DECIPHERING ROLES OF GDNF-GFRA1 RET SIGNALING IN NORMAL AND INJURED KIDNEY

描述(申请人提供)：先天性、慢性和急性肾脏疾病对我们的社会造成严重的发病率、死亡率和经济负担。通过确定导致肾脏疾病的致病机制，将在所有这些领域产生重大影响。由于人类和小鼠都有许多肾脏发育和功能的特征，而肾脏损伤的动物模型概括了与人类相似的事件，因此基因工程小鼠模型为描绘肾脏疾病的分子机制提供了重要的工具。一种流行的观点认为，这些疾病中的许多是由基因功能异常引起的，这些基因在早期肾脏形成中也很重要。由于从肾脏发育开始就缺乏这些基因，往往会导致肾脏或其他异常和死亡，因此缺乏动物模型来研究这些基因在成年后肾脏疾病中的作用。胶质细胞源性神经营养因子(GDNF)及其辅助受体GFRA1和受体Ret构成了对肾脏发育至关重要的信号系统。它还调节几个组织特异性祖细胞的功能，并参与修复、再生和治疗许多疾病。然而，在正常和异常情况下，该信号系统在出生后肾脏中的生物学作用尚不清楚，因为它的缺失会导致肾脏发育不全和死亡。我们推测GDNF-GFRA1-Ret信号是成年小鼠肾脏维持所必需的，它在急性肾损伤(AKI)后的肾脏保护和再生中起重要作用，GDNF通过GFRA1-Ret信号通路对不同类型的损伤所致的AKI肾脏具有保护作用。我们已经生成了一些动物模型，这些模型对于检验这些假说是必要的。例如，为了克服致命性，我们开发了独特的小鼠模型，在最初的肾脏发育(条件小鼠)后，这种模型将使GFRA1或Ret信号失活。为了鉴定GFRA1或Ret在正常和疾病状态下的表达细胞，我们产生了“报告”小鼠。为了确定GDNF的治疗潜力，我们建立了对脑缺血再灌注损伤具有保护作用的系统给药方法。这项提案中特定目标1的目标将是使用GDNF、GFRA1和Ret报告小鼠来确定这些蛋白在正常出生后肾脏中的表达位置，以及它们在AKI期间的表达变化，特别是与修复和再生有关的变化。在具体目标2中，我们将使用GFRA1和Ret条件性小鼠来确定它们的缺失对成年小鼠和AKI正常肾功能的影响。在目标3中，我们将确定在不同的AKI模型中使用GFLS的外源性治疗是否具有肾脏保护作用，刺激再生，并通过GFRA1发生。通过这些研究，我们将确定这一途径在肾脏健康和疾病中的重要性。收集到的见解将对基础科学家、临床医生和受肾脏疾病影响的患者的广泛群体感兴趣。公共卫生相关性：GDNF-GFRA1-Ret信号通路对肾脏发育至关重要，并调节许多组织特异性干细胞的功能。在这项建议中，我们将利用新的动物模型来调节这一途径，并确定其在正常和疾病状态下出生后肾脏功能中的作用，并检查GDNF治疗是否可以保护肾脏免受肾功能衰竭的影响。这些研究将为损伤肾脏的修复和再生机制以及治疗提供新的见解。

DESCRIPTION (provided by applicant): Congenital, chronic and acute kidney diseases cause significant morbidity, mortality and economic burden to our society. A significant impact in all these areas will be made by identifying pathogenetic mechanisms that cause kidney diseases. Because humans and mice share a number of characteristics of kidney development and function, and animal models of renal injury recapitulate events that are similar to humans, genetically engineered mouse models offer an important tool to delineate molecular mechanisms of renal diseases. A prevailing idea is that a number of these conditions are caused by aberrant function of genes that are also important in early kidney formation. Because absence of these genes from the beginning of kidney development often results in renal or other abnormalities and lethality, there is scarcity of animal models to examine the role of these genes in kidney disease in adulthood. Glial cell line-derived neurotrophic factor (GDNF), its coreceptor Gfra1 and receptor Ret constitute a signaling system that is critical for kidney development. It also regulates function of several tissue specific progenitors and is implicated in repair, regeneration and therapy of a number of conditions. However, biological roles of this signaling system in the postnatal kidney in normal and abnormal conditions are not known since its absence results in renal agenesis and lethality. We hypothesize that GDNF-Gfra1-Ret signaling is required for kidney maintenance in adult mice, it is important in kidney protection and regeneration after acute kidney injury (AKI), and GDNF can protect kidneys from AKI due to different types of insults by acting through Gfra1-Ret pathway. We have generated a number of animal models that are necessary to test these hypotheses. For example, to overcome lethality we have developed unique mouse models that will inactivate Gfra1 or Ret signaling after initial kidney development (conditional mice). To identify Gfra1 or Ret expressing cells in normal and disease states, we have generated "reporter" mice. To determine therapeutic potential of GDNF, we have established systemic delivery method that has shown protection in ischemic-reperfusion injury to the brain. The objective of specific Aim 1 in this proposal will be to use GDNF, Gfra1 and Ret reporter mice to identify where these proteins are expressed in the normal postnatal kidney and how their expression changes during AKI particularly in relation to repair and regeneration. In Specific Aim 2 we will use Gfra1 and Ret conditional mice to determine the impact of their loss on normal renal function in adult mice, and in AKI. In Aim 3, we will determine if exogenous treatment with GFLs in different AKI models is renoprotective, stimulates regeneration and occurs through Gfra1. Through these studies we will determine the importance of this pathway in renal health and disease. The insights gleaned will be of interest to a broad group of basic scientist, clinicians and patients affected by renal diseases. PUBLIC HEALTH RELEVANCE: GDNF-Gfra1-Ret signaling pathway is critical for kidney development and regulates function of a number of tissue specific stem cells. In this proposal we will utilize novel animal models to modulate this pathway and determine its role in the function of postnatal kidney in normal and disease states and examine if treatment with GDNF can protect kidneys from renal failure. The studies will provide novel insights into mechanisms of repair and regeneration and therapy in injured kidneys.