**2. NAPlr and Plasmin Activity in Glomeruli as Biomarkers of PSAGN**

NAPlr is a 43-kDa protein that was originally isolated from the cytoplasmic fraction of group A streptococcus as a candidate nephritogenic protein for PSAGN [8]. Glomerular NAPlr deposition is detected by immunostaining, and is frequently observed in the early phase of PSAGN; all patients within 2 weeks of disease onset have been reported to show NAPlr deposition [2].

NAPlr was also found to be the same molecule as streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [8]. Although GAPDH is a well-known housekeeping gene, it also has pleiotropic functions, such as energy production (glycolysis), regulation of gene expression, and autophagy [10]. In addition, GAPDH from some bacteria, including streptococci, has been shown to have plasmin-binding activity [11,12].

NAPlr binds plasmin and maintains plasmin activity by protecting it from its physiological inhibitors. Plasmin activity can be detected by *in situ* zymography using a plasmin-sensitive synthetic substrate, which is resistant to the addition of α2-antiplasmin but is completely abrogated by aprotinin, a serine protease inhibitor [9]. Plasmin is considered to cause glomerular damage directly by degrading extracellular matrix proteins and indirectly by activating pro–matrix metalloproteases. Additionally, plasmin can exert proinflammatory function by activating and accumulating inflammatory cells.

NAPlr is also known to convert complement component C3 to C3b, indicating its involvement in the activation of the alternative complement pathway [8]. However, it should be noted that NAPlr deposition is observed mainly in glomerular neutrophils, mesangial cells, and endothelial cells, and its distribution in glomeruli is different from that of C3 and IgG, which are considered to localize within the subepithelial hump [13]. In this regard, NAPlr, which also contains a urokinase-type plasminogen activator receptor (uPAR)-binding site [11], may bind with uPAR expressed on neutrophils, thereby inducing prominent endocapillary inflammation in early phase PSAGN, or NAPlr may be phagocytosed by neutrophils as exogenous material. Thus, glomerular damage from the disease may initially occur in the inner side of the glomerular capillary walls by NAPlr deposition, rather than subepithelial immune complexes.

Streptococcal pyrogenic exotoxin B (SPEB), which is another potential nephritogenic protein of PSAGN with cationic character, has been considered to pass through the glomerular basement membrane and be deposited in the subepithelial area [14]. However, a subsequent study showed that the glomerular distribution of NAPlr and SPEB were essentially similar and that NAPlr staining was dominant [13]. Importantly, as with NAPlr, SPEB has plasmin-binding activity [5]. Thus, it is possible that these 2 (or more) proteins are cooperatively involved in the disease pathogenesis of PSAGN. Figure 1 shows a scheme of the mechanisms involved in the development of PSAGN.

**Figure 1.** Putative mechanism for the development of poststreptococcal acute glomerulonephritis. Streptococcal infection induces the release of nephritogenic proteins, such as nephritis-associated plasmin receptor (NAPlr), into the circulation. Circulating NAPlr accumulates on the inner side of the glomerular capillary walls, and then traps and maintains the activity of plasmin, which induces glomerular damage by the degradation of extracellular matrix proteins or by activating and accumulating inflammatory cells. Thereafter, immune complexes, formed either *in situ* or in the circulation, pass through the altered glomerular basement membrane (GBM). Accumulation of immune complexes, complements, and plasma proteins forms "humps" on the outer side of the glomerular capillary walls. This scheme is based on the figure from Oda et al. [2]. Ab: antibody; C: complement; Endo: endothelial cell; Mes: mesangial cell; MΦ: macrophage; PMN: polymorphonuclear cell.

Some streptococcal strains have been isolated from PSAGN patients, and such strains have been considered to be "nephritogenic". However, NAPlr, as well as SPEB, have been found in virtually all streptococcal strains [14]. In addition, gene sequences of NAPlr were highly conserved and its protein expression levels were similar between various streptococcal strains [15]. Therefore, it is reasonable to think that any strains expressing NAPlr can be nephritogenic. Another possibility also remains that although NAPlr (and SPEB) are essential molecules, other factors, from both bacteria and the hosts, play important roles in the onset/progression of PSAGN.

There have been several reports showing the occurrence of IRGN in renal transplant recipients, suggesting that IRGN may be a cause of renal allograft injury [16,17]. We have recently encountered a renal transplant recipient who developed PSAGN; notably, NAPlr deposition and plasmin activity were observed in the glomeruli of the patient's transplanted kidney (manuscript in preparation).
