Discussion
In this study, we showed that oral administration of P.g directly induced periodontitis and CKD in mice. Oral administration of P.grecruited macrophages to the kidney, induced inflammatory factors, M1 macrophage polarization, ferroptosis and activation of the NF‑κB/NLRP3 pathway in vivo . Moreover, ferroptosis in GMCs was activated during CKD through the NF‑κB/NLRP3 pathway after stimulation by the supernatant of M1 macrophages polarized by P.g supernatant in vitro.
Periodontitis and CKD are chronic conditions with high prevalence worldwide. Periodontitis is also a noncommunicable disease with a high prevalence of 45%–50% and is the sixth most common human disease(Kassebaum, Bernabe, Dahiya, Bhandari, Murray and Marcenes 2014). There is now a significant body of evidence to support independent associations between periodontitis and systemic diseases, including diabetes, cardiovascular disease, chronic obstructive pulmonary disease and CKD(Tonetti, Van Dyke and Working group 1 of the joint 2013). CKD affects approximately 13% of the global population and is associated with increased morbidity and mortality(Jha et al. 2013). A recent systematic review reported an association between periodontitis and CKD; there was an increased prevalence of CKD in patients with periodontitis compared with patients without periodontitis(Sharma et al. 2014). Successful periodontal treatment can reduce the levels of systemic inflammation in patients with CKD(D’Aiuto et al. 2004, Fang et al. 2015, Siribamrungwong, Yothasamutr and Puangpanngam 2014, Vilela, Bastos, Fernandes, Ferreira, Chaoubah and Bastos 2011). Patients with CKD have been shown to have high levels of periodontitis(Parsegian, Randall, Curtis and Ioannidou 2022). Our study showed that oral administration ofP.g reduced renal function, promoted the inflammatory response in the kidneys and periodontitis, and induced the occurrence of CKD (Figure 1). The results showed that periodontitis-associated pathogenic bacteria promoted the development of CKD in mice. In periodontal tissues, macrophages make significant contributions to tissue homeostasis and defense. Bacteria and their products promote M1 polarization in macrophages and upregulate proinflammatory factors that trigger an inflammatory response in local organs(Sun, Gao, Meng, Lu, Zhang and Chen 2021). M1 macrophages play an important role in the occurrence and development of periodontitis and early phases of CKD. In murine CKD models exposed to LPS, robust macrophage accumulation and increased M1 polarization were observed, and M1 macrophages produced large amounts of proinflammatory factors(Wen et al. 2019). In our study, macrophages accumulated in kidney tissue, and the high expression of CD86 and iNOS proved the M1 polarization of macrophages (Figure 2A-D). Moreover, proinflammatory factors (IL-6 and IL-17) were highly expressed, and an anti-inflammatory factor (IL-10) was expressed at low levels in vivo (Figure 3A-D). Furthermore, P.g supernatant promoted M1 macrophage polarization in vitro (Figure 2E-H), increased proinflammatory factors (IL-6 and IL-17) and decreased an anti-inflammatory factor (IL-10) (Figure 3E-H), which were released from M1 macrophages, and these results were similar to those of previous studies. These results suggest that P.g promoted the development of CKD by recruiting macrophages and promoting M1 macrophage polarization and the inflammatory response in vivo and in vitro .
The NF-кB signaling pathway is the key regulator of inflammatory diseases, including CKD(Bao and Peng 2016). NF-κB, which is a major transcription factor associated with immune regulation, is widely expressed in biological systems. Inhibitory proteins of the κb family (IκB) play a critical role in regulating the normal balance of NF-κB. The activation of NF-κB induced by phosphorylated IκB can disrupt the balance of transcription to activate NLRP3 and pro-IL-1β. Numerous studies have indicated that the NF-κB signaling pathway has a strong correlation with NLRP3 inflammasome activation in various diseases, including CKD. NLRP3, which is downstream of the NF-кB signaling pathway and plays a key role in host defense against pathogens, mediates vascular smooth muscle cell programmed cell death in CKD (Pang et al. 2022). NLRP3 inflammasome activation has been shown to mediate many mechanisms of CKD through the regulation of proinflammatory cytokines, tubulointerstitial injury, glomerular diseases, renal inflammation, and fibrosis (Wang et al. 2020). Our study showed that P.g increased the expression of P65, phosphorylated-p65 (p-p65) and NLRP3 and decreased the phosphorylation level of the NF‑κB inhibitory protein IkBa (p-IkBa) in kidney tissue (Figure 4A-F). GMCs are stromal cells that are important for kidney glomerular homeostasis and the glomerular response to injury. Through direct crosstalk with neighboring immune cells and indirectly through the remodeling of the matrix, GMCs can regulate a variety of processes, such as immunity, inflammation, and regeneration, which play important roles in CKD. Therefore, GMCs were chosen for subsequent in vitro experiments in our study. The progression of CKD cannot occur without the interaction between immune cells and GMCs. To simulate the in vivo  environment, GMCs were cocultured with MΦCM. We found that MΦCM from P.g supernatant increased the expression of P65, p-p65 and NLRP3 and decreased p-IkBa expression in GMCs (Figure 4I-M). These results showed that P.g promoted inflammation in CKD by triggering the NF‑κB/NLRP3 pathway.
