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Commun Biol
Commun Biol
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10.1038/s42003-024-06826-x
Article
Contribution of collagen-binding protein Cnm of Streptococcus mutans to induced IgA nephropathy-like nephritis in rats
Naka Shuhei 1
Matsuoka Daiki 1
Misaki Taro 23
Nagasawa Yasuyuki 4
Ito Seigo 5
Nomura Ryota 6
http://orcid.org/0000-0002-2209-2685
Nakano Kazuhiko 7
http://orcid.org/0009-0007-8650-0477
Matsumoto-Nakano Michiyo mnakano@okayama-u.ac.jp

1
1 https://ror.org/02pc6pc55 grid.261356.5 0000 0001 1302 4472 Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama Japan
2 https://ror.org/036pfyf12 grid.415466.4 0000 0004 0377 8408 Division of Nephrology, Seirei Hamamatsu General Hospital, Hamamatsu, Shizuoka Japan
3 https://ror.org/02cd6sx47 grid.443623.4 0000 0004 0373 7825 Department of Nursing, Faculty of Nursing, Seirei Christopher University, Hamamatsu, Shizuoka Japan
4 https://ror.org/001yc7927 grid.272264.7 0000 0000 9142 153X Department of General Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo Japan
5 Department of Internal Medicine, Japan Self-Defense Force Iruma Hospital, Saitama, Japan
6 https://ror.org/03t78wx29 grid.257022.0 0000 0000 8711 3200 Department of Pediatric Dentistry, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
7 https://ror.org/035t8zc32 grid.136593.b 0000 0004 0373 3971 Department of Pediatric Dentistry, Graduate School of Dentistry, The University of Osaka, Suita, Osaka Japan
14 9 2024
14 9 2024
2024
7 11417 11 2023
3 9 2024
© The Author(s) 2024
2024
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IgA nephropathy (IgAN), the most common primary glomerulonephritis, is considered an intractable disease with unknown pathogenic factors. In our previous study, Streptococcus mutans, the major causative bacteria of dental caries, which expresses Cnm, was related to the induction of IgAN-like nephritis. In the present study, the Cnm-positive S. mutans parental strain, a Cnm-defective isogenic mutant strain, its complementation strain, and recombinant Cnm (rCnm) protein were administered intravenously to Sprague Dawley rats, and the condition of their kidneys was evaluated focusing on the pathogenicity of Cnm. Rats treated with parental and complement bacterial strains and rCnm protein developed IgAN-like nephritis with mesangial proliferation and IgA and C3 mesangial deposition. Scanning immunoelectron microscopy revealed that rCnm was present in the electron-dense deposition area of the mesangial region in the rCnm protein group. These results demonstrated that the Cnm protein itself is an important factor in the induction of IgAN in rats.

Collagen-binding protein (Cnm), a surface protein possessed by Streptococcus mutans, is a possible pathogenic factor in IgA nephropathy development, as IgA nephropathy-like nephritis was induced in rats following Cnm administration.

Subject terms

Clinical microbiology
Cellular microbiology
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pmcIntroduction

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is considered an intractable disease with various pathogenetic factors1–3. Over approximately 20 years from the onset of disease, 30–40% of patients develop terminal renal failure1–4. Common clinical findings include proteinuria and hematuria, but a renal biopsy is considered essential to confirm a diagnosis5–7. The diagnostic hallmark of IgAN is the predominance of IgA deposits, either alone or with IgG, IgM, or both, in the glomerular mesangial regions8. More than 90% of IgAN patients have complement C3 deposition in their glomeruli9. Most C3 is present in the mesangial and paramesangial regions including some vascular endothelial cells2,10. Typical histopathological findings in patients with IgAN include increased numbers of mesangial cells and matrix in the mesangial region as well as the deposition of immune complexes11. Patients with IgAN sometimes present with macroscopic hematuria when they have an upper respiratory tract infection, such as tonsillitis12. Several bacterial species have been reported to be potential factors involved in the pathogenesis of IgAN13–17, including dental caries-related bacteria18–25 and periodontitis-related bacteria26–28.

Streptococcus mutans, the major causative bacteria of dental caries, is a Gram-positive, facultative anaerobic bacteria29, which can induce infective endocarditis by invading the bloodstream during invasive dental procedures, such as tooth extractions30. A collagen-binding protein (Cnm) of approximately 120 kDa is expressed on the surface of some S. mutans strains31 and is involved in adhesion to and invasion of vascular endothelial cells, indicating it might be an important factor that causes infective endocarditis32–34. It was reported that S. mutans expressing Cnm was detected more frequently in patients with IgAN than in healthy subjects18. Indeed, the intravenous administration of S. mutans expressing Cnm isolated from the oral cavity of IgAN patients induced IgAN-like nephritis in rats21. In addition, when Cnm-positive S. mutans was inoculated into the oral cavity of a rat dental caries model, IgAN-like lesions developed with severe dental caries extending to the pulp space, which can cause bacteremia22.

These results led us to consider that Cnm protein on the surface of S. mutans, but not S. mutans itself, might be an important virulence factor involved in S. mutans-related IgAN. In the present study, we focused on the ability of Cnm on S. mutans to cause IgAN-like nephritis in rats using Cnm-deficient mutant strains and recombinant Cnm (rCnm) protein.

