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Published ahead of print on December 1, 2004
J Am Soc Nephrol 16: 247-254, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2004040341
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Clinical Immunology and Pathology

Glomerular Plasmin-Like Activity in Relation to Nephritis-Associated Plasmin Receptor in Acute Poststreptococcal Glomerulonephritis

Takashi Oda*, Kazuo Yamakami*, Fumihiro Omasu*, Shigenobu Suzuki{dagger}, Soichiro Miura{dagger}, Tetsuzo Sugisaki{ddagger} and Nobuyuki Yoshizawa*

* Department of Public Health and {dagger} Second Department of Internal Medicine, National Defense Medical College, Saitama; and {ddagger} Department of Nephrology, Showa University School of Medicine, Tokyo, Japan

Address correspondence to: Dr. Takashi Oda, Department of Public Health, National Defense Medical College, 3-2 Namiki, Tokorozawa-shi, Saitama 359-8513, Japan. Phone: +81-4-2995-1575; Fax: +81-4-2996-5196; E-mail: takashio{at}cc.ndmc.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A nephritogenic antigen for acute poststreptococcal glomerulonephritis (APSGN) was isolated recently from group A streptococcus and termed nephritis-associated plasmin receptor (NAPlr). In vitro experimental data indicate that the pathogenic role of NAPlr occurs through its ability to bind to plasmin and maintain its proteolytic activity. However, the mechanism whereby this antigen induces glomerular damage in vivo has not been fully elucidated. Renal biopsy tissues from 17 patients with APSGN, 8 patients with rapidly progressive glomerulonephritis, and 10 normal kidneys were analyzed in this study. Plasmin-like activity was assessed on cryostat sections by in situ zymography with a plasmin-sensitive synthetic substrate. Serial sections were simultaneously assessed for NAPlr deposition by immunofluorescence staining. Glomerular plasmin-like activity was absent or weak in normal controls and in patients with rapidly progressive glomerulonephritis, although tubulointerstitial activity was occasionally detected. Prominent glomerular plasmin-like activity was found in patients who had APSGN and in whom glomerular NAPlr was positive, whereas it was absent or weak in patients who had APSGN and in whom glomerular NAPlr was negative. The distribution of glomerular plasmin-like activity was identical to that of NAPlr deposition but was generally different from that of fibrin(ogen) deposition as assessed by double staining. The activity was abolished by the addition of aprotinin to the reaction mixture but was not altered by the addition of a matrix metalloprotease inhibitor, a cysteine protease inhibitor, or inhibitors of plasminogen activators. Thus, upregulated glomerular plasmin-like activity in relation to NAPlr deposition in APSGN was identified. This result supports the nephritogenic character of NAPlr and offers insight into the mechanism whereby this antigen induces nephritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute poststreptococcal glomerulonephritis (APSGN), a renal disease that occurs after group A streptococcus (GAS) infection, is the prototype of acute nephritic syndrome, which also includes several types of chronic glomerulonephritis such as IgA nephropathy, lupus nephritis, and rapidly progressive glomerulonephritis (RPGN). This disease is generally considered to be induced by the glomerular deposition of nephritogenic streptococcal antigen and the subsequent formation of immune complexes in situ and/or by deposition of circulating antigen-antibody complexes (1). However, involvement of other factors, such as cell-mediated immunity (2), cell proliferation and apoptosis (2–4), and injurious complement components (5), has been suggested, and the complete pathogenic mechanism of this disease has not been established (1). Even the identity of the nephritogenic antigen remains a matter of debate. Many proteins such as endostreptosin (6), preabsorbing antigen (7), nephritis strain–associated protein (8), and streptococcal pyrogenic exotoxin B (SPEB) (9) have been reported as potent nephritogenic antigens in APSGN.

We recently isolated and characterized a novel nephritogenic antigen from GAS that we termed nephritis-associated plasmin receptor (NAPlr) because the nucleotide sequence shows extremely high homology to that of the plasmin receptor (Plr) of GAS (10,11). Indeed, NAPlr exhibits functions such as specific binding to plasmin(ogen) and expression of glyceraldehyde-3-phosphate dehydrogenase activity that are similar to Plr (11,12). Actually, Plr was shown to be the same entity as streptococcal glyceraldehyde-3-phosphate dehydrogenase (13). These findings indicate that NAPlr is identical to Plr, even though the two were isolated from different cell fractions. NAPlr was isolated from the cytoplasmic fraction, and Plr was isolated from the cell-surface fraction (12,14). The most characteristic feature of Plr in vitro is that it binds to plasmin and maintains plasmin’s proteolytic activity by protecting it from physiologic inhibitors, such as {alpha}2-antiplasmin ({alpha}2-AP) (14–16). We found significant glomerular NAPlr deposition in the early phase of APSGN, which led us to speculate that deposited NAPlr traps and maintains plasmin in the active form and induces glomerular damage in vivo (11). However, we had no direct evidence of this.

