Regulation of Intestinal Sodium Absorption in Health and Disease

我们的退伍军人患者群体非常容易患上慢性疾病，如与炎症相关的 肠道疾病（IBD），由于他们的年龄和许多共病。尽管医学取得了重大进步， IBD相关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相关的最衰弱的症状，其病理生理学中涉及的机制。 腹泻是由于肠分泌增加和/或水和电解质吸收减少所致。一 人体肠道电解质吸收的主要途径涉及Na+/H+（NHE）和Cl-的耦合作用 /HCO 3-交换器。研究表明，NHE 3在介导肠钠吸收中发挥关键作用 因为NHE 3敲除小鼠表现出结肠炎表型。此外，已经表明，NHE 3 KO小鼠比NHE 3敲除小鼠更容易被感染。 易患结肠炎。然而，到目前为止，对参与的分子机制知之甚少。 NHE 3在炎症相关性肠炎中的下调。为了阐明本 IBD相关腹泻中NHE 3表达下调的潜在机制，我们目前的研究表明， 研究主要集中在转录因子、HNF 4 β（肝核因子）和microRNA（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HNF 4 β辅激活因子调节HNF 4 β介导的NHE 3表达，p300 [具有内在的 组蛋白乙酰化酶（HAT）活性]和组蛋白脱乙酰酶（HDAC）的抑制。我们的初步研究还 显示了肠道NHE 3通过miRNA的调节。基于这些数据，我们假设HNF 4是一种 通过转录和转录后机制的相关辅调节因子和miRNA 在调节NHE 3基因表达中起重要作用，而NHE 3基因表达是脑梗死病理生理学的基础。 腹泻因此，本申请旨在研究NHE 3基因表达的调控 通过HNF 4 κ B、其相关的共调节因子和miRNA，利用体外细胞培养、离体人/小鼠 肠源性单层（目的1和2）和体内小鼠模型（目的3）。具体目标是：1。 阐明共激活子、共阻遏子和HNF 4 β乙酰化在HNF 4 β-乙酰化调节中的作用。 介导的NHE 3基因表达，并确定HNF 4 β减弱IFN β/TNF β的机制。 NHE 3; 2的诱导阻遏：研究NHE 3 3 ′ UTR（3 ′非翻译区）在响应NHE 3; 2的诱导阻遏中的作用。 miRNA模拟NHE 3并确定miR结合NHE 3的3个UTR在调节NHE 3中的作用 响应于miR结合位点中的突变或通过相应antigomirs的miR沉默的3： 检查HNF 4 β水平升高的治疗作用（通过喂食富含共轭亚油酸的饮食）， 特异性miRNA模拟物或antigomirs对正常和炎症条件下NHE 3表达和功能的影响 利用TNBS诱导的结肠炎小鼠模型在条件下进行。这些研究的结果不仅将提高我们的 了解NHE 3作为IBD相关腹泻治疗靶点的重要性，但也将 首次建立了HNF 4 β，其相关的辅助调节因子和miRNA在调节细胞凋亡中的作用。 肠NHE 3在正常和炎症条件下。

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.