Results

Systemic condition of rats

We evaluated proteinuria, hematuria, and renal function, which are the clinical characteristics of IgAN, in rats. Animal experiments were performed using S. mutans strain SN74 (serotype e, Cnm-positive) isolated from the oral cavity of a patient with severe IgAN, SN74CND strain (CND), a Cnm deficient mutant of the SN74 strain, SN74CNDcomp strain (Comp), a Cnm complement SN74CND strain, and rCnm protein. Phosphate-buffered saline (PBS) was used as a control. Serum albumin (ALB), blood urea nitrogen (BUN), and creatinine (CRE) levels in all groups at the time of sacrifice were not significantly different (Table 1). Furthermore, there were no significant differences in serum IgA concentrations among the PBS, SN74, and rCnm groups (Supplementary Table 1). On the other hand, serum IgG concentrations were significantly higher in the rCnm group compared to the PBS group (P < 0.0167) (Supplementary Table 1). There was no significant difference in the urine protein/creatinine ratio in each group (Fig. 1a). Furthermore, hematuria was not observed in all rats in the PBS group and in 1 of 14 rats in the CND group. In contrast, 7 of 19 rats in the SN74 group, 3 of 20 rats in the Comp group, and 4 of 18 rats in the rCnm protein group developed hematuria. The hematuria positivity rate was significantly higher in the SN74 groups than in the PBS and CND groups (P < 0.01, P < 0.05) (Fig. 1b).Table 1 Serum levels of kidney markers in rats inoculated with PBS, SN74, SN74CND, SN74CNDcomp, or rCnm

Groups	Serum levels (mean ± SEM)	
ALB (g/dL)	BUN (mg/dL)	CRE (mg/gL)	
PBS (n = 14)	3.80 ± 0.06	19.29 ± 0.54	0.31 ± 0.01	
SN74 (n = 19)	3.97 ± 0.03	20.72 ± 0.76	0.33 ± 0.01	
SN74CND (n = 20)	3.96 ± 0.04	19.65 ± 0.68	0.31 ± 0.01	
SN74CNDcomp (n = 20)	3.80 ± 0.04	17.07 ± 0.61	0.30 ± 0.01	
rCnm (n = 18)	3.80 ± 0.05	20.80 ± 0.49	0.31 ± 0.01	
Statistical significance was determined by analysis of variance with Bonferroni’s correction. There were no significant differences in levels between the groups.

ALB albumin, BUN blood urea nitrogen, CRE creatinine, SEM standard error of the mean.

Fig. 1 Analysis of urine components in rats.

a Urinary protein ratio (urinary protein/urinary creatinine). b Hematuria. Each column represents the mean ± standard errors of the means of the PBS group (n = 14), SN74 group (n = 19), SN74CND group (n = 20), SN74CNDcomp group (n = 20), and rCnm group (n = 18). a Statistical significance was determined by analysis of variance with Bonferroni’s correction. b Statistical significance was determined using Fisher’s exact test. *P < 0.05, **P < 0.01.

These results suggested no change in renal function or proteinuria in any of the experimental groups. However, hematuria was present in the Cnm-positive strain group, Comp group, and rCnm protein group, but not the CND group. These results suggest that Cnm protein or S. mutans with Cnm protein induced hematuria in the early stages of IgAÑ.

Histopathological analyses of kidney tissues

We evaluated mesangial cell and matrix proliferation, which are important features of IgAN. Histopathological analyses of period acid-Schiff (PAS)-stained sections revealed the prominent proliferation of mesangial cells and mesangial matrix in rats in the SN74, Comp, and rCnm protein groups (Fig. 2a). Mesangial proliferation scores were significantly higher in the SN74 and Comp groups than in the PBS and CND groups, and the score of the rCnm protein group was significantly higher than that of the CND group (P < 0.01) (Fig. 2b). These results suggest that the presence of Cnm protein induces mesangial cell and matrix proliferation. The mesangial proliferation score of the hematuria-positive rat group was higher than of the hematuria-negative rat group (Supplementary Fig. 1a). Furthermore, no obvious atrophy was seen in the tubules, though fibrosis was observed at the inner brush border of the tubules in Masson’s trichrome (MT) stained images of rats in the SN74, Comp, and rCnm groups (Supplementary Fig. 2). In addition, α-SMA expression was observed in the immunofluorescent (IF) staining at the same area (Supplementary Fig. 3).Fig. 2 Histopathological appearance of kidney tissues following PAS staining.

a Representative image of PAS staining. Scale bars, 50 μm (all panels). b Mesangial proliferation scores. Each column represents the mean ± standard errors of the means of the control group (n = 14), SN74 group (n = 19), SN74CND group (n = 20), SN74CNDcomp group (n = 20), and rCnm group (n = 18). Statistical significance was determined by analysis of variance with Bonferroni’s correction. *P < 0.01.

Immunohistochemical analyses of kidney tissues

We evaluated the deposition of IgA in the mesangial region, the most important and defining finding of IgAN. We also evaluated C3 and IgG which is highly stained in tissues from patients with IgAN. Immunohistochemical analyses using IgA-, C3-, and IgG specific antibodies showed prominent positive reactions in the mesangial regions of rats inoculated with SN74, Comp, or rCnm protein groups (Figs. 3, 4). In contrast, these deposits were not observed in the PBS and SN74CND groups. Immunochemical analysis using IgG-specific antibodies demonstrated IgG deposition in some rats in the rCnm group (Fig. 5). The positive rate of IgA deposition was significantly higher in the SN74, Comp, and rCnm groups than in the PBS and CND groups (Fig. 6a). The positive rate of C3 deposition was significantly higher in the SN74 group than in the PBS and CND groups, and significantly higher in the rCnm protein group than in the PBS group (Fig. 6b). The positive rate of IgG deposition was 5 out of 18 rats in the rCnm protein group, but there was no significant difference between each group (Fig. 6c). The positive rates for IgA and C3 deposition were significantly higher in the SN74 and Comp groups than in the PBS and CND groups, and significantly higher in the rCnm protein group than in the PBS group (Fig. 6d). These results suggest that the presence of Cnm protein induces the deposition of IgA, C3, and IgG in the mesangial region of the renal glomerulus. The positive rate of deposition of IgA, C3, and/or IgG in the hematuria-positive rat group was higher than in the hematuria-negative rat group (Supplementary Fig. 1b).Fig. 3 Representative histopathological appearance of kidney tissues by immunohistochemistry with IgA-specific antibodies.

