High Urinary Phosphate Induces TLR4-mediated Inflammation and Cystogenesis in Polycystic Kidney Disease

项目概要/摘要 常染色体显性多囊肾病（ADPKD）是一种遗传性疾病，其特征是存在 大量充满液体的囊肿、间质炎症和纤维化导致肾脏功能进行性衰退 功能。新的证据表明尿磷酸盐 (Pi) 促进囊肿发生并激活先天性 免疫反应。在之前的研究中，高 Pi 饮食被证明会增加肾脏 CaPi 纳米晶体沉积 并在 ADPKD 非直系同源啮齿动物模型中加速囊肿生长；而饮食Pi的限制 减缓疾病进展。 ADPKD 中囊肿的持续生长会导致功能性肾单位的丧失和 未受影响的肾单位的代偿性超滤增加单个肾单位 Pi 排泄以保护 Pi 平衡。过饱和的肾小管内 Pi 水平可导致形成 CaPi 纳米晶体，从而导致肾小管 损伤和炎症反应。最近，先天免疫的关键组成部分 Toll 样受体 4 (TLR4) 已被认为介导高尿 Pi 诱导的 NF-κB 激活和肾小管损伤；然而，角色 TLR4 作为高 Pi 诱导的 PKD 加速的主要介质尚未被研究。决心，一个家庭 内源性脂质衍生物，通过抑制 NF-κB 停止损伤后的先天免疫反应，并减少 通过增加巨噬细胞的吞噬作用来沉积肾矿物质晶体。目前，治疗效果 的解析素尚未在 PKD 中进行评估。在初步研究中，人类 ADPKD 肾脏升高 与正常人肾脏相比，囊性衬里细胞中有晶体沉积和更高的 TLR4 表达。 将人 ADPKD 细胞与 CaPi 纳米晶体一起孵育会增加 TNF-α mRNA，表明 CaPi 纳米晶体 刺激TLR4/NF-κB信号通路。挑战 Pkd1RC/RC 小鼠，一种直系同源 ADPKD 模型，具有 高Pi饮食加速囊肿生长并增加损伤、炎症和纤维化标志物。我们还发现 短期施用 resolvin D1 降低了肾脏炎症标志物的表达，包括 TNF-α， Pkd1RC/RC 小鼠中的 IL-1β 和 MCP-1。因此，我们假设高尿 Pi 导致 TLR4/NF-κB- ADPKD 中依赖的先天免疫反应，并且使用 resolvins 抑制 NF-κB 可减轻疾病 进展。拟议的实验将确定：（1）TLR4 在介导高饮食影响中的作用 Pi 对囊肿发生的影响，以及 (2) 使用消退素抑制先天免疫反应是否可以预防 PKD 进展。 PI 将与负责研究的副总裁办公室密切合作，并协调战略 由负责多元化和包容性的副总裁领导，制定并实施强有力的招聘计划 多样化的本科生。 PI具有极大的热情和成功的培训经验 本科生和研究生。该提案将：（1）为本科生提供更多机会 密歇根理工大学的学生参加生物医学研究，（2）奠定基础 PI 未来对 PKD 领域的贡献，以及 (3) 通过引入使大学研究基础设施多样化 先天免疫和 PKD。

PROJECT SUMMARY/ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the presence of numerous fluid-filled cysts, interstitial inflammation, and fibrosis leading to a progressive decline in kidney function. Emerging evidence suggests that urinary phosphate (Pi) promotes cystogenesis and activates innate immune responses. In previous studies, a high Pi diet was shown to increase renal CaPi nanocrystal deposition and accelerated cyst growth in a non-orthologous rodent model of ADPKD; whereas the restriction of dietary Pi attenuated disease progression. Relentless cyst growth in ADPKD leads to the loss of functional nephrons and compensatory hyperfiltration of unaffected nephrons increases single nephron Pi excretion to preserve Pi balance. Supersaturated intratubular Pi levels can lead to the formation of CaPi nanocrystals that cause tubule damage and inflammatory responses. Recently, toll-like receptor 4 (TLR4), a key component of innate immunity, has been suggested to mediate high urinary Pi-induced NF-κB activation and tubule damage; however, the role of TLR4 as the primary mediator in high Pi-induced PKD acceleration has not been studied. Resolvins, a family of endogenous lipid derivatives, cease innate immune responses after injury through inhibiting NF-κB and reduce renal mineral crystal deposition by increasing phagocytosis of macrophages. Currently, the therapeutic effects of resolvins have not been evaluated in PKD. In preliminary studies, human ADPKD kidneys have elevated crystal deposition and higher TLR4 expression in cystic lining cells compared with normal human kidneys. Incubation of human ADPKD cells with CaPi nanocrystals increased TNF-α mRNA suggesting CaPi nanocrystals stimulate TLR4/NF-κB signaling pathway. Challenging Pkd1RC/RC mice, an orthologous ADPKD model, with a high Pi diet accelerated cyst growth and increased injury, inflammation, and fibrosis markers. We also found that short-term administration of resolvin D1 reduced the renal expression of inflammation markers including TNF-α, IL-1β, and MCP-1, in Pkd1RC/RC mice. Thus, we hypothesized that high urinary Pi leads to TLR4/NF-κB- dependent innate immune responses in ADPKD, and that inhibition of NF-κB using resolvins attenuates disease progression. The proposed experiments will determine: (1) the role of TLR4 in mediating the effect of high dietary Pi on cystogenesis, and (2) if inhibiting innate immune response using resolvins can prevent PKD progression. The PI will work closely with the Office of the Vice President for Research and in coordination with the strategies led by the Vice President for Diversity and Inclusion to develop and implement a robust recruitment plan for diverse undergraduate students. The PI has tremendous enthusiasm and successful experience in training undergraduate and graduate students. This proposal will: (1) provide more opportunities for undergraduate students at Michigan Technological University to participate in Biomedical Research, (2) lay the groundwork for the PI’s future contributions to the fields of PKD, and (3) diversify university research infrastructure by introducing innate immunity and PKD.