Regulation of NHE3 by the mTOR/autophagy pathways in diarrheal diseases

描述（由申请人提供）：慢性腹泻和体重减轻伴发作性重度急性腹泻经常发生在器官移植受者中，与使用免疫抑制剂有关。严重形式的腹泻需要多次住院治疗，费用昂贵，影响患者的生活质量，并显著增加移植物丢失和患者死亡的风险。西罗莫司（雷帕霉素）在器官移植中经常用作免疫抑制剂。与使用雷帕霉素有关的腹泻发生率从25%到42%不等。在腹泻的各种原因（如感染）中，很大一部分器官受者出现与使用雷帕霉素相关的腹泻，原因不明。因此，了解雷帕霉素诱发腹泻的机制至关重要。雷帕霉素的作用是通过抑制哺乳动物雷帕霉素靶蛋白通路（mTOR）介导的。雷帕霉素抑制mTOR可刺激自噬活性。因此，mTOR和自噬途径是介导雷帕霉素诱导的腹泻的有力候选途径，但mTOR和/或自噬在腹泻疾病中的确切机制仍完全不清楚。刷状边界Na+/H+交换器3 （NHE3）主要位于肠的顶端膜，已知负责大部分Na和水的吸收。NHE3的缺失足以引起腹泻。因此，我们假设雷帕霉素治疗改变了mTOR和自噬途径，这反过来又影响了NHE3，导致腹泻。事实上，我们的初步数据证明了雷帕霉素在腹泻患者和动物中抑制NHE3的直接作用。本研究的目的是利用转基因小鼠模型进一步确定雷帕霉素对NHE3的调节是否通过肠上皮细胞的mTOR和自噬途径介导。我们将使用肠上皮细胞特异性mTOR和Atg7敲除（mTOR-/-和Atg7-/-）小鼠，我们将研究NHE3在mTOR-/-和Atg7-/-小鼠（Aim IA和IIA）中的表达是否改变，以及雷帕霉素在mTOR-/-和Atg7-/-小鼠（Aim IB和IIB）中是否失去作用。本研究结果为mTOR和自噬通路在调控根尖膜转运蛋白NHE3中的作用提供了新的见解，并可能有助于开发新的腹泻疾病治疗策略。

DESCRIPTION (provided by applicant): Chronic diarrhea and weight loss with episodic profound acute diarrhea frequently occur in organ transplant recipients related to the use of immunosuppressants. Severe forms of diarrhea require multiple hospitalizations, which are costly, affect patients' quality of life and significantly increase the risk of graft loss and patint death. Sirolimus (rapamycin) is frequently used as an immunosuppressant in organ transplantations. The incidence of diarrhea related to rapamycin use varies from 25% to 42%. Among the various causes of diarrhea (e.g., infection), a significant portion of organ recipients experience diarrhea associated with the use of rapamycin without a clear cause. It is therefore of paramount importance to understand the mechanisms underlying rapamycin-induced diarrhea. Rapamycin's action is mediated by inhibiting the mammalian Target of Rapamycin pathway (mTOR). Inhibition of mTOR by rapamycin stimulates autophagy activity. Thus, the mTOR and autophagy pathways are strong candidates for mediating rapamycin- induced diarrhea, but the exact mechanism of mTOR and/or autophagy in diarrheal disease remains entirely unclear. The brush border Na+/H+ exchanger 3 (NHE3), primarily residing at the apical membrane in the intestine, is known to be responsible for the majority of Na and water absorption. Deletion of NHE3 is sufficient to cause diarrhea. We thus hypothesize that rapamycin treatment alters the mTOR and autophagy pathways, which in turn impinge on NHE3, resulting in diarrhea. Indeed, our preliminary data demonstrated a direct role of suppression of NHE3 by rapamycin in diarrheal patients as well as in animals. The objective of this proposal is to use genetically modified mouse models to further pinpoint if NHE3 regulation by rapamyicn is mediated by the mTOR and autophagy pathways in intestinal epithelial cells. We will use the intestinal epithelial cell- specific mTOR and Atg7 knockout (mTOR-/- and Atg7-/-) mice, with which we will ask if NHE3 expression is altered in mTOR-/- and Atg7-/- mice (Aim IA and IIA) and if rapamycin loses its effect in mTOR-/- and Atg7-/- mice (Aim IB and IIB). The results from this proposal could provide new insights into the roles of the mTOR and autophagy pathways in regulation of the apical membrane transporter NHE3, and may aid in the development of new therapeutic strategies for diarrheal diseases.