The first image shows staining with an anti-IgA antibody. The second image shows staining with an anti-CD34 antibody. The third image shows nuclear staining. The fourth row of images shows the left three images superimposed. Scale bars, 50 μm (all panels).

Fig. 4 Representative histopathological appearance of kidney tissues by immunohistochemistry with C3-specific antibodies.

The first image shows staining with an anti-C3 antibody. The second image shows staining with an anti-CD34 antibody. The third image shows nuclear staining. The fourth row of images shows the left three images superimposed. Scale bars, 50 μm (all panels).

Fig. 5 Representative histopathological appearance of kidney tissues by immunohistochemistry with IgG-specific antibodies.

The first image shows staining with an anti-IgG antibody. The second image shows nuclear staining. The third row of images shows the left two images superimposed. Scale bars, 50 μm (all panels).

Fig. 6 Frequencies of positive immunochemical staining.

a Frequencies of positive immunochemical staining with anti-IgA, (b) anti-C3, (c) anti-IgG, and (d) anti-IgA and anti-C3. P-values of less than 0.05 were considered to indicate significant differences. Statistical significance was determined using Fisher’s exact test. *P < 0.05, **P < 0.01, ***P < 0.001.

Immunoelectron microscopy of Cnm protein in renal glomeruli

We evaluated the deposition of electron dense deposits (EDD) in the mesangial region, which are an important feature of IgAN. Furthermore, the presence of Cnm protein in renal glomeruli was evaluated by immunoelectron microscopy using Cnm antibodies. Electron microscopic analysis showed EDD in the rCnm protein groups. Furthermore, immunoelectron microscopy analysis using Cnm antibodies demonstrated colloidal deposition in the EDD region in the rCnm protein groups (Fig. 7). These results suggest that Cnm protein is associated with the initiation of EDD deposition in renal glomeruli.Fig. 7 Identification of rCnm proteins in the mesangial region by immunoelectron microscopy.

a, b Representative images of the kidneys of rats administrated with rCnm. Black arrows indicate electron-dense deposits. White arrows indicate colloidal deposition.

Discussion

Our previous studies examined the onset of IgAN-like renal lesions using two rat animal models21,22. First, Cnm-positive S. mutans strain SN74 was administered intravenously to 4‐week‐old specific pathogen-free Sprague Dawley rats, in which bacteremia induced by invasive dental procedures, such as tooth extractions, was simulated21. Second, the SN74 strain was inoculated into the oral cavities of 2-week-old specific pathogen-free Sprague Dawley rats fed a high-sucrose diet for 32 weeks, which produced severe dental caries extending to the pulp space leading to the intravenous entry of oral bacteria from the lesion22. Both models developed IgAN-like glomerulonephritis, which led us to consider that Cnm protein on the surface of S. mutans, not S. mutans, might be an important factor in the development of IgAN-like glomerulonephritis.

For the present study, it was considered important to determine the number of bacteria to be administered to the IgA model rats. It has been estimated that following invasive dental procedures, approximately 60% of treated patients possess greater than 1 × 104 CFU/mL of bacteria in systemic blood35. When converted to rats, that value is more than 1 × 108 CFU, which was an important factor for determining the amount of bacteria administered in the present study.

In rat jugular vein administration models used in our previous studies, following administration of 1 × 107 CFU of an S. mutans MT8148 strain, bacteria were isolated from blood obtained up to 24 h later36, while bacteria were isolated up to 48 h later after administration of 1 × 109 CFU37,38. In a rat model of infective endocarditis, 1 × 108 CFU of S. mutans TW295 was administered and then the bacteria were isolated from blood obtained up to 7 days later39. Nevertheless, there is a possibility that such bacteria could be isolated after longer periods in these models with a heart valve injury induced beforehand as compared to normal rats. Furthermore, the strains used in those studies are different from S. mutans SN74 used in this study, a PA- and Cnm-positive strain, while MT8148 is PA-positive and Cnm-negative, and TW295 is PA-negative and Cnm-positive.

In our preliminary experiments conducted for a prior study, IgA model rats died when treated with 1 × 109 CFU of S. mutans SN74, whereas no clear findings related to development of IgAN were observed at concentrations below 1 × 107 CFU21. Those results were referred to when deciding the dose of S. mutans SN74 at 1 × 108 CFU for the present experiments.

In addition, though only one intravenous administration was used in this study, it has been reported that 1 mg of dental plaque contains more than 1 × 107 bacteria and the organisms can enter the bloodstream at a high frequency when invasive dental procedures are performed40. Therefore, a single administration was considered adequate to allow the bacteria to enter the bloodstream.

In the present study, the rate of hematuria and IgA and complement C3 deposition in the mesangial regions of the SN74 and Comp groups were significantly higher than in the CND and PBS groups; however, there were no significant differences in proteinuria or the levels of any serum parameter between the groups. These results suggest that rats in the SN74 and Comp groups reached the early stage of IgAN. Furthermore, the PAS proliferation scores the SN74 and Comp groups were significantly higher than those of the PBS and CND groups. These histopathological findings suggest that the typical important features of IgAN were present in the SN74 and Comp groups, but not the CND group. Therefore, the present study demonstrated that the Cnm protein has a strong relationship with the development of IgAN.

The mesangial proliferation score and the positive rate of IgA, C3, and/or IgG deposition in the hematuria-positive group were higher than in the hematuria-negative group. Hematuria was suggested to be associated with mesangial proliferation and deposition of IgA, C3, and/or IgG in glomerular mesangial regions. In clinical practice, hematuria is an important indicator of IgAN, but it has become clear that hematuria may also be an important indicator in a model IgAN-like nephritis.

To clarify whether IgAN was caused by Cnm, we administered rCnm protein intravenously. Surprisingly, when the rCnm protein was administered directly to rats, they developed IgAN-like nephritis based on clinical and histopathological findings. Immunoelectron microscopy images of the rCnm protein groups clearly demonstrated Cnm deposition in the EDD region. These findings indicated that Cnm deposits in the mesangial region were related to the deposition of IgA or C3.