Plasmin is converted from plasminogen by two types of plasminogen activators, urokinase type (uPA) and tissue type (tPA). This conversion is inhibited by a primary physiologic inhibitor, plasminogen activator inhibitor-1 (PAI-1) (17). The central role of the plasminogen activator/plasmin cascade in fibrinolysis has been well characterized. However, recent studies (17,18) suggest more broad pathophysiologic roles of this cascade in various processes such as embryonic development, ovulation, cell migration, wound heeling, angiogenesis, and neoplasia. In the renal field, this pathway is attracting considerable attention as a potent modulator of renal fibrosis in relation to its effect on extracellular matrix turnover (19–23). Indeed, plasmin can degrade laminin and fibronectin and can activate latent matrix metalloprotease (MMP) in vitro. However, it is rapidly inhibited and tightly regulated by physiologic inhibitors such as {alpha}2-AP and hence is not normally found in an active form in vivo (14,18,24). Probably because of such unstable characteristics, few researchers have attempted to detect plasmin activity in vivo, despite the considerable interest in it.

In the present study, we attempted to confirm our hypothesis that renal glomerular plasmin activity is upregulated in relation to NAPlr deposition in patients with APSGN. For this purpose, we used an in situ zymography method with a plasmin-sensitive synthetic substrate that we found to be a simple, stable, and highly sensitive procedure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Renal tissues that were collected from 17 patients with APSGN were used in this study. Patient characteristics are given in Table 1. All patients showed overt symptoms of APSGN, such as facial edema, hypertension, and hematuria. Percutaneous needle biopsies were performed for diagnostic purposes over a period of 24 yr (1979 to 2003) at the National Defense Medical College (Saitama, Japan) to rule out progressive renal disease with acute nephritic syndrome (e.g., IgA nephropathy, lupus nephritis, RPGN). Informed consent was obtained from each patient. A diagnosis of APSGN was made according to serologic and bacteriologic evidence of acute streptococcal infection before the onset of nephritis as well as from characteristic histologic features of the renal tissue under light microscopy, immunofluorescence (IF), and electron microscopy. Eight patients with RPGN (defined as the presence of crescents in >60% of glomeruli) and 10 normal kidneys that had been removed for localized tumors served as disease controls and normal controls, respectively.


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  		Table 1. Characteristics of APSGN patientsa

 
In Vitro Zymography for Plasmin Activity
To confirm the suitability of a zymographic assay for evaluation of plasmin activity, we conducted preliminary in vitro experiments. Ten microliters of plasmin (Sigma Chemical Co., St. Louis, MO) at increasing concentrations (0, 1, 2, 5, and 10 x 10–2 µg/µl) in 0.067 M sodium phosphate buffer (PB; pH 7.1) were incubated in 96-well microtiter plates with 100 µl of reaction mixture that contained 0.1% Fast Violet B and 0.5 mM p-toluenesulfonyl-l-lysine {alpha}-naphthyl ester (Tos-Lys-NE) in 0.067 M PB for 10 min at 27°C. Tos-Lys-NE was obtained from Torii Pharmaceutical Co., Ltd. (Tokyo, Japan). Reaction color as a result of cleavage of Tos-Lys-NE by plasmin was evaluated by absorbance at 500 nm with a microplate reader after addition of 0.1% SDS to dissolve the precipitated reaction product. A control assay in which the substrate was omitted was performed. In addition, the effects of {alpha}2-AP (5 µg/ml; Merck, Darmstadt, Germany) and aprotinin (0.1 U/µl) as plasmin inhibitors (16,20), EDTA as an MMP inhibitor (25), and E-64 as a cysteine protease inhibitor were examined by running the same reaction with the addition of these inhibitors. To determine the sensitivity of Tos-Lys-NE to plasminogen activators, we performed similar in vitro zymographic assays in which plasmin was replaced by either tPA or uPA (Merck) at comparable concentrations. All chemicals, unless otherwise stated, were purchased from WAKO Pure Chemical Industries (Osaka, Japan).