Programmed cell death such as ferroptosis leads to an inflammatory response(Qiao et al. 2022), and ferroptosis plays a critical role in CKD(Ye et al. 2022). Studies have reported that ferroptotic cells release the proteoglycan decorin and trigger the production of proinflammatory cytokines in an NF-кB-dependent manner. These results show that ferroptosis is a form of inflammatory cell death related to the NF-кB signaling pathway. Most patients with CKD exhibit varying degrees of iron metabolism and lipid metabolism disorders. Renal iron deposition occurs spontaneously in CKD and is related to ischemia-, hypoxia-, and cytotoxicity-induced release of catalytic iron(Wang, Liu, Wang and Sun 2021). There were significant changes in ferroptosis-associated markers, including decreased expression of GPX4 and increased expression of ACSL4, as well as lipid peroxidation products and iron levels in CKD mice(Giuliani et al. 2022). Our results showed that ACSL4 was upregulated and xCT and GPX4 were downregulated in the kidney tissue of mice in the P.g group (Figure 5A-E). Cell viability was decreased (Figure 4G and H), and ferroptosis markers (ACSL4, xCT and GPX4) showed the same effects as in vivo (Figure 5F-J), indicating ferroptosis activation in CKD. Moreover, the viability of GMCs was reversed by Fer-1 (a ferroptosis inhibitor), and ferroptosis marker repression (ACSL4, xCT and GPX4) was partially inhibited, which suggested that ferroptosis was activated in GMCs in vivo andin vitro (Figure 6C-J) in CKD. More importantly, fer-1 inhibited p-p65, promoted p-IkBa and downregulated downstream NLRP3, Caspase 1, and IL-1β expression (Figure 6H-K), which indicated that fer-1 inhibited inflammation in GMCs via the NF-κB/NLRP3 signaling pathway. These observations indicated that P.g induced CKD pathogenesis through activation of the NF‑κB/NLRP3 pathway, which depended on ferroptosis in GMCs. Recent evidence indicates crosstalk between ferroptosis and the NF-κB/NLRP3 pathway in inflammatory diseases(Yao, Lan, Li, Wang and Qi 2023). Ferroptotic cells recruit immune cells by releasing damage-associated molecular patterns (DAMPs), which in turn activate the NF-κB pathway. Fer-1 improved sepsis-induced cardiac dysfunction via the NF-κB signaling pathway(Xiao et al. 2021). The iron chelator deferasirox inhibited NF-κB activity in hepatoma cells in a ferroptosis-dependent manner(Jomen et al. 2022). Overexpression of GPX4 in human T47D cells reduced NF-κB activation by inhibiting IκB phosphorylation(Kretz-Remy, Mehlen, Mirault and Arrigo 1996). To further clarify the mechanism, QNZ (NF-κB inhibitor) was used after GMCs were cocultured with MΦCM, and the results showed that the change in cell activity was reversed (Figure 7A-B) and that the NF-κB/NLRP3 pathway was inhibited in GMCs (Figure 7C-F). More importantly, the expression of ACSL4 was decreased, and the expression of GPX4 and xCT was increased, suggesting that ferroptosis in GMCs is regulated by the NF-κB/NLRP3 pathway (Figure 7G-I). Mechanistically, these findings suggest that periodontitis induced byP.g can activate crosstalk between the NF‑κB/NLRP3 pathway and ferroptosis in GMCs by stimulating M1 macrophage polarization during the progression of CKD.
In summary, our results indicate that P.g induces the pathological process of CKD by recruiting macrophages, promoting M1 macrophage polarization and releasing inflammatory factors, resulting in crosstalk between ferroptosis and the NF-κB/NLRP3 signaling pathway in GMCs. The study suggests the role of periodontitis in promoting CKD, which provides evidence of the importance of periodontitis therapy in the prevention and treatment of CKD. However, there are some limitations in our study. First, the mitochondrial function of GMCs was abnormal, which needs to be further studied. Second, the role of macrophages in the interaction between CKD and periodontal disease was unclear.