Infection of the mucosal epithelium of the upper respiratory tract of IgAN patients by bacteria or viruses is common. Antigenic proteins from viruses, including herpes simplex virus41, adenovirus42, hepatitis B virus43, cytomegalovirus44, or bacteria, including Escherichia coli strain 0745, Pseudomonas aeruginosa46, Haemophilus parainfluenzae13, MRSA47–49 are associated with IgAN. However, there have been no reports of the protein deposited in the lesions. This is the first report to show the deposition of Cnm protein in a lesion.

It is considered possible that of S. mutans Cnm proteins may be separated from the bacterium surface, then bind to IgA and other immunoglobulins in blood, leading to induction of glycosylated abnormal IgA (Gd-IgA) expression. On the other hand, the present results revealed that Cnm proteins exist in the dens-deposit region of the kidney. Therefore, those proteins may have an auxiliary role in binding of IgA to the mesangial region or are possibly related to formation of immune complexes by IgA attached with IgG and C3. In a future study, we intend to examine binding of Cnm protein to other immunoglobulins, including IgA, to clarify the mechanism of IgAN development related to Cnm.

In summary, the present study clearly demonstrated that the Cnm protein itself is an important factor in the development of the IgAN. Further studies are needed to clarify the novel potential mechanism of the onset of IgAN to the Cnm protein.

Materials and methods

Bacterial strains

S. mutans strain SN74 (serotype e) was isolated from the oral cavity of a patient with severe IgAN21. Strain SN74CND, a Cnm-inactivated isogenic mutant strain of SN74, and SN74CND-comp, a Cnm-complemented strain of SN74CND, were constructed in our previous study50. In addition, we used rCnm, a recombinant protein extracted after the transformation of Escherichia coli BL21 strain with a plasmid containing the cnm gene inserted into a pGEX 6P-1 vector50.

Animal experiments

All rats were treated humanely, in accordance with National Institutes of Health and AERI-BBRI Animal Care and Use Committee guidelines. All procedures used in the present study were approved by the Animal Care and Use Committee of Okayama University (approval number: OKU2020864). The effects of the intravenous administration of S. mutans were analyzed in a rat model, as described previously, with some modifications21,51. Briefly, specific pathogen-free Sprague–Dawley rats (male, 4-week-old; Japan CLEA, Tokyo, Japan) were randomly divided into PBS, SN74, CND, Comp, and rCnm groups. Rats were allowed free access to water and food throughout the experimental period. Rats were fed an MF diet (ORIENTAL YEAST Co., Ltd, Tokyo, Japan). Rats received intravenous injections (through the jugular vein) of each S. mutans type (1 × 108 colony-forming units) suspended in 100 μl PBS or PBS alone (i.e., without the addition of bacteria) or 200 μg of rCnm.

The rats were euthanized 45 days after infection and their kidneys were removed. Urinary levels of protein and CRE were measured by Nagahama Lifescience (ORIENTAL YEAST Co., Ltd.). Serum levels of CRE, ALB, and BUN were measured by Nagahama Lifescience. Serum IgA concentrations were measured using the Rat IgA ELISA Kit (BETHYL Laboratories Inc, Texas, USA), and serum IgG concentrations were measured using the Rat IgG ELISA Kit (BETHYL Laboratories Inc). Hematuria was defined as more than five red blood cells per field of view at ×400 magnification22.

Histological analyses of kidneys

Histological evaluations of kidneys were performed by using the following methods20,21. Excised kidney samples were fixed in 3.7% formaldehyde (diluted in PBS), embedded in paraffin, and cut into 3 μm-thick sections for histopathological analysis. PAS staining was performed to evaluate increases in the numbers of mesangial cells and mesangial matrix in glomeruli. Mesangial proliferation scores were then calculated based on the proportion of glomeruli with mesangial cells and matrix proliferation among 50 glomeruli in PAS-stained sections21,22. MT staining was performed to evaluate fibrosis in the tubules and interstitial area. Additionally, alterations in IgA, C3, IgG, CD34, and α-SMA expression patterns in tissue samples were detected using standard immunohistochemical techniques with IgA-, C3-, IgG-, CD34 (vascular endothelial cell marker), α-SMA -specific antibodies. The primary antibodies used were Purified Mouse Anti-Rat IgA (BD Biosciences, Franklin Lakes, NJ, USA), anti-C3 (B-9) (sc-28294; Santa Cruz Biotechnology, Dallas, TX, USA), anti-rat IgG (H + L), (Alexa Fluor 488 Conjugate) (#4416; Cell Signaling TECHNOLOGY, MA, USA), anti-CD34 (EP373Y) (ab81289; Abcam, Cambridge, MA, USA), and α-Smooth Muscle Actin (D4K9N) XP Rabbit mAb (#19245; Cell Signaling TECHNOLOGY) antibodies. Secondary antibodies were Donkey Anti-Mouse IgG H&L (Alexa Fluor 488) preadsorbed (ab150109; Abcam) and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 647) (ab150075; Abcam). Fluorescence immunostaining was performed using these antibodies. Stained sections were observed using a semi-motorized fluorescence microscope (BX53; OLYMPUS, Tokyo, Japan).

Immunogold transmission electron microscopy

Transmission electron microscopy was performed in accordance with the method of Naka et al.21. For pre-fixation, excised kidney tissue specimens were immersed in a solution of 2% glutaraldehyde and 2% paraformaldehyde in PBS (0.1 M, pH 7.4) for 16–18 h. Post-fixation was performed in 2% osmium tetroxide for 1.5 h. After specimens were washed with PBS, they were dehydrated through a graded ethanol series and embedded in low-viscosity resin (Spurr resin; Polysciences, Warrington, PA, USA). Sections (80 nm) were mounted on a 100-mesh nickel grid, incubated with PBS containing 10% goat serum (GEMINI Bio, San Carlos, CA, USA) and 1% BSA. Sections were incubated with anti-Cnm antibody overnight at 4 °C and washed with PBS containing 0.1% BSA more than five times. Then, they were incubated with gold colloid [Anti-IgG (H + L), Rabbit, Goat-Poly, Gold 15 nm, EM; BBI solutions, Crumlin, UK] conjugate goat anti-rat IgG antibody (BioLegend), washed three times with PBS containing 0.1% BSA, and then washed once with distilled water. Specimens were finally fixed with 2% glutaraldehyde. Specimens were observed under a transmission electron microscope (H-7650; HITACHI, Tokyo, Japan).