In Situ Zymography for Plasmin-Like Activity
Plasmin-like activity in cryostat sections (4 µm) of renal tissues was assessed by in situ zymography according to the method of Takuma et al. (26) with a few modifications. Briefly, after being washed with PBS, the sections were incubated for 30 min at 27°C with the same reaction mixture used for the in vitro assay. The sections were then counterstained with methylgreen. The specificity of the reaction was investigated by including the same protease inhibitors used for the in vitro assay (aprotinin, {alpha}2-AP, EDTA, or E-64) in the reaction mixture. To rule out the possibility that plasminogen activators contribute to the reaction by generating plasmin in situ during the incubation period, we also investigated the effect of a tPA inhibitor (rabbit anti-human tPA inhibiting antibody; ICN, Irvine, CA) and a uPA inhibitor (1 mM amiloride; Sigma Chemical Co.) on the in situ zymographic reaction by including these inhibitors in the reaction mixture.

Light microscopic images of all glomeruli in each renal section were acquired with a digital camera. Measurement of the positive area relative to the total area of each glomerulus was calculated with LuminaVision Ver. 2.04 image analysis software (Mitani Corp., Fukui, Japan), and the average numbers were regarded as the relative glomerular plasmin-like activity for each patient. The number of glomeruli analyzed for each APSGN patient ranged from 2 to 11 (4.1 ± 2.7), and the number of glomeruli analyzed for each RPGN patient ranged from 3 to 17 (8.0 ± 5.5). In normal control kidneys, 10 glomeruli were selected randomly and were analyzed similarly.

IF Detection of NAPlr
Preparation of FITC-conjugated rabbit anti-NAPlr antibody and direct IF staining were performed as reported previously (12). Briefly, serial sections were washed with PBS and incubated with anti-NAPlr antibody for 30 min at 27°C. After being washed with PBS, the sections were mounted and observed by fluorescence microscopy. To evaluate the relation between NAPlr deposition and plasmin activity, we assigned APSGN patients to either of two groups, depending on the glomerular positivity for NAPlr: NAPlr positive (14 patients) and NAPlr negative/weak (3 patients).

Double Staining for Plasmin-Like Activity and NAPlr IF or Plasmin(ogen) IF
To verify the co-localization of plasmin-like activity and NAPlr deposition, we performed the zymographic assay and NAPlr IF staining sequentially on the same sections obtained from several NAPlr-positive APSGN patients. To clarify the relation between plasmin(ogen) deposition and plasmin-like activity, we also performed double staining for plasmin-like activity and plasmin(ogen) IF on sections from several NAPlr-positive APSGN patients. After incubation with the reaction mixture for the plasmin assay, sections were washed with PBS without counterstaining and then stained for either NAPlr or plasmin(ogen) with an FITC-conjugated rabbit anti-NAPlr antibody or a rabbit anti-human plasmin(ogen) antibody (Nordic Immunological Laboratories, Tilburg, Netherlands) that was prelabeled with fluorescein. Labeling of the anti-plasmin(ogen) antibody with fluorescein was performed with the EZ-Label Fluorescein Protein Labeling Kit (Pierce Biotechnology, Rockford, IL) by following the manufacturer’s instructions. Sections were incubated with the antibodies for 30 min at 27°C. Sections were then washed with PBS, mounted, and observed by light microscopy for plasmin-like activity and by fluorescence microscopy for either NAPlr or plasmin(ogen) staining.

Double Staining for Fibrin(ogen) IF and NAPlr IF or Plasmin-Like Activity
The relation between fibrin(ogen) deposition and NAPlr deposition or plasmin-like activity was evaluated by double staining. For co-localization of fibrin(ogen) and NAPlr, we labeled the anti-NAPlr antibody with Alexa Fluor 594 (Molecular Probes, Eugene, OR), according to the manufacturer’s instructions, and applied the labeled antibody and FITC-conjugated goat anti-human fibrin(ogen) antibody (ICN) simultaneously to sections from several NAPlr-positive APSGN patients. Double staining for plasmin-like activity and fibrin(ogen) IF was performed in the same manner as the double staining for plasmin-like activity and NAPlr IF.

