Regulation of Intestinal Sodium Absorption in Health and Disease

我们的资深患者群体非常容易患上与炎症性疾病相关的腹泻疾病 肠道疾病(IBD)由于他们的年龄和许多并存的疾病。尽管医学上取得了重大进步， IBD相关性腹泻的治疗仍然具有挑战性。因此，重要的是要了解 腹泻是与IBD相关的最虚弱的症状，其机制与腹泻的病理生理学有关。 腹泻是由于肠道分泌物增加和/或水和电解质吸收减少所致。一个 人体肠道电解质吸收的主要途径是Na/H(NHE)和Cl-的耦合作用 /HCO3-交换器。研究表明，NHE3在调节肠道钠吸收中起关键作用 AS NHE3基因敲除小鼠表现为腹泻型。此外，已有研究表明，NHE3KO小鼠 易患结肠炎的。然而，到目前为止，人们对涉及到的分子机制知之甚少。 NHE3在腹泻中的下调与炎症相关。为了澄清这个问题 IBD相关性腹泻中NHE3表达下调的机制 研究集中在转录因子、HNF4(肝细胞核因子)和microRNAs(MiRNAs)的作用上。 已知在IBD的发病机制中起关键作用。我们广泛的初步数据提供了强有力的证据 对于HNF4共激活子对HNF4介导的NHE3表达的调节，p300[具有内在的 组蛋白乙酰酶(HAT)活性]和组蛋白脱乙酰酶(HDAC)的抑制。我们的初步研究也 显示了miRNAs对肠道NHE3的调节作用。根据这些数据，我们假设HNF4，其 转录和转录后机制中的相关共调控因子和miRNAs 在调节NHE3基因表达中发挥重要作用，而NHE3基因表达是NHE3基因表达的病理生理基础 拉肚子。因此，目前的应用旨在研究NHE3基因表达的调节 通过HNF4miRNA、其相关的协同调控因子和利用体外细胞培养、体外人/鼠 肠样来源的单层(目标1和2)和活体小鼠模型(目标3)。具体目标是：1. 阐明共激活子(S)、共抑制子(S)和HNF4乙酰化在HNF4- HNF4抑制干扰素/肿瘤坏死因子的机制 NHE3诱导的抑制；2：研究NHE3 3非翻译区(3非翻译区)在反应中的作用 MiRNA在NHE3上的模拟及其与NHE3的3非编码区的结合在调节NHE3中的作用 对miR结合位点突变或miR沉默的反应的表达和3： 研究增加HNF4水平的治疗作用(通过饲喂高共轭亚油酸饮食)和 特异性miRNA对正常和炎症状态下NHE3表达和功能的影响 条件利用TNBS诱导的小鼠结肠炎模型。这些研究的结果不仅将加强我们的 了解NHE3作为IBD相关性腹泻治疗靶点的重要性，但也将 首次确定了HNF4、其相关的协同调节因子和miRNA在调节 正常和炎症条件下的肠道NHE3。

Our veteran patient population is very vulnerable to diarrheal illnesses such as associated with inflammatory bowel diseases (IBD) due to their age and numerous co-morbidities. In spite of significant medical advances, the treatment of IBD-associated diarrhea still remains challenging. Therefore, it is important to understand the mechanisms involved in the pathophysiology of diarrhea, the most debilitating symptom associated with IBD. Diarrhea results from increased intestinal secretion and/or decreased absorption of water and electrolytes. A major route of electrolyte absorption in the human intestine involves coupled operation of Na+/H+ (NHE) and Cl- /HCO3- exchangers. Studies have shown NHE3 to play a critical role in mediating intestinal sodium absorption as NHE3 knockout mice exhibit diarrheal phenotype. Also, it has been shown that NHE3 KO mice are more susceptible to colitis. To date, however, very little is known about the molecular mechanisms involved in the downregulation of NHE3 in diarrhea-associated with inflammation. In order to elucidate the mechanisms underlying the down-regulation of NHE3 expression in IBD-associated diarrhea, our current studies focused on the role of transcription factor, HNF4 (hepatic nuclear factor) and microRNAs (miRNAs) known to play a crucial role in the pathogenesis of IBD. Our extensive preliminary data provide strong evidence for the regulation of HNF4-mediated NHE3 expression by HNF4 co-activator, p300 [possess intrinsic histone acetylase (HAT) activity] and inhibition of histone deacetylases (HDACs). Our preliminary studies also showed the modulation of intestinal NHE3 by miRNAs. Based on these data, we hypothesize that HNF4, its associated co-regulators and miRNAs via both transcriptional and post-transcriptional mechanisms play important roles in modulating NHE3 gene expression that underlie the pathophysiology of diarrhea. The current application is, therefore, designed to investigate the regulation of NHE3 gene expression by HNF4, its associated co-regulators and miRNAs utilizing both in vitro cell culture, ex-vivo human/mouse enteroid-derived monolayers (Aims 1 & 2) and in vivo mouse models (Aim 3). The Specific Aims are: 1. Elucidate the role of co-activator (s), co-repressor (s) and HNF4 acetylation in the modulation of HNF4- mediated NHE3 gene expression and determine the mechanisms by which HNF4 attenuates IFN/TNF- induced repression of NHE3; 2: Investigate the role of NHE3 3UTR (3 untranslated region) in response to miRNA mimics on NHE3 and establish the role of miR binding to 3UTR of NHE3 in regulating NHE3 expression in response to mutations in the miR-binding sites or miR silencing by respective antigomirs and 3: Examine the therapeutic role of increased HNF4 levels (by feeding conjugated linoleic acid rich diet) and specific miRNA mimics or antigomirs on NHE3 expression and function under normal and inflammatory conditions utilizing TNBS-induced colitis mouse model. The outcome of these studies will not only enhance our understanding of the importance of NHE3 as a therapeutic target for IBD-associated diarrhea, but will also establish for the first time, the role of HNF4, its associated co-regulators and miRNAs in the modulation of intestinal NHE3 under normal and inflammatory conditions.