Statistical analyses

Statistical analyses were performed using GraphPad Prism 8 Statistics Software (GraphPad, Inc., La Jolla, CA, USA). All results are presented as the mean ± standard error. Differences in whole-body weight serum levels, urine components, and mesangial proliferation scores were assessed by analysis of variance with Bonferroni’s correction. P-values less than 0.01 were considered statistically significant. Differences in serum IgA and IgG levels were assessed by analysis of variance with Bonferroni’s correction. P-values less than 0.0167 were considered statistically significant. Positive immunohistochemical staining results and relationship between hematuria and IgA, C3, and/or IgG positive rate results were compared using Fisher’s exact test. P-values less than 0.05 were considered statistically significant. Relationship hematuria and mesangial proliferation scores results were compared using Student t-test. P-values less than 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Peer Review File

Supplemental Material

Description of Additional Supplementary File

Supplementary Data 1

Reporting Summary

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-024-06826-x.

Acknowledgements

This work was supported by funding from JSPS KAKENHI (grant numbers 20K10225, 21KK0160, 21H03149, 23K09146, 23K09435, 23K27805 24K02650). We thank Yumiko Morishita, Mika Monobe, and Miki Kajino (Central Research Laboratory, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences) for assistance with preparing tissue segments. We thank Masumi Furutani and Moemi Tsukano (Central Research Laboratory, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences) for assistance with electron microscopic analyses. We thank Mitchell Arico and J. Ludovic Croxford, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Author contributions

S.N. and D.M. designed the study under the supervision of M.M.N. and K.N. S.N., D.M., T.M., Y.N., S.I. and M.M.N. performed the animal experiments. Statistical analyses and data interpretation were performed by S.N., D.M., T.M., S.I., R.N. and K.N. S.N., D.M., T.M., K.N. and M.M.N. wrote the manuscript and all authors approved the final version.

Peer review

Peer review information

Communications Biology thanks Jianbo Qing and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editors: Jesmond Dalli and Tobias Goris. A peer review file is available.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The source data underlying Figs. 1, 2, 6, Supplementary Fig. 1, and Table 1, Supplementary Table 1. These source data are listed in Supplementary Data 1.

Materials availability

Correspondence and requests for materials should be addressed to Michiyo Matsumoto-Nakano.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Shuhei Naka, Daiki Matsuoka.
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References

