**4. Glomerular NAPlr Deposition and Plasmin Activity as Candidates of General Biomarkers of Bacterial IRGN**

The diagnosis of IRGN is made based on a combination of clinical and pathological findings. Nasr et al. [5] proposed the diagnostic criteria for IRGN as follows, in which at least 3 of the 5 items are required for a positive diagnosis: (1) clinical or laboratory evidence of infection preceding or at the onset of GN, (2) decrease in serum complement levels, (3) endocapillary proliferative and exudative glomerulonephritis, (4) C3-dominant or codominant glomerular immunofluorescence staining, and (5) hump-shaped subepithelial deposits on electron microscopy. The most common pathogen causing bacterial IRGN is staphylococcus, followed by streptococcus and Gram-negative bacteria. Infection sites are diverse, including the upper respiratory tract, skin, lung, and urinary tract, and the identification of foci is sometimes difficult. In addition, disease-specific diagnostic biomarkers have not been identified to date, and there are usually several differential diagnoses that show confounding clinical or pathological characteristics. Therefore, the prompt and accurate diagnosis of IRGN is often difficult.

Interestingly, glomerular NAPlr deposition and plasmin activity have recently been demonstrated in patients with IRGN induced by some bacterial strains, such as *Streptococcus pneumoniae* [28], *Aggregatibacter actinomycetemcomitans* (a Gram-negative coccobacillus that sometimes causes periodontal disease and infectious endocarditis) [29], *Mycoplasma pneumoniae* [30], or *S. aureus* (both methicillin-sensitive and -resistant strains; unpublished observations). The sequences of *S. pneumoniae* GAPDH share high identity with NAPlr, and the C-terminal sequences of *S. pneumoniae* GAPDH, which are most likely to be associated with the plasmin-binding activity, are completely identical to those of streptococcal GAPDH [28]. *M. pneumoniae* GAPDH has been shown to not only have cross-immunoreactivity to the anti-NAPlr antibody but also to have a plasmin-binding function. In addition, *M. pneumoniae* GAPDH has been reported to bind to plasminogen and convert it to plasmin [31]. As shown in Table 1, sequences of GAPDH from *A. actinomycetemcomitans* and *S. aureus* also show high similarity to that of NAPlr at the amino acid level, and *S. aureus* GAPDH has been reported to bind to enzymatically active plasmin [32]. As stated above, GAPDH sequences are highly preserved between streptococcal species [15], and it is, therefore, reasonable that GAPDH from these bacteria have plasmin-binding ability and that its deposition in glomeruli is detected by the anti-NAPlr antibody. However, it should be noted that although the glomerular staining pattern of NAPlr is essentially the same among patients with IRGN, the staining intensity in patients with non-streptococcal IRGN is generally weaker than in those with PSAGN. In this regard, the timing of

when a renal biopsy is performed might affect the staining intensity; it could be possible that, in patients with non-streptococcal IRGN, performing renal biopsy during the acute phase is often avoided because of comorbidities or ongoing infection. Another possibility is that positive immunostaining of NAPlr in patients with non-streptococcal IRGN is caused by cross-immunoreactivity to the anti-NAPlr antibody, and is, therefore, weaker than that in patients with PSAGN.

**Table 1.** Identity and similarity of bacterial GAPDH and streptococcal GAPDH (nephritis-associated plasmin receptor; NAPlr).


Data are presented as percentages. Nucleotide and amino acid sequences of bacterial GAPDH registered with Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/kegg\_ja.html) were used, and identities and similarities were evaluated by Dr. Masayuki Fujino (the AIDS Research Center, National Institute of Infectious Diseases) using genetic information processing software (GENETYX-MAC ver. 18, GENETYX Corporation, Tokyo, Japan).

On the other hand, even if the overall amino acid sequence similarity is not very high, it is possible that some types of bacterial GAPDH, which have a similar steric structure at the antibody-binding site, show cross-immunoreactivity to the anti-NAPlr antibody. Indeed, positive staining for NAPlr and plasmin activity has been used as a marker of IGRN in patients with various forms of glomerulonephritis, such as proliferative glomerulonephritis with monoclonal immunoglobulin G deposits [33] and eosinophilic proliferative glomerulonephritis [34], even if neither the pathogens nor the infection sites could be identified. As the binding site of the anti-NAPlr antibody and that of plasmin in the GAPDH sequence would be different, the GAPDH of some bacteria are expected to bind with plasmin but not react with the anti-NAPlr antibody. In this regard, whether there are IRGN cases in which glomerular plasmin activity is observed without immunoreactivity to the anti-NAPlr antibody needs to be investigated in the future.

Collectively, GAPDH from various bacteria appear to react with the anti-NAPlr antibody and to have plasmin-binding ability, and positivity of the anti-NAPlr antibody and plasmin activity in glomeruli may act as both diagnostic and pathogenetic biomarkers of bacterial IRGN in general. Putative pathogenic mechanisms of IRGN, focusing on NAPlr and plasmin activity as biomarkers, are depicted in Figure 2.

**Figure 2.** Possible scheme for the pathogenic mechanisms of bacterial infection-related glomerulonephritis (IRGN), focusing on the glomerular deposition of nephritis-associated plasmin receptor (NAPlr) and related plasmin activity. Ab: antibody; ANCA: antineutrophil cytoplasmic antibody; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MPGN: membranoproliferative glomerulonephritis; PSAGN: poststreptococcal acute glomerulonephritis.
