Identification of new therapeutic targets for ADPKD

常染色体显性遗传性多囊肾病(ADPKD)影响着全球超过1200万人，目前 终末期肾病(ESKD)的常见原因。ADPKD是由两个基因中的一个基因突变引起的， PKD1或PKD2，分别编码多囊蛋白1(PC)和PC2。丢失PC1或PC2会导致 激活许多激酶和下游信号通路，这是致病机制的核心。 ADPKD中的囊性生长。此外，从药物上抑制一些不同的激酶在 在PKD的动物模型中，PKD肾脏被证明可以减缓囊泡的生长，PKD产生的激酶抑制剂包括 治疗ADPKD患者最有希望的候选药物。然而，虽然人类亲属 由500多个激酶组成，其中只有一小部分经过测试以确定它们是否发挥了 在ADPKD发病机制中