1. D’Amico G The commonest glomerulonephritis in the world: IgA nephropathy Q. J. Med. 1987 64 709 727 3329736
D’Amico, G. The commonest glomerulonephritis in the world: IgA nephropathy. Q. J. Med. 64, 709–727 (1987).3329736
2. Julian BA Waldo FB Rifai A Mestecky J IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States? Am. J. Med 1988 84 129 132 10.1016/0002-9343(88)90019-8 3337116
Julian, B. A., Waldo, F. B., Rifai, A. & Mestecky, J. IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States? Am. J. Med 84, 129–132 (1988).3337116 10.1016/0002-9343(88)90019-8
3. Koyama A Igarashi M Kobayashi M Natural history and risk factors for immunoglobulin A nephropathy in Japan. Research group on progressive renal diseases Am. J. Kidney Dis. 1997 29 526 532 10.1016/S0272-6386(97)90333-4 9100040
Koyama, A., Igarashi, M. & Kobayashi, M. Natural history and risk factors for immunoglobulin A nephropathy in Japan. Research group on progressive renal diseases. Am. J. Kidney Dis. 29, 526–532 (1997).9100040 10.1016/S0272-6386(97)90333-4
4. Chauveau D Droz D Follow-up evaluation of the first patients with IgA nephropathy described at Necker Hospital Contrib. Nephrol. 1993 104 1 5 10.1159/000422388 8325022
Chauveau, D. & Droz, D. Follow-up evaluation of the first patients with IgA nephropathy described at Necker Hospital. Contrib. Nephrol. 104, 1–5 (1993).8325022 10.1159/000422388
5. Hotta O Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy Am. J. Kidney Dis. 2001 38 736 743 10.1053/ajkd.2001.27690 11576876
Hotta, O. et al. Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am. J. Kidney Dis. 38, 736–743 (2001).11576876 10.1053/ajkd.2001.27690
6. Hwang HS Predictors for progression in immunoglobulin A nephropathy with significant proteinuria Nephrology 2010 15 236 241 10.1111/j.1440-1797.2009.01196.x 20470285
Hwang, H. S. et al. Predictors for progression in immunoglobulin A nephropathy with significant proteinuria. Nephrology 15, 236–241 (2010).20470285 10.1111/j.1440-1797.2009.01196.x
7. Tatematsu M Complete remission within 2 years predicts a good prognosis after methylprednisolone pulse therapy in patients with IgA nephropathy Clin. Exp. Nephrol. 2012 16 883 891 10.1007/s10157-012-0644-0 22618296
Tatematsu, M. et al. Complete remission within 2 years predicts a good prognosis after methylprednisolone pulse therapy in patients with IgA nephropathy. Clin. Exp. Nephrol. 16, 883–891 (2012).22618296 10.1007/s10157-012-0644-0
8. Wyatt RJ Julian BA IgA nephropathy N. Engl. J. Med. 2013 368 2402 2414 10.1056/NEJMra1206793 23782179
Wyatt, R. J. & Julian, B. A. IgA nephropathy. N. Engl. J. Med. 368, 2402–2414 (2013).23782179 10.1056/NEJMra1206793
9. Hara M Urinary excretion of podocalyxin indicates glomerular epithelial cell injuries in glomerulonephritis Nephron 1995 69 397 403 10.1159/000188509 7777103
Hara, M. et al. Urinary excretion of podocalyxin indicates glomerular epithelial cell injuries in glomerulonephritis. Nephron 69, 397–403 (1995).7777103 10.1159/000188509
10. Levy M Berger’s disease in children. Natural history and outcome Medicine 1985 64 157 180 10.1097/00005792-198505000-00002 3887094
Levy, M. et al. Berger’s disease in children. Natural history and outcome. Medicine 64, 157–180 (1985).3887094 10.1097/00005792-198505000-00002
11. Sugiyama H Japan renal biopsy registry and Japan kidney disease registry: committee report for 2009 and 2010 Clin. Exp. Nephrol. 2013 17 155 173 10.1007/s10157-012-0746-8 23385776
Sugiyama, H. et al. Japan renal biopsy registry and Japan kidney disease registry: committee report for 2009 and 2010. Clin. Exp. Nephrol. 17, 155–173 (2013).23385776 10.1007/s10157-012-0746-8
12. Donadio JV Grande JP IgA nephropathy N. Engl. J. Med. 2002 347 738 748 10.1056/NEJMra020109 12213946
Donadio, J. V. & Grande, J. P. IgA nephropathy. N. Engl. J. Med. 347, 738–748 (2002).12213946 10.1056/NEJMra020109
13. Suzuki S Nakatomi Y Sato H Tsukada H Arakawa M Haemophilus parainfluenzae antigen and antibody in renal biopsy samples and serum of patients with IgA nephropathy Lancet 1994 343 12 16 10.1016/S0140-6736(94)90875-3 7905040
Suzuki, S., Nakatomi, Y., Sato, H., Tsukada, H. & Arakawa, M. Haemophilus parainfluenzae antigen and antibody in renal biopsy samples and serum of patients with IgA nephropathy. Lancet 343, 12–16 (1994).7905040 10.1016/S0140-6736(94)90875-3
14. Kusano K Tokunaga O Ando T Inokuchi A Helicobacter pylori in the palatine tonsils of patients with IgA nephropathy compared with those of patients with recurrent pharyngotonsillitis Hum. Pathol. 2007 38 1788 1797 10.1016/j.humpath.2007.04.012 17714758
Kusano, K., Tokunaga, O., Ando, T. & Inokuchi, A. Helicobacter pylori in the palatine tonsils of patients with IgA nephropathy compared with those of patients with recurrent pharyngotonsillitis. Hum. Pathol. 38, 1788–1797 (2007).17714758 10.1016/j.humpath.2007.04.012
15. Iwama H Horikoshi S Shirato I Tomino Y Epstein-Barr virus detection in kidney biopsy specimens correlates with glomerular mesangial injury Am. J. Kidney Dis. 1998 32 785 793 10.1016/S0272-6386(98)70134-9 9820448
Iwama, H., Horikoshi, S., Shirato, I. & Tomino, Y. Epstein-Barr virus detection in kidney biopsy specimens correlates with glomerular mesangial injury. Am. J. Kidney Dis. 32, 785–793 (1998).9820448 10.1016/S0272-6386(98)70134-9
16. Koyama A Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy Kidney Int. 2004 66 121 132 10.1111/j.1523-1755.2004.00714.x 15200419
Koyama, A. et al. Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy. Kidney Int. 66, 121–132 (2004).15200419 10.1111/j.1523-1755.2004.00714.x
17. Rollino C Vischini G Coppo R IgA nephropathy and infections J. Nephrol. 2016 29 463 468 10.1007/s40620-016-0265-x 26800970
Rollino, C., Vischini, G. & Coppo, R. IgA nephropathy and infections. J. Nephrol. 29, 463–468 (2016).26800970 10.1007/s40620-016-0265-x
