cell supernatant, which is the starting material for the isolation of NAPlr.
The ability of NAPlr to activate complement was measured asconversion of C3 (Figure 3A). C3 conversion was observed inthe presence or absence of chelating reagent. Thus, NAPlr activatedthe alternate complement pathway. In addition, we found thatNAPlr induced the formation of iC3b in a dose-dependent manner(Figure 3B).
Figure 3. Complement activation by NAPlr. (A) Immunoelectrophoresis shows conversion of C3 after incubation of normal human serum (NHS) with NAPlr with or without Mg2+ and EGTA (middle). As a positive control, zymosan was added to NHS and indicates activated C3 (top). NHS not incubated with NAPlr shows a single arc of C3 (bottom). (B) Formation of iC3b from C3 in NHS incubated with various amounts of NAPlr. The plots use average values from triplicate assays.
Measurement of Serum Anti-NAPlr Antibody
Sera from patients with APSGN, pediatric and adult patientswith streptococcal infection without renal involvement, andcontrol subjects were tested to determine the titers of anti-NAPlrantibody. Anti-NAPlr antibody was detected more frequently inthe sera of patients with APSGN than in sera from other subjects(Table 3), and significantly increased levels of anti-NAPlrantibody were found in the sera of patients with APSGN (Figure 4A).It is noteworthy that as much as 72% of the adult II group(mean age 53.2 yr) possessed anti-NAPlr antibody, whereas only26% of the nonrenal pediatric patients (mean age 7.2 yr) possessedanti-NAPlr antibody.
Table 3. Anti-NAPlr antibody in patients with APSGN, patients with group A streptococcal infection without renal involvement, children, and normal adults
Figure 4. (A) Levels of anti-NAPlr antibody titers in acute poststreptococcal glomerulonephritis (APSGN), streptococcal infection without renal involvement (SI), nonrenal pediatric patients, and normal adults. Values are mean ± SEM. P < 0.05 for APSGN versus SI, pediatrics, and normal adults by t test. (B) Levels of ASO titers from the same group of patients compared with anti-NAPlr antibody titers. The APSGN and SI groups show significantly elevated ASO titers in comparison to those in nonrenal pediatric patients and normal adults. Values are mean ± SEM. P < 0.001 for titers in APSGN and SI versus those in other groups by t test.
ASO titers compared with the anti-NAPlr antibody titers areshown on Figure 4B. ASO titers were significantly higher inthe APSGN and streptococcal infection groups than in the controlgroups, indicating a serologic response to a streptococcal product,regardless of renal involvement. Over the 10 yr that the seraof the 50 patients with documented APSGN were monitored, theanti-NAPlr antibody titers tended to increase during the acutephase of the disease. After the acute phase, titers decreasedbut remained significantly higher in patients with APSGN thanin age-matched control adults (Figure 5). The rate of anti-NAPlrantibody positivity was also higher than that of controls duringthe 10-yr follow-up period.
Figure 5. Serial anti-NAPlr antibody levels in the sera of patients with acute poststreptococcal glomerulonephritis (APSGN). Values are mean ± SEM, and the figure indicates the rate of anti-NAPlr antibody detection at each time point. The shaded area is the mean ± SEM of normal adults (mean age 30 yr). P < 0.05 for titers of the serial sera of patients with APSGN versus age-matched controls by t test.
Immunofluorescence Studies of Kidney Biopsy Specimens
Thirty-six (72%) of 50 APSGN renal biopsy specimens were positivefor glomerular NAPlr with anti-NAPlr antibody (Table 4). All25 renal biopsy specimens obtained in the early disease stage(1 to 14 d after APSGN onset) and 11 (61%) of 18 biopsy specimensobtained in the middle disease stage (15 to 30 d after onset)were positive for glomerular NAPlr. The antigen was localizedmainly to the mesangium and part of the GBM, and infiltratingleukocytes were observed in a ringlike pattern (Figure 6A).However, no staining was observed 31 d after onset. Pretreatmentof sections with unlabeled anti-NAPlr antibody or with serumfrom a convalescing patient abolished the staining with FITC-labeledanti-NAPlr-antibody. In addition, preabsorption of FITC-labeledanti-NAPlr antibody with recombinant streptococcal Plr abolishedglomerular staining of NAPlr. All APSGN renal biopsy specimensobtained within 30 d of onset showed intense and extensive depositionof C3 along the GBM and/or in the mesangium (Figure 6B). IgGstaining was present in the glomeruli of 64% and 61% of thesections representing the early and middle disease stages, respectively,and it was not always colocalized with C3. Staining of IgA andIgM ranged from blush to faint. Colocation studies of NAPlrwith C3 or IgG revealed that the distribution of NAPlr differsfrom that of C3 or IgG (Figure 7).
