Environmentally relevant doses of fluoride exposure mediate kidney injury by affecting Tregs/Teffs homeostasis.

Introduction

Fluorine is the 13th most abundant chemical element on Earth and is widely distributed in geological environments. It is released into groundwater through the gradual weathering of fluoride-containing rocks and minerals [1]. A total of 25 countries and regions worldwide are affected by fluorosis due to the consumption of excessively fluoridated water. Drinking water type of fluorosis impacts over 70,000 villages across 28 provincial-level administrative regions in China [2,3]. The World Health Organization (WHO) recommends a permissible limit of 1.5 mg/L for fluoride in drinking water [4]; however, this standard is based on historical evidence of skeletal tissue damage. Recent studies indicate that even exposure to fluoride levels below 1.5 mg/L can lead to adverse health outcomes in non-skeletal tissues [5]. Among these, the kidney—a target organ of fluoride—has received considerable attention regarding its injury response [[6], [7], [8], [9]]. However, current research has largely focused on the effects of fluoride on renal function in adolescents, while potential risks to adult kidney health under contemporary exposure conditions have not been adequately addressed [9]. Fluoride that accumulates in the kidneys can stimulate the expression of inflammatory cytokines such as IL-1B, IL-6, and TNF-I, triggering inflammatory responses. If left uncontrolled, persistent fluoride exposure may exacerbate renal pathological damage and dysfunction, creating a vicious cycle [10,11]. Therefore, it is crucial to identify risk factors that increase susceptibility to renal injury among residents in fluorosis-endemic areas, enabling early screening, timely diagnosis, and intervention to reduce disease incidence and slow the progression of kidney impairment.

The kidneys and the immune system are interdependent, an interaction that also makes the kidney susceptible to damage resulting from immune homeostasis imbalance [12,13]. Excessive activation of the immune system and inflammatory infiltration are significant contributors to renal functional impairment [14,15]. Multiple immune cell types, including T cells, macrophages, collectively participate in the complex process of kidney injury [16]. In animal models of fluorosis, fluoride can promote a shift in the renal Th1/Th2 balance toward Th1 polarization, while simultaneously increasing the number of macrophages in the kidney and promoting their differentiation toward the M1 phenotype [17]. Our previous studies have shown that fluoride exposure increases the risk of imbalance in the proportions of peripheral blood CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and regulatory T cells (Tregs) among residents in fluoride-endemic areas [18,19]. In addition, adoptive transfer of Tregs can alleviate fluoride-induced kidney injury in rats [20]. These findings suggest that immune cells play a critical role in maintaining renal immune homeostasis. As important members of this network, Tregs play a central role in preserving renal immune homeostasis.

Tregs are defined as immune cells that exert anti-inflammatory functions by suppressing the pro-inflammatory effects of effector T cells (Teffs). The balance between anti-inflammatory and pro-inflammatory responses is crucial for maintaining systemic immune homeostasis [21]. Tregs inhibit Teffs activation and proliferation through multiple mechanisms, thereby suppressing inflammation and promoting tissue repair [22]. Studies have shown that the expansion of functionally stable Tregs in vivo using IL-2 complexes (IL-2C) effectively inhibits the development of renal fibrosis in mice [23]. Moreover, a randomized controlled trial demonstrated that blocking IL-6 effectively enhanced the ability of Tregs to suppress Teffs, thereby controlling inflammation in kidney transplantation [24]. However, abnormal Tregs activation can impair their capacity to restrain Teffs-mediated inflammatory responses in the kidneys [25]. Thus, maintaining Tregs development, numerical homeostasis, and suppressive function toward Teffs is critical for mitigating renal injury and promoting recovery. Our previous studies have shown that long-term exposure to fluoride in drinking water at concentrations ?4?mg/L can induce peripheral recruitment of Tregs and disrupt Tregs/Teffs balance, thereby contributing to inflammatory processes [18,19].

The Nuclear Factor kappa B (NF-kB) family has been demonstrated to participate in the development, activation, and functional maintenance of Tregs [26]. NF-kB complexes containing the P65 or c-Rel subunits play key roles in immune responses and immune cell activation [27]. Deficiency of P65 in Tregs leads to systemic autoimmune syndrome, while loss of c-Rel further exacerbates this condition, indicating that the P65 and C-rel subunits of NF-kB are involved in regulating Tregs homeostasis and thereby influence self-immune tolerance. Numerous animal studies have shown a direct relationship between fluoride accumulation and renal injury. Fluoride rapidly transitions NF-kB from an inhibited to an activated state and induces its downstream pathways, contributing to the process of kidney damage [11,28].

This study aims to dynamically monitor the regulation of Tregs/Teffs balance across different stages of renal injury through field investigations and animal experiments with varying fluoride exposure doses and durations. It further seeks to elucidate the molecular mechanisms by which Tregs/Teffs imbalance mediates fluoride-induced renal dysfunction, thereby providing epidemiological and experimental evidence for identifying precise diagnostic and therapeutic targets for kidney injury caused by environmentally relevant fluoride exposure. Additionally, a risk prediction model for renal dysfunction mediated by Tregs/Teffs imbalance will be established to offer a quantitative tool for early warning of renal abnormalities in fluoride-exposed populations. Ultimately, this research aims to propose novel strategies for developing diagnostic biomarkers and targeted therapies for kidney injury induced by low-to-moderate fluoride exposure.