IMCD phosphodiesterase in pregnancy; role in ECFVE

描述（由申请人提供）：大的、持续的血浆容量扩张（PVE）是正常、成功妊娠的必要特征。次优PVE与不良胎儿结局相关，是子痫前期的一个组成部分。该项目的目标是确定正常妊娠期PVE的主要机制，从而最终预防与PVE失败相关的妊娠并发症。这一建议的动机是，正常妊娠的肾脏和血流动力学反应与不适当PVE状态（如肾病或肝硬化）的肾脏和血流动力学反应有几个显著的相似之处。病理性钠潴留的主要肾脏机制是依赖cGMP作为第二信使的尿钠机制的失败，由于磷酸二酯酶（PDE）对cGMP的水解增加。在这里，我们将验证这样一种假设，即妊娠期间肾小管PDE系统（尤其是髓内集管，IMCD）的选择性上调，导致对利钠刺激（如急性容量扩张和心房利钠肽）的广泛难耐，从而导致PVE。我们还将描述参与肾脏对正常P反应的PDE的亚型和位置，并确定妊娠期间肾脏PDE活性的增加是由翻译事件还是翻译后事件引起的。我们将研究血管紧张素II和肾神经活动（主要肾钠保留系统）是如何参与的；我们将检验妊娠期PVE的“欠填充”假说，并确定母体激素黄体酮、催乳素或松弛素是否对肾PDE活性和p的PVE升高起主要作用。最后，我们将在妊娠期间慢性和局部抑制相关PDE，以确定其对母体血流动力学和胎儿结局的影响。所有的研究都将在大鼠身上进行，使用体内技术来测量肾脏钠处理能力，并结合体外技术对分离的肾小球、近端小管和髓内收集管细胞进行检测，包括酶活性测定、Western blot、免疫细胞化学、Northern blot或RNAse保护测定和原位杂交。

DESCRIPTION (provided by applicant): A large, sustained plasma volume expansion (PVE) is a necessary feature of normal, successful pregnancy. Suboptimal PVE is associated with adverse fetal outcome, and is a component of preeclampsia. The goal of this project is to determine the primary mechanism of the normal gestational PVE, so that ultimately, complications of pregnancy associated with failure to PVE can be prevented. This proposal is motivated by several marked similarities between the renal and hemodynamic responses to normal pregnancy, and those seen in states of inappropriate PVE, such as nephrosis or cirrhosis. The primary renal mechanism in pathological sodium retention is a failure of natriuretic mechanisms that rely on cGMP as second messenger, due to increased cGMP hydrolysis by phosphodiesterase (PDE). Here, we will test the hypothesis that selective upregulation of the renal tubular PDE system (particularly the inner medullary collecting duct, IMCD) occurs in pregnancy and causes widespread refractoriness to natriuretic stimuli, such as acute volume expansion and administered atrial natriuretic peptide, leading to PVE. We will also characterize the isoforms and location in the kidney of the PDEs involved in the renal response to normal P and determine whether increased renal PDE activity in pregnancy results from translational or posttranslational events. We will investigate how angiotensin II and renal nerve activity (major renal sodium retaining systems) are involved; we will test the "underfill" hypothesis of gestational PVE and we will determine whether the maternal hormones progesterone, prolactin or relaxin are primarily responsible for the increased renal PDE activity and PVE of P. Finally, we will chronically and locally inhibit the relevant PDE(s) during pregnancy to establish the impact on maternal hemodynamics and fetal outcome. All studies will be in rat, using a combination of in vivo techniques to measure renal sodium handling capacity and in vitro techniques on isolated glomeruli, proximal tubules and inner medullary collecting duct cells, to include enzyme activity assays, Western blot, immunocytochemistry, Northern blot or RNAse protection assay and in situ hybridization.