Porphyromonas gingivalis Induces Chronic Kidney Disease through Crosstalk between the NF-κB/NLRP3 Pathway and Ferroptosis in GMCs

Abstract

Objective

Porphyromonas gingivalis (P.gingivalis) is a gram-negative bacterium found in the human oral cavity and is a recognized pathogenic bacterium associated with chronic periodontitis and systemic diseases, including chronic kidney disease (CKD), but the roles and molecular mechanism of P.gingivalis in CKD pathogenesis are unclear.

Methods

In this study, an animal model of oral P.gingivalis administration and glomerular mesangial cells (GMCs) cocultured with M1-polarized macrophages and P.gingivalis supernatant were constructed. After seven weeks of P.gingivalis gavaged, peripheral blood was collected to detect the changes in renal function. By collecting the teeth and kidneys of mice, H&E staining and IHC were used to analyze the expression of periodontal inflammatory factors in mice, PAS staining was used to analyze glomerular lesions. The supernatant of macrophages was treated with 5% P.gingivalis supernatant. H&E staining, IHC, Western blot and RT-PCR were applied to analyze renal inflammatory factors, macrophage M1 polarization, NF-κB, NLRP3 and ferroptosis changes in vitro.

Results

We found that oral P.gingivalis administration induced CKD in mice. P.gingivalis supernatant induced macrophage polarization and inflammatory factor upregulation, which triggered the activation of the NF-κB/NLRP3 pathway and ferroptosis in GMCs. By inhibiting the NF-κB/NLRP3 pathway and ferroptosis in GMCs, cell viability and the inflammatory response were partially alleviated in vitro.

Conclusion

We demonstrated that P.gingivalis induced CKD in mice by triggering crosstalk between the NF κB/NLRP3 pathway and ferroptosis in GMCs. Overall, our study suggested that periodontitis can promote the pathogenesis of CKD in mice, which provides evidence of the importance of periodontitis therapy in the prevention and treatment of CKD.

References

[1] Lv JC, Zhang LX. Prevalence and Disease Burden of Chronic Kidney Disease. Adv Exp Med Biol, 2019, 1165: 3-15

[2] Wang L, Xu X, Zhang M, et al.. Prevalence of Chronic Kidney Disease in China: Results From the Sixth China Chronic Disease and Risk Factor Surveillance. JAMA Intern Med, 2023, 183(4): 298-310

[3] Coresh J, Selvin E, Stevens LA, et al.. Prevalence of chronic kidney disease in the United States. JAMA, 2007, 298(17): 2038-2047

[4] Jepsen S, Suvan J, Deschner J. The association of periodontal diseases with metabolic syndrome and obesity. Periodontol 2000, 2020, 83(1): 125-153

[5] Jiao J, Jing W, Si Y, et al.. The prevalence and severity of periodontal disease in Mainland China: Data from the Fourth National Oral Health Survey (2015–2016). J Clin Periodontol, 2021, 48(2): 168-179

[6] Jepsen S, Suvan J, Deschner J. Working group 1 of the joint, Periodontitis and atherosclerotic cardiovascular disease: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic Diseases. J Clin Periodontol, 2013, 40(Suppl14): S24-S29

[7] Genco RJ, Borgnakke WS. Risk factors for periodontal disease. Periodontol 2000, 2013, 62(1): 59-94

[8] Bui FQ, Almeida-da-Silva CLC, Huynh B, et al.. Association between periodontal pathogens and systemic disease. Biomed J, 2019, 42(1): 27-35

[9] Deschamps-Lenhardt S, Martin-Cabezas R, Hannedouche T, et al.. Association between periodontitis and chronic kidney disease: Systematic review and meta-analysis. Oral Dis, 2019, 25(2): 385-402

[10] Kapellas K, Singh A, Bertotti M, et al.. Periodontal and chronic kidney disease association: A systematic review and meta-analysis. Nephrology (Carlton), 2019, 24(2): 202-212

[11] Fisher MA, Taylor GW, Papapanou PN, et al.. Clinical and serologic markers of periodontal infection and chronic kidney disease. J Periodontol, 2008, 79(9): 1670-1678

[12] Navarrete M, Garcia J, Dutzan N, et al.. Interferon-gamma, interleukins-6 and -4, and factor XIII-A as indirect markers of the classical and alternative macrophage activation pathways in chronic periodontitis. J Periodontol, 2014, 85(5): 751-760

[13] Lew JH, Naruishi K, et al.. High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes. Cell Physiol Biochem, 2018, 50(3): 973-986

[14] Wang Y, Li C, Wan Y, et al.. Quercetin-Loaded Ceria Nanocomposite Potentiate Dual-Directional Immunoregulation via Macrophage Polarization against Periodontal Inflammation. Small, 2021, 17(41): e2101505

