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.