Statistical Analyses
Data are expressed as the mean ± SD. A t test (parametric assay) or the Mann-Whitney U test (nonparametric assay) was used to evaluate differences between mean values. Differences were considered significant when the two-tailed P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vitro Zymography for Plasmin Activity
In vitro assay confirmed that the Tos-Lys-NE substrate was sensitive for plasmin activity (significant positive linear regression of the assay for plasmin, Y = 0.042 + 0.017 x X, r = 0.998, P < 0.0001) and that the assay system was stable (no reaction was observed in the absence of either substrate or plasmin; Figure 1). Hydrolyzing activity was completely suppressed by the addition of aprotinin or {alpha}2-AP but was not affected by the addition of either EDTA or E-64 (data not shown). Neither tPA nor uPA showed the Tos-Lys-NE hydrolyzing activity in this assay system (data not shown).



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  		Figure 1. In vitro plasmin assay with chromogenic substrate. Incubation of serial dilutions of plasmin with reaction mixture that contained p-toluenesulfonyl-l-lysine {alpha}-naphthyl ester (Tos-Lys-NE) resulted in a positive linear regression, confirming the sensitivity of the substrate for plasmin. Activity was completely suppressed by the addition of aprotinin or {alpha}2-antiplasmin ({alpha}2-AP).

 
In Situ Zymography for Plasmin-Like Activity and IF Detection of NAPlr
Weak or no plasmin-like activity was found in the glomeruli of normal control subjects. However, occasional tubulointerstitial activity was identified (Figure 2A). In RPGN patients, occasional glomerular plasmin-like activity was identified, predominantly in crescents, but in general, glomerular activity was weak or absent. However, strong tubulointerstitial activity was present in tubular epithelial cells and in interstitial infiltrating cells (Figure 2B). In NAPlr-positive APSGN patients, prominent activity was identified in glomeruli (Figure 2C). Activity was localized predominantly in the mesangium and infiltrating leukocytes and partially along the glomerular basement membrane (GBM); the distribution was almost identical to that of NAPlr deposition as assessed by IF staining of serial sections (data not shown). The activity was completely abolished by the addition of aprotinin (Figure 2D), whereas addition of {alpha}2-AP (Figure 2E), EDTA, E-64, or inhibitors of plasminogen activators (anti-tPA antibody, amiloride) did not affect the reaction color (data not shown). Glomerular plasmin-like activity was weak or absent in APSGN patients who were negative for glomerular NAPlr (Figure 2F). Relative glomerular plasmin-like activity as assessed by the measurement of positive glomerular areas was significantly greater in NAPlr-positive APSGN (9.9 ± 3.4%) than in NAPlr-negative APSGN (0.84 ± 0.95%; P < 0.01) or in RPGN (0.57 ± 0.33%; P < 0.0001) or in normal control tissues (0.30 ± 0.19%; P < 0.0001) (Figure 3).



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  		Figure 2. Representative photomicrographs of in situ zymography for plasmin-like activity. Weak or no activity was found in glomeruli from normal kidneys (A) or from patients with rapidly progressive glomerulonephritis (RPGN), although strong tubulointerstitial positivity was present in patients with RPGN (B). Prominent glomerular plasmin-like activity was identified in a nephritis-associated plasmin receptor (NAPlr)-positive patient who had acute poststreptococcal glomerulonephritis (APSGN; 18 d after onset; C). Addition of aprotinin completely inhibited this activity (D; serial section to C), whereas addition of {alpha}2-AP did not inhibit the activity (E). Glomerular activity was weak or absent in a NAPlr-negative APSGN patient (26 d after onset) (F). Magnification, x200.

 


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  		Figure 3. Relative glomerular plasmin-like activity as assessed by measurement of positive glomerular areas by in situ zymography. The activity was significantly greater in NAPlr-positive APSGN than in NAPlr-negative APSGN (P < 0.01) or in RPGN (P < 0.0001) or in normal kidneys (P < 0.0001).

 
Double Staining for Plasmin-Like Activity and NAPlr IF or Plasmin(ogen) IF
The similar distribution of glomerular plasmin-like activity and NAPlr deposition in serial sections was examined further by double staining in the same sections. The merged image (Figure 4C) clearly showed that the distributions of the activity (Figure 4A) and NAPlr deposition (Figure 4B) were almost identical, although zymography tended to detect a slightly larger area than did NAPlr IF staining. This slight difference in distribution may be due to a difference in the sensitivities of these methods.



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  		Figure 4. Representative light and fluorescence photomicrographs of double staining for plasmin-like activity (A and D; in situ zymography) and NAPlr (B; immunofluorescence [IF] staining) or plasmin(ogen) (E; IF staining) from a patient with APSGN (15 d after onset). The same fields were observed under light microscopy (A and D), fluorescence microscopy (B and E), and merged (C and F). Merged images clearly show the identical distribution of glomerular plasmin-like activity and NAPlr deposition (C) and that the activity is localized to subsets of plasmin(ogen)-positive sites (F). Magnification, x200.