[15] Yang J, Zhu Y, Duan D, et al.. Enhanced activity of macrophage M1/M2 phenotypes in periodontitis. Arch Oral Biol, 2018, 96: 234-242

[16] Lech M, Grobmayr R, Ryu M, et al.. Macrophage phenotype controls long-term AKI outcomes—kidney regeneration versus atrophy. J Am Soc Nephrol, 2014, 25(2): 292-304

[17] Anders HJ, Ryu M. Renal microenvironments and macrophage phenotypes determine progression or resolution of renal inflammation and fibrosis. Kidney Int, 2011, 80(9): 915-925

[18] Kon V, Linton MF, Fazio S. Atherosclerosis in chronic kidney disease: the role of macrophages. Nat Rev Nephrol, 2011, 7(1): 45-54

[19] Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and repair. J Clin Invest, 2008, 118(11): 3522-3530

[20] Li C, Ding XY, Xiang DM, et al.. Enhanced M1 and Impaired M2 Macrophage Polarization and Reduced Mitochondrial Biogenesis via Inhibition of AMP Kinase in Chronic Kidney Disease. Cell Physiol Biochem, 2015, 36(1): 358-372

[21] Mussbacher M, Salzmann M, Brostjan C, et al.. Cell Type-Specific Roles of NF-kappaB Linking Inflammation and Thrombosis. Front Immunol, 2019, 10: 85

[22] Sucajtys-Szulc E, Debska-Slizien A, Rutkowski B, et al.. Hepatocyte Nuclear Factor 1alpha Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor-kappaB in Experimental Model of Chronic Kidney Disease. Int J Mol Sci, 2022, 23(16): 8883

[23] Bhargava S, Merckelbach E, Noels H, et al.. Homeostasis in the Gut Microbiota in Chronic Kidney Disease. Toxins (Basel), 2022, 14(10): 648

[24] Mahendra J, Palathingal P, Mahendra L, et al.. Impact of Red Complex Bacteria and TNF-alpha Levels on the Diabetic and Renal Status of Chronic Kidney Disease Patients in the Presence and Absence of Periodontitis. Biology (Basel), 2022, 11(3): 451

[25] Palm E, Demirel I, Bengtsson T, et al.. The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalis. Cytokine, 2015, 76(2): 424-432

[26] Baek KJ, Ji S, Kim YC, et al.. Association of the invasion ability of Porphyromonas gingivalis with the severity of periodontitis. Virulence, 2015, 6(3): 274-281

[27] Huang Y, Tian C, Li QM, et al.. TET1 Knockdown Inhibits Porphyromonas gingivalis LPS/IFN-gamma-Induced M1 Macrophage Polarization through the NF-kappaB Pathway in THP-1 Cells. Int J Mol Sci, 2019, 20(8): 2023

[28] Holden JA, Attard TJ, Laughton KM, et al.. Porphyromonas gingivalis lipopolysaccharide weakly activates M1 and M2 polarized mouse macrophages but induces inflammatory cytokines. Infect Immun, 2014, 82(10): 4190-4203

[29] Lam RS, O’Brien-Simpson NM, et al.. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis. PLoS One, 2016, 11(7): e0158629

[30] Liu LL, Guo HM, Song AM, et al.. Progranulin inhibits LPS-induced macrophage M1 polarization via NF-small ka, CyrillicB and MAPK pathways. BMC Immunol, 2020, 21(1): 32

[31] Iwasaki M, Taylor GM, Manz MC, et al.. Serum antibody to Porphyromonas gingivalis in chronic kidney disease. J Dent Res, 2012, 91(9): 828-833

[32] Agostini L, Martinon F, Burns K, et al.. NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity, 2004, 20(3): 319-325

[33] Gonçalves AC, Ferreira LS, Manente FA, et al.. The NLRP3 inflammasome contributes to host protection during Sporothrix schenckii infection. Immunology, 2017, 151(2): 154-166

[34] Lv XF, Fan C, Jiang ZX, et al.. Isoliquiritigenin alleviates P.gingivalis-LPS/ATP-induced pyroptosis by inhibiting NF-kappaB/NLRP3/GSDMD signals in human gingival fibroblasts. Int Immunopharmacol, 2021, 101: 108338 Pt B

[35] Dixon SJ, Lemberg KM, Lamprecht MR, et al.. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 2012, 149(5): 1060-1072

[36] Yao C, Lan D, Li X, et al.. Porphyromonas gingivalis is a risk factor for the development of nonalcoholic fatty liver disease via ferroptosis. Microbes Infect, 2023, 25(1–2): 105040