18. Misaki T Distribution of Streptococcus mutans strains with collagen-binding proteins in the oral cavity of IgA nephropathy patients Clin. Exp. Nephrol. 2015 19 844 850 10.1007/s10157-014-1072-0 25492252
Misaki, T. et al. Distribution of Streptococcus mutans strains with collagen-binding proteins in the oral cavity of IgA nephropathy patients. Clin. Exp. Nephrol. 19, 844–850 (2015).25492252 10.1007/s10157-014-1072-0
19. Misaki T Presence of Streptococcus mutans strains harbouring the cnm gene correlates with dental caries status and IgA nephropathy conditions Sci. Rep. 2016 6 36455 10.1038/srep36455 27811984
Misaki, T. et al. Presence of Streptococcus mutans strains harbouring the cnm gene correlates with dental caries status and IgA nephropathy conditions. Sci. Rep. 6, 36455 (2016).27811984 10.1038/srep36455
20. Ito S Specific strains of Streptococcus mutans, a pathogen of dental caries, in the tonsils, are associated with IgA nephropathy Sci. Rep. 2019 9 20130 10.1038/s41598-019-56679-2 31882880
Ito, S. et al. Specific strains of Streptococcus mutans, a pathogen of dental caries, in the tonsils, are associated with IgA nephropathy. Sci. Rep. 9, 20130 (2019).31882880 10.1038/s41598-019-56679-2
21. Naka S Intravenous administration of Streptococcus mutans induces IgA nephropathy-like lesions Clin. Exp. Nephrol. 2020 24 1122 1131 10.1007/s10157-020-01961-1 32909181
Naka, S. et al. Intravenous administration of Streptococcus mutans induces IgA nephropathy-like lesions. Clin. Exp. Nephrol. 24, 1122–1131 (2020).32909181 10.1007/s10157-020-01961-1
22. Naka S Streptococcus mutans induces IgA nephropathy-like glomerulonephritis in rats with severe dental caries Sci. Rep. 2021 11 5784 10.1038/s41598-021-85196-4 33707585
Naka, S. et al. Streptococcus mutans induces IgA nephropathy-like glomerulonephritis in rats with severe dental caries. Sci. Rep. 11, 5784 (2021).33707585 10.1038/s41598-021-85196-4
23. Nagasawa, Y. et al. Title IgA nephropathy and oral bacterial species related to dental caries and periodontitis. Int. J. Mol. Sci. 23, 725 (2022).
24. Misaki, T. et al. Simultaneous presence of Campylobacter rectus and Cnm-positive Streptococcus mutans in the oral cavity is associated with renal dysfunction in IgA nephropathy patients: 5-year follow-up analysis. Nephron 147, 1–10 (2022).
25. Misaki T cnm-positive Streptococcus mutans is associated with galactose-deficient IgA in patients with IgA nephropathy PLoS One 2023 18 e0282367 10.1371/journal.pone.0282367 36862654
Misaki, T. et al. cnm-positive Streptococcus mutans is associated with galactose-deficient IgA in patients with IgA nephropathy. PLoS One 18, e0282367 (2023).36862654 10.1371/journal.pone.0282367
26. Misaki T Campylobacter rectus in the oral cavity correlates with proteinuria in immunoglobulin A nephropathy patients Nephron 2018 139 143 149 10.1159/000487103 29428934
Misaki, T. et al. Campylobacter rectus in the oral cavity correlates with proteinuria in immunoglobulin A nephropathy patients. Nephron 139, 143–149 (2018).29428934 10.1159/000487103
27. Nagasawa, Y. et al. Relationship between IgA Nephropathy and Porphyromonas gingivalis; red complex of periodontopathic bacterial species. Int. J. Mol. Sci. 22, 13022 (2021).
28. Nomura, R. et al. Distribution of periodontopathic bacterial species between saliva and tonsils. Odontology. 111, 719–727 (2022).
29. Hamada S Slade HD Biology, immunology, and cariogenicity of Streptococcus mutans Microbiol. Rev. 1980 44 331 384 10.1128/mr.44.2.331-384.1980 6446023
Hamada, S. & Slade, H. D. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44, 331–384 (1980).6446023 10.1128/mr.44.2.331-384.1980
30. Nakano K Ooshima T Serotype classification of Streptococcus mutans and its detection outside the oral cavity Future Microbiol. 2009 4 891 902 10.2217/fmb.09.64 19722842
Nakano, K. & Ooshima, T. Serotype classification of Streptococcus mutans and its detection outside the oral cavity. Future Microbiol. 4, 891–902 (2009).19722842 10.2217/fmb.09.64
31. Sato Y Streptococcus mutans strains harboring collagen-binding adhesin J. Dent. Res. 2004 83 534 539 10.1177/154405910408300705 15218042
Sato, Y. et al. Streptococcus mutans strains harboring collagen-binding adhesin. J. Dent. Res. 83, 534–539 (2004).15218042 10.1177/154405910408300705
32. Abranches J The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells Infect. Immun. 2011 79 2277 2284 10.1128/IAI.00767-10 21422186
Abranches, J. et al. The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells. Infect. Immun. 79, 2277–2284 (2011).21422186 10.1128/IAI.00767-10
33. Nomura R Potential involvement of collagen-binding proteins of Streptococcus mutans in infective endocarditis Oral. Dis. 2013 19 387 393 10.1111/odi.12016 22998492
Nomura, R. et al. Potential involvement of collagen-binding proteins of Streptococcus mutans in infective endocarditis. Oral. Dis. 19, 387–393 (2013).22998492 10.1111/odi.12016
34. Nomura R Potential involvement of Streptococcus mutans possessing collagen binding protein Cnm in infective endocarditis Sci. Rep. 2020 10 19118 10.1038/s41598-020-75933-6 33154489
Nomura, R. et al. Potential involvement of Streptococcus mutans possessing collagen binding protein Cnm in infective endocarditis. Sci. Rep. 10, 19118 (2020).33154489 10.1038/s41598-020-75933-6
35. Seymour RA Lowry R Whitworth JM Martin MV Infective endocarditis, dentistry and antibiotic prophylaxis; time for a rethink? Br. Dent. J. 2000 189 610 616 10.1038/sj.bdj.4800845 11132691
Seymour, R. A., Lowry, R., Whitworth, J. M. & Martin, M. V. Infective endocarditis, dentistry and antibiotic prophylaxis; time for a rethink? Br. Dent. J. 189, 610–616 (2000).11132691 10.1038/sj.bdj.4800845
36. Nomura R Nakano K Ooshima T Contribution of glucan-binding protein C of Streptococcus mutans to bacteremia occurrence Arch. Oral. Biol. 2004 49 783 788 10.1016/j.archoralbio.2004.04.001 15308422