 
A plasmin-like activity and plasmin(ogen) IF co-localization assay was performed to confirm that the activity detected by in situ zymography truly represented that of plasmin(ogen). The glomerular staining pattern of in situ zymography (Figure 4D) differed from that of plasmin(ogen) IF (Figure 4E), but the overall distribution was similar; the staining pattern of plasmin(ogen) was generally diffuse, whereas that of in situ zymography was punctate and relatively restricted. The merged image showed that glomerular plasmin-like activity was localized within plasmin(ogen)-positive sites (Figure 4F). This result is reasonable because the anti-plasmin(ogen) antibody should detect both active and inactive plasmin as well as plasminogen, whereas zymography should detect only active plasmin.

Double Staining for Fibrin(ogen) IF and NAPlr IF or Plasmin-Like Activity
Merged images (Figure 5, C and F) showed that the distributions of fibrin(ogen) deposition (Figure 5, B and E) and of NAPlr deposition (Figure 5A) or plasmin-like activity (Figure 5D) were generally different. Positive areas for NAPlr deposition or plasmin-like activity seemed to be free of fibrin(ogen) deposition.



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  		Figure 5. Representative photomicrographs of double staining for NAPlr (A; IF staining, Alexa Fluor 594) or plasmin-like activity (D; in situ zymography) and fibrin(ogen) (B and E; IF staining, FITC) from a patient with APSGN (20 d after onset). The same fields were observed under fluorescence microscopy with filters for different colors (A, red; B, green) and merged (C) or under light microscopy (D), fluorescence microscopy (E), and merged (F). Merged images show the different distribution of NAPlr deposition or plasmin-like activity with fibrin(ogen)-positive sites. Magnification, x264.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous finding that the nephritogenic antigen for APSGN is the same entity as Plr of GAS led us to speculate that the antigen might be able to maintain the proteolytic activity of plasmin in the same way as shown in vitro and contribute to the development of APSGN in vivo (11). Poon-King et al. (27) reported the in vitro plasmin(ogen)-binding ability of nephritis plasmin binding protein, which is identical to SPEB, another potent nephritogenic antigen of GAS, and suggested a similar mechanism for the development of APSGN. However, to the best of our knowledge, there have been no reports showing upregulated glomerular plasmin activity in APSGN or in any type of human glomerulonephritis in situ.

Here, we showed prominent intraglomerular plasmin-like activity in NAPlr-positive APSGN patients by in situ zymography with a plasmin-sensitive synthetic substrate. We selected Tos-Lys-NE as the substrate for this assay because the C-terminal end of lysine is most vulnerable to plasmin activity (24), and this substrate is applicable to histologic analysis (26). D-Val-Leu-Lys-p-nitroanilide (25) and Tos-Gly-Pro-Lys-p-nitroanilide (19,21) are also commercially available synthetic peptides for plasmin assay, but these peptides are not applicable to histologic analysis. Because of the extremely high sensitivity of naphthyl ester, the histologic localization obtained with the present method was sharp and hence could be used for serial section assays or for double staining. However, the definitive identification of the proteolytic activity detected by this method as plasmin in vivo is difficult. To the best of our knowledge, no substrate is completely specific for plasmin. Tos-Lys-NE, D-Val-Leu-Lys-p-nitroanilide (25), and Tos-Gly-Pro-Lys-p-nitroanilide (19,21) have high affinity for and are sensitive to plasmin, but they are also sensitive to other proteases. We attempted to specify the enzyme with the use of several inhibitors. The addition of EDTA, E-64, tPA antibody, or amiloride did not suppress the activity; thus, we could tell that the protease was not an MMP, a cysteine protease, tPA, or uPA but could not exactly specify that it was plasmin. E-64 was used to assess the contribution of cysteine proteases because SPEB, another potent nephritogenic antigen, is a cysteine protease that is deposited in the glomeruli of APSGN patients (9). Our results indicate that cysteine proteases did not contribute to the activity assayed. Aprotinin is a widely used plasmin inhibitor and did suppress the activity in vivo, but it potentially inhibits other proteases such as trypsin or chymotrypsin. {alpha}2-AP inhibited plasmin activity in vitro (Figure 1), but it did not inhibit the glomerular plasmin-like activity in vivo in the present study (Figure 2E). However, this result was in keeping with our concept that deposited NAPlr can trap and maintain plasmin activity in vivo, because receptor-bound plasmin should be protected from inactivation by {alpha}2-AP, according to the results of Boyle et al. (16). {epsilon}-Aminocaproic acid and tranexamic acid are lysine analogs that are also popularly used as plasmin inhibitors. However, neither sufficiently inhibited plasmin activity in vitro in our assay system (data not shown), probably because of the extremely high affinity of the substrate for plasmin that we used. Thus, we did not identify a specific inhibitor of in vivo activity. However, the identical distribution of proteolytic activity with the plasmin receptor (NAPlr); the relation of the proteolytic activity with plasmin(ogen) deposition; complete inhibition of the activity by aprotinin; and exclusion of MMP, cysteine proteases, tPA, and uPA strongly suggest that it represents plasmin. Nonetheless, we used the term "plasmin-like" instead of "plasmin."