Figure 6. Immunofluorescence microscopy of glomeruli from a patient with acute poststreptococcal glomerulonephritis (APSGN) 11 d after onset. (A) Localization of NAPlr. Staining sites, which are thought to represent free antigen, are localized primarily in the mesangium and part of the glomerular basement membrane (GBM), and infiltrating leukocytes show a ring-like granular pattern (original magnification, x200). (B) Deposition of C3. Intense, diffuse, fine granular deposition of C3 is seen mainly along the GBM (original magnification, x200). (C) Deposition of fibrinogen. Fibrinogen is deposited primarily along the inner side of the GBM (original magnification, x200). (D) Deposition of plasminogen. Plasminogen is deposited predominantly in the mesangium and along part of the GBM (original magnification, x200).
Figure 7. Immunofluorescence microscopy of glomeruli from a patient with acute poststreptococcal glomerulonephritis (APSGN) 18 d after onset. The distributions of NAPlr (Alexa Fluor 594, red) and C3 (FITC, green) (top panels) and of NAPlr (FITC, green) and IgG (Alexa Fluor 594, red) (bottom panels) are essentially different, as shown by the respective double immunofluorescence stainings (original magnification, x200).
Fibrinogen was stained intensely in the glomeruli in 15 (60%)of 25 patients 1 to 14 d after onset. Fibrinogen was observedmainly on the endothelial side of the GBM (Figure 6C), and thefrequency of staining was relatively consistent throughout thecourse of the disease. In contrast, plasminogen was observedin the glomeruli in 10 (40%) of 25 patients in the early stageand less frequently in the later stage. The localization ofplasminogen was predominantly in the mesangium and part of theGBM (Figure 6D), and when present, it was always colocalizedwith NAPlr. Most complement components, except C1q and C4, weredetected frequently in glomeruli. We observed intense stainingof C3, P, C5, C9, S, and MAC, particularly in the early stage(Table 4). Staining of C1q and C4 was weak and infrequent.
Characterization of NAPlr has been incomplete (7). The availablepartial amino acid sequence for purified NAPlr was identicalto that of streptococcal Plr (10) and similar to that of streptococcalGAPDH, suggesting that NAPlr has GAPDH activity (11). Thus,NAPlr is implicated as a virulence factor. However, the roleof cytoplasmic GAPDH in APSGN was not clear. In the study presentedhere, we found that purified NAPlr has GAPDH activity on zymograms.Characteristics of NAPlr that are similar to characteristicsof GAPDH include adhesion to fibronectin, myosin, and actinand plasmin receptor activity (10,19,20). Thus, NAPlr is expectedto interact with these molecules in the pathogenesis of APSGN.
We previously detected anti-NAPlr antibody in sera at a relativelyearly stage of APSGN (7). In the study presented here, analysisof sera from patients with APSGN showed a significantly higherfrequency of anti-NAPlr antibody than in other subjects, includingthose with streptococcal infection alone. In addition, anti-NAPlrantibody titers tended to be highest during the first week ofinfection and decreased thereafter. However, the titers didnot decrease to the baseline levels of age-matched controls,and they remained significantly higher than those of the controlsubjects over the 10-yr follow-up period. These findings suggestthat recurrence of APSGN is rare and that a single infectionconfers life-long immunity. Anti-NAPlr antibody was presentin only 26% of subjects in the youngest control group (age 0.2to 10 yr), and the rate increased to 72% (age 52 to 59 yr).This may explain why younger children have a greater tendencyto suffer from this disease. Individuals appear to acquire immunitygradually through repeated streptococcal infection and thus,older people seldom develop APSGN (21).