Nomura, R., Nakano, K. & Ooshima, T. Contribution of glucan-binding protein C of Streptococcus mutans to bacteremia occurrence. Arch. Oral. Biol. 49, 783–788 (2004).15308422 10.1016/j.archoralbio.2004.04.001
37. Nakano K Fujita K Nishimura K Nomura R Ooshima T Contribution of biofilm regulatory protein A of Streptococcus mutans, to systemic virulence Microbes Infect. 2005 7 1246 1255 10.1016/j.micinf.2005.04.012 16039152
Nakano, K., Fujita, K., Nishimura, K., Nomura, R. & Ooshima, T. Contribution of biofilm regulatory protein A of Streptococcus mutans, to systemic virulence. Microbes Infect. 7, 1246–1255 (2005).16039152 10.1016/j.micinf.2005.04.012
38. Nakano K Tsuji M Nishimura K Nomura R Ooshima T Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia Microbes Infect. 2006 8 114 121 10.1016/j.micinf.2005.06.005 16442486
Nakano, K., Tsuji, M., Nishimura, K., Nomura, R. & Ooshima, T. Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia. Microbes Infect. 8, 114–121 (2006).16442486 10.1016/j.micinf.2005.06.005
39. Otsugu M Nomura R Matayoshi S Teramoto N Nakano K Contribution of Streptococcus mutans strains with collagen-binding proteins in the presence of serum to the pathogenesis of infective endocarditits Infect. Immun. 2017 85 e00401 e00417 10.1128/IAI.00401-17 28947650
Otsugu, M., Nomura, R., Matayoshi, S., Teramoto, N. & Nakano, K. Contribution of Streptococcus mutans strains with collagen-binding proteins in the presence of serum to the pathogenesis of infective endocarditits. Infect. Immun. 85, e00401–e00417 (2017).28947650 10.1128/IAI.00401-17
40. Gibbons RJ Socransky SS Vanhoute Dearujo WCJ Studies of the predominant cultivable microbiota of dental plaque Arch. Oral. Biol. 1964 9 365 370 10.1016/0003-9969(64)90069-X 14170653
Gibbons, R. J., Socransky, S. S. & Vanhoute, Dearujo W. C. J. Studies of the predominant cultivable microbiota of dental plaque. Arch. Oral. Biol. 9, 365–370 (1964).14170653 10.1016/0003-9969(64)90069-X
41. Nagy J Uj M Szucs G Trinn C Burger T Herpes virus antigens and antibodies in kidney biopsies and sera of IgA glomerulonephritic patients Clin. Nephrol. 1984 21 259 262 6329570
Nagy, J., Uj, M., Szucs, G., Trinn, C. & Burger, T. Herpes virus antigens and antibodies in kidney biopsies and sera of IgA glomerulonephritic patients. Clin. Nephrol. 21, 259–262 (1984).6329570
42. Tomino Y Yagame M Omata F Nomoto Y Sakai H A case of IgA nephropathy associated with adeno- and herpes simplex viruses Nephron 1987 47 258 261 10.1159/000184520 2827043
Tomino, Y., Yagame, M., Omata, F., Nomoto, Y. & Sakai, H. A case of IgA nephropathy associated with adeno- and herpes simplex viruses. Nephron 47, 258–261 (1987).2827043 10.1159/000184520
43. Lai KN Lai FM Lo S Ho CP Chan KW IgA nephropathy associated with hepatitis B virus antigenemia Nephron 1987 47 141 143 10.1159/000184477 3320776
Lai, K. N., Lai, F. M., Lo, S., Ho, C. P. & Chan, K. W. IgA nephropathy associated with hepatitis B virus antigenemia. Nephron 47, 141–143 (1987).3320776 10.1159/000184477
44. Gregory MC Hammond ME Brewer ED Renal deposition of cytomegalovirus antigen in immunoglobulin-A nephropathy Lancet 1988 1 11 14 10.1016/S0140-6736(88)91000-8 2891887
Gregory, M. C., Hammond, M. E. & Brewer, E. D. Renal deposition of cytomegalovirus antigen in immunoglobulin-A nephropathy. Lancet 1, 11–14 (1988).2891887 10.1016/S0140-6736(88)91000-8
45. Woodroffe AJ Immunologic studies in IgA nephropathy Kidney Int. 1980 18 366 374 10.1038/ki.1980.147 7463948
Woodroffe, A. J. et al. Immunologic studies in IgA nephropathy. Kidney Int. 18, 366–374 (1980).7463948 10.1038/ki.1980.147
46. Endo Y Hara M Glomerular IgA deposition in pulmonary diseases Kidney Int. 1986 29 557 562 10.1038/ki.1986.34 3702213
Endo, Y. & Hara, M. Glomerular IgA deposition in pulmonary diseases. Kidney Int. 29, 557–562 (1986).3702213 10.1038/ki.1986.34
47. Koyama A Glomerulonephritis associated with MRSA infection: a possible role of bacterial superantigen Kidney Int 1995 47 207 216 10.1038/ki.1995.25 7731148
Koyama, A. et al. Glomerulonephritis associated with MRSA infection: a possible role of bacterial superantigen. Kidney Int 47, 207–216 (1995).7731148 10.1038/ki.1995.25
48. Shimizu Y Staphylococcal cell membrane antigen, a possible antigen in post-methicillin resistant Staphylococcus aureus (MRSA) infection nephritis and IgA nephropathy, exhibits high immunogenic activity that is enhanced by superantigen J. Nephrol. 2005 18 249 256 16013011
Shimizu, Y. et al. Staphylococcal cell membrane antigen, a possible antigen in post-methicillin resistant Staphylococcus aureus (MRSA) infection nephritis and IgA nephropathy, exhibits high immunogenic activity that is enhanced by superantigen. J. Nephrol. 18, 249–256 (2005).16013011
49. Sharmin S Shimizu Y Hagiwara M Hirayama K Koyama A Staphylococcus aureus antigens induce IgA-type glomerulonephritis in Balb/c mice J. Nephrol. 2004 17 504 511 15372411
Sharmin, S., Shimizu, Y., Hagiwara, M., Hirayama, K. & Koyama, A. Staphylococcus aureus antigens induce IgA-type glomerulonephritis in Balb/c mice. J. Nephrol. 17, 504–511 (2004).15372411
50. Naka S Cnm of Streptococcus mutans is important for cell surface structure and membrane permeability Front. Cell Infect. Microbiol 2022 12 994014 10.3389/fcimb.2022.994014 36176579
Naka, S. et al. Cnm of Streptococcus mutans is important for cell surface structure and membrane permeability. Front. Cell Infect. Microbiol 12, 994014 (2022).36176579 10.3389/fcimb.2022.994014
51. Nomura R Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis Infect. Immun. 2014 82 5223 5234 10.1128/IAI.02164-14 25287921
Nomura, R. et al. Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis. Infect. Immun. 82, 5223–5234 (2014).25287921 10.1128/IAI.02164-14