We selected RPGN as a disease control because it is one of the few types of human glomerulonephritis with prominent glomerular infiltration of neutrophils and macrophages, similar to APSGN (28,29). Glomerular infiltrating cells are suspected to affect the results of the zymographic assay because these cells are known to secrete uPA and to express uPA receptor (17,30). However, glomerular plasmin-like activity was minimal in RPGN patients. This may be due to the instability of plasmin or to the upregulated expression of PAI-1 in the glomeruli of RPGN patients, as reported previously (31–33).

The general difference in distribution between fibrin(ogen) deposition and NAPlr deposition or plasmin-like activity is not surprising, because plasmin, which is suspected to be bound by and co-localized with NAPlr, should have the ability to degrade fibrin(ogen), thereby decreasing the fibrin(ogen) deposition. Fibrin deposition has been shown to be an important mediator of glomerular injury in progressive renal diseases, particularly crescentic glomerulonephritis (34). In this sense, the fibrin(ogen)olysis in situ shown in this study may indicate the contribution of plasmin also to the resolution of APSGN, although further investigation will be required to confirm this.

APSGN is believed to be mediated by an immune complex that may be formed either in situ or in the circulation (1). NAPlr is a potential candidate for the nephritogenic antigen because it is highly antigenic (12). However, this study clearly showed that NAPlr not only acts as a component of the immune complex but also has a direct, nonimmunologic function as a plasmin receptor and contributes to pathogenesis by maintaining proteolytic activity in situ. This is somewhat consistent with previous clinical findings that proteinuria and microscopic hematuria are occasionally found in the dormant phase of the disease, when antibody against the nephritogenic antigen has not yet developed. Patients with streptococcal infection and the above manifestations have a higher incidence of APSGN (35). We previously suggested a mechanism for glomerular damage by the nephritogenic antigen through direct activation of the complement system in situ (7). However, in our recent study, we found different distributions of C3 and NAPlr deposition in the glomeruli of APSGN patients (11), in clear contrast to the similarity of plasmin-like activity and NAPlr deposition in the present study. We suspect that direct complement activation by NAPlr is mediated predominantly in the circulation rather than in situ.

From these findings, we propose the following APSGN induction mechanism. Infection of the throat or skin with GAS induces the release of cytoplasmic NAPlr into the circulation. Circulating NAPlr binds to the renal glomeruli on the mesangial matrix and GBM, probably through its adhesive character (12). Bound NAPlr then traps plasmin and maintains its activity, which may induce glomerular damage in situ by degrading the GBM by itself or by activating pro-MMP. Plasmin activity may also mediate inflammation by activating and accumulating monocytes and neutrophils in situ (36,37). Such glomerular damage may induce urine abnormalities during the latent period of the disease. Finally, the developed antibody forms immune complexes that can readily pass through the altered GBM and thereafter accumulate in the subepithelial space as humps. This final step of immune complex deposition, accompanied by the activation of complement and immune cell accumulation, leads to the full-blown and overt disease state.

In summary, we identified upregulated glomerular plasmin-like activity in relation to NAPlr deposition in human APSGN. Instead of immunohistochemical detection, we used an in situ zymography method and identified functional proteolytic activity within glomeruli in situ. Our results suggest the important role of plasmin in the development of APSGN and strongly support the idea that NAPlr isolated from GAS is a nephritogenic agent for APSGN.


   Acknowledgments
 
We are grateful to our colleagues Satoko Kiyono and Mami Morisugi for expert secretarial assistance and to Tatsuyo Harasawa, Central Research Laboratory, National Defense Medical College, for excellent technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 30, 2004. Accepted for publication September 28, 2004.




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