As we reported previously, immunohistochemistry showed thatNAPlr was present in glomeruli in APSGN renal biopsy specimens(7). In the study presented here, all specimens obtained 1 to14 d after APSGN onset were positive for glomerular NAPlr, whereasno specimen obtained 31 to 90 d after onset was positive forthe antigen. Thus, glomerular NAPlr tended to decrease overtime in patients with APSGN. Furthermore, the difference inthe localization of NAPlr in comparison to that of IgG or C3indicated that NAPlr exists as a free antigen with or withoutplasmin(ogen). We suspect that during the early phase of APSGN,the antigenic sites are not fully saturated and can interactwith anti-NAPlr antibody, whereas later in the course of thedisease, the sites are saturated.
We previously reported that complement components were depositedin affected glomeruli 1 to 32 d after onset of APSGN (7). Inthe study presented here, the majority of biopsy specimens showedfrequent and intense staining for C3, P, C5, C9, S, and MAC,particularly at 1 to 90 d after onset. NAPlr was deposited in100% of the specimens obtained from early in the disease course(1 to 14 d after onset). Thus, NAPlr as well as complement componentsare associated with APSGN (22–24). In the study presentedhere, the deposition of C3 without IgG in glomeruli in 9 of25 patients 1 to 14 d after APSGN onset and the lack of circulatinganti-NAPlr antibody in 4 of 50 patients suggest that complementcomponents are associated with the initial inflammatory reaction(25–27). In addition, NAPlr cleaved C3 to C3b in humanserum in vitro. Thus, NAPlr may activate the complement cascadein circulation (28,29).
NAPlr was detected in glomeruli of all early APSGN biopsy specimens,and anti-NAPlr antibody was detected in the majority of serumsamples from patients with APSGN. Because NAPlr has plasminogen-bindingactivity (7,19), NAPlr on the mesangial matrix and GBM is expectedto interact with plasmin(ogen). Plasmin may induce glomerulardamage by degrading the GBM through activation of matrix metalloproteinaseprecursors. In fact, we recently observed significant glomerularplasmin activity that reflected the distribution of NAPlr depositionin the early phase of APSGN (T. Oda et al., unpublished data).Circulating immune complexes may readily pass through the alteredGBM and accumulate in the subepithelial space (30). Taken together,our findings suggest that NAPlr is a virulence factor for APSGNand that the presence of a high titer of anti-NAPlr antibodyshould prevent autoimmune sequelae. Further studies regardingthe role of NAPlr will allow us to better understand the pathologyof APSGN.
Acknowledgments
We thank Dr. Takayuki Fujita (Second Department of InternalMedicine, Nihon University School of Medicine) for discussionof the complement analysis.
Dillon HC Jr: Post-streptococcal glomerulonephritis following pyoderma. Rev Infect Dis 1: 935–945, 1979[Medline]
Kaplan EL, Anthony BF, Chapman SS, Wannamaker LW: Epidemic acute glomerulonephritis associated with type 49 streptococcal pyoderma. I. Clinical and laboratory findings. Am J Med 48: 9–27, 1970[CrossRef][Medline]
Silva FG: Acute postinfectious glomerulonephritis and glomerulonephritis complicating persistent bacterial infection. In: Heptinstall’s Pathology of the Kidney, 5th ed., edited by Jennette JC, Olson JL, Schwartz MM, Silva FG, Philadelphia, Lippincott-Raven, 1998, pp 389–453
Poon-King R, Bannan J, Viteri A, Cu G, Zabriskie JB: Identification of an extracellular plasmin binding protein from nephritogenic streptococci. J Exp Med 178: 759–763, 1993[Abstract/Free Full Text]
Yoshizawa N, Oshima S, Sagel I, Shimizu J, Treser G: Role of a streptococcal antigen in the pathogenesis of acute poststreptococcal glomerulonephritis: Characterization of the antigen and a proposed mechanism for the disease. J Immunol 148: 3110–3116, 1992[Abstract]
Cu GA, Messano S, Bannan JD, Zabriskie JB: Immunohistochemical and serological evidence for the role of streptococcal proteinase in acute post-streptococcal glomerulonephritis. Kidney Int 54: 819–826, 1998[CrossRef][Medline]
Yamakami K, Yoshizawa N, Wakabayashi K, Takeuchi A, Tadakuma T, Boyle MD: The potential role for nephritis-associated plasmin receptor in acute poststreptococcal glomerulonephritis. Methods 21: 185–197, 2000[CrossRef][Medline]