[37] Qiao SW, Li BS, Cai Q, et al.. Involvement of ferroptosis in Porphyromonas gingivalis lipopolysaccharide-stimulated periodontitis in vitro and in vivo. Oral Dis, 2023, 29(8): 3571-3582

[38] Ye YZ, Chen A, Li L, et al.. Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification. Kidney Int, 2022, 102(6): 1259-1275

[39] Zhou L, Xue X, Hou Q, et al.. Targeting Ferroptosis Attenuates Interstitial Inflammation and Kidney Fibrosis. Kidney Dis (Basel), 2022, 8(1): 57-71

[40] Xu MY, Tao J, Yang YD, et al.. Ferroptosis involves in intestinal epithelial cell death in ulcerative colitis. Cell Death Dis, 2020, 11(2): 86

[41] Yao C, Lan DM, Li X, et al.. Porphyromonas gingivalis triggers inflammation in hepatocyte depend on ferroptosis via activating the NF-kappaB signaling pathway. Oral Dis, 2024, 30(3): 1680-1694

[42] Kassebaum NJ, Bernabe E, Li X, et al.. Global burden of severe periodontitis in 1990–2010: a systematic review and metaregression. J Dent Res, 2014, 93(11): 1045-1053

[43] Siribamrungwong M, Yothasamutr K, Puangpanngam K. Periodontal treatment reduces chronic systemic inflammation in peritoneal dialysis patients. Ther Apher Dial, 2014, 18(3): 305-308

[44] D’Aiuto F, Parkar M, Andreou G, et al.. Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res, 2004, 83(2): 156-160

[45] Fang F, Wu B, Qu Q, et al.. The clinical response and systemic effects of non-surgical periodontal therapy in end-stage renal disease patients: a 6-month randomized controlled clinical trial. J Clin Periodontol, 2015, 42(6): 537-546

[46] Vilela EM, Bastos JA, Fernandes N, et al.. Treatment of chronic periodontitis decreases serum prohepcidin levels in patients with chronic kidney disease. Clinics (Sao Paulo), 2011, 66(4): 657-662

[47] Parsegian KD, Randall D, Curtis M, et al.. Association between periodontitis and chronic kidney disease. Periodontol 2000, 2022, 89(1): 114-124

[48] Sun X, Gao J, Meng X, et al.. Polarized Macrophages in Periodontitis: Characteristics, Function, and Molecular Signaling. Front Immunol, 2021, 12: 763334

[49] Wen Y, Lu XH, Ren JF, et al.. KLF4 in Macrophages Attenuates TNFalpha-Mediated Kidney Injury and Fibrosis. J Am Soc Nephrol, 2019, 30(10): 1925-1938

[50] Bao H, Peng A. The Green Tea Polyphenol(-)-epigallocatechin-3-gallate and its beneficial roles in chronic kidney disease. J Transl Int Med, 2016, 4(3): 99-103

[51] Pang Q, Wang P, et al.. Irisin protects against vascular calcification by activating autophagy and inhibiting NLRP3-mediated vascular smooth muscle cell pyroptosis in chronic kidney disease. Cell Death Dis, 2022, 13(3): 283

[52] Wang YJ, Chen YY, et al.. Induction of Autophagy by Pterostilbene Contributes to the Prevention of Renal Fibrosis via Attenuating NLRP3 Inflammasome Activation and Epithelial-Mesenchymal Transition. Front Cell Dev Biol, 2020, 8: 436

[53] Bostanci N, Emingil G, Saygan B, et al.. Expression and regulation of the NALP3 inflammasome complex in periodontal diseases. Clin Exp Immunol, 2009, 157(3): 415-422

[54] Hamedi M, Belibasakis GN, Cruchley AT, et al.. Porphyromonas gingivalis culture supernatants differentially regulate interleukin-1beta and interleukin-18 in human monocytic cells. Cytokine, 2009, 45(2): 99-104

[55] Yilmaz O, Sater AA, Yao LY, et al.. ATP-dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis. Cell Microbiol, 2010, 12(2): 188-198

[56] Giuliani KTK, Grivei A, Nag P, et al.. Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c(+) dendritic cells. Cell Death Dis, 2022, 13(8): 739

[57] Xiao Z, Kong B, Fang J, et al.. Ferrostatin-1 alleviates lipopolysaccharide-induced cardiac dysfunction. Bioengineered, 2021, 12(2): 9367-9376

[58] Jomen W, Ohtake T, Akita T, et al.. Iron chelator deferasirox inhibits NF-kappaB activity in hepatoma cells and changes sorafenib-induced programmed cell deaths. Biomed Pharmacother, 2022, 153: 113363

[59] Kretz-Remy C, Mehlen P, Mirault ME, et al.. Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression. J Cell Biol, 1996, 133(5): 1083-1093