Ringdahl U, Sjobring U: Analysis of plasminogen-binding M proteins of streptococcus pyogenes. Methods 21: 143–150, 2000[CrossRef][Medline]
Pancholi V, Fischetti VA: -Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273: 14503–14515, 1998[Abstract/Free Full Text]
Lottenberg R, Broder CC, Boyle MD, Kain SJ, Schroeder BL, Curtis R 3rd: Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor. J Bacteriol 174: 5204–5210, 1992[Abstract/Free Full Text]
Winram SB, Lottenberg R: The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase. Microbiology 142: 2311–2320, 1996[Abstract]
Lange K, Seligson G, Cronin W: Evidence for the in situ origin of poststreptococcal GN: Glomerular localization of endostreptosin and the clinical significance of the subsequent antibody response. Clin Nephrol 19: 3–10, 1983[Medline]
Vogt A, Batsford SR, Rodriguez-Iturbe B, Garcia R: Cationic antigens in poststreptococcal glomerulonephritis. Clin Nephrol 20: 271–279, 1983[Medline]
Seligson G, Lange K, Majeed HA, Deol H, Cronin W, Bovie R: Significance of endostreptosin antibody titers in poststreptococcal glomerulonephritis. Clin Nephrol 24: 69–75, 1985[Medline]
Parra G, Rodriguez-Iturbe B, Batsford S, Vogt A, Mezano S, Olavarria F, Exeni R, Laso M, Orta N: Antibody to streptococcal zymogen in the serum of patients with acute glomerulonephritis: A multicentric study. Kidney Int 54: 509–517, 1998[CrossRef][Medline]
Fischetti VA: Streptococcal M protein: Molecular design and biological behavior. Clin Microbiol Rev 2: 285–314, 1989[Abstract/Free Full Text]
Treser G, Semar M, Mcvicar M, Franklin A, Ty A, Sagel I, Lange K: Antigenic streptococcal components in acute glomerulonephritis. Science 163: 676–677, 1969[Abstract/Free Full Text]
Yoshizawa N, Treser G, Sagel I, Ty A, Ahmed U, Lange K: Demonstration of antigenic sites in glomeruli of patients with acute poststreptococcal glomerulonephritis by immunofluorescein and immunoferritin techniques. Am J Pathol 70: 131–150, 1973[Medline]
D’Costa SS, Boyle MD: Interaction of group A streptococci with human plasmin(ogen) under physiological conditions. Methods 21: 165–177, 2000[CrossRef][Medline]
Pancholi V, Fischetti VA: A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate dehydrogenase with multiple binding activity. J Exp Med 176: 415–426, 1992[Abstract/Free Full Text]
Peake PW, Pussell BA, Karplus TE, Riley EH, Charslesworth JA: Post-streptococcal glomerulonephritis: Studies on the interaction between nephritis strain-associated protein (NSAP), complement and the glomeruli. APMIS 99: 460–466, 1991[Medline]
Yoshizawa N, Oshima S, Takeuchi A, Kondo S, Oda T, Shimizu J, Nishiyama J, Ishida A, Nakabayashi I, Tazawa K: Experimental acute glomerulonephritis induced in the rabbit with a specific streptococcal antigen. Clin Exp Immunol 107: 61–67, 1997[CrossRef][Medline]
Westberg NG, Naff GB, Boyer JT, Michael AF: Glomerular deposition of properdin in acute and chronic glomerulonephritis with hypocomplementemia. J Clin Invest 50: 642–649, 1971
Kirschfink M, Borsos T: Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes. Mol Immunol 25: 505–512, 1988[CrossRef][Medline]
Yoshizawa N, Oda T, Oshikawa Y, Akashi Y, Suzuki Y, Shimizu J, Shimizu E, Niwa H, Treser G: Cell-mediated immune response in acute poststreptococcal glomerulonephritis. Jpn J Nephrol 36: 322–330, 1994
Oda T, Yoshizawa N, Takeuchi A, Nakabayashi I, Nishiyama J, Ishida A, Tazawa K, Murayama M, Hotta O, Taguma Y: Glomerular proliferating cell kinetics in acute post-streptococcal glomerulonephritis (APSGN). J Pathol 183: 359–368, 1997[CrossRef][Medline]
Couser WG: Pathogenesis of glomerular damage in glomerluronephritis. Nephrol Dial Transplant 13 [Suppl 1]: 10–15, 1998[Free Full Text]
Kobrin S, Madaio MP: Acute poststreptococcal glomerulonephritis and other bacterial infection-related glomerulonephritides. In: Diseases of the Kidney, 6th ed., edited by Schrier RW, Gottschalk CW, Boston, Little Brown, 1997, pp 1579–1593
Vogt A, Schmiedeke T, Stockl F, Sugisaki Y, Mertz A, Batsford SR: The role of cationic proteins in the pathogenesis of immune complex glomerulonephritis. Nephrol Dial Transplant 5 [Suppl 1]: 6–9, 1990
Received for publication August 26, 2003.
Accepted for publication April 10, 2004.
This article has been cited by other articles:
B. Rodriguez-Iturbe and J. M. Musser The Current State of Poststreptococcal Glomerulonephritis
J. Am. Soc. Nephrol.,
October 1, 2008;
19(10):
1855 - 1864.
[Abstract][Full Text][PDF]
T. Oda, K. Tamura, N. Yoshizawa, T. Sugisaki, K. Matsumoto, M. Hattori, T. Sawai, T. Namikoshi, M. Yamada, Y. Kikuchi, et al. Elevated urinary plasmin activity resistant to {alpha}2-antiplasmin in acute poststreptococcal glomerulonephritis
Nephrol. Dial. Transplant.,
July 1, 2008;
23(7):
2254 - 2259.
[Abstract][Full Text][PDF]
T. Oda, N. Yoshizawa, K. Yamakami, A. Ishida, O. Hotta, S. Suzuki, and S. Miura Significance of glomerular cell apoptosis in the resolution of acute post-streptococcal glomerulonephritis
Nephrol. Dial. Transplant.,
March 1, 2007;
22(3):
740 - 748.
[Abstract][Full Text][PDF]
A. Servais, V. Fremeaux-Bacchi, M. Lequintrec, R. Salomon, J. Blouin, B. Knebelmann, J.-P. Grunfeld, P. Lesavre, L.-H. Noel, and F. Fakhouri Primary glomerulonephritis with isolated C3 deposits: a new entity which shares common genetic risk factors with haemolytic uraemic syndrome
J. Med. Genet.,
March 1, 2007;
44(3):
193 - 199.
[Abstract][Full Text][PDF]
M. R. Graham, K. Virtaneva, S. F. Porcella, D. J. Gardner, R. D. Long, D. M. Welty, W. T. Barry, C. A. Johnson, L. D. Parkins, F. A. Wright, et al. Analysis of the Transcriptome of Group A Streptococcus in Mouse Soft Tissue Infection
Am. J. Pathol.,
September 1, 2006;
169(3):
927 - 942.
[Abstract][Full Text][PDF]
T. Oda, K. Yamakami, F. Omasu, S. Suzuki, S. Miura, T. Sugisaki, and N. Yoshizawa Glomerular Plasmin-Like Activity in Relation to Nephritis-Associated Plasmin Receptor in Acute Poststreptococcal Glomerulonephritis
J. Am. Soc. Nephrol.,
January 1, 2005;
16(1):
247 - 254.
[Abstract][Full Text][PDF]
B. Rodriguez-Iturbe Nephritis-Associated Streptococcal Antigens: Where Are We Now?
J. Am. Soc. Nephrol.,
July 1, 2004;
15(7):
1961 - 1962.
[Full Text][PDF]
Top
Abstract
Introduction
-hemolytic streptococci belonging to T types 1, 4, and 12 and M types 12 and 49 were isolated from the pharynx of five patients with APSGN. Growth conditions and purification of NAPlr were as described previously (7). We had prepared NAPlr in the presence of protease inhibitors and had confirmed the absence of proteolytic activity in the purified fraction (7). 
-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273: 14503–14515, 1998
P < 0.05 for APSGN versus SI, pediatrics, and normal adults by t test. (B) Levels of ASO titers from the same group of patients compared with anti-NAPlr antibody titers. The APSGN and SI groups show significantly elevated ASO titers in comparison to those in nonrenal pediatric patients and normal adults. Values are mean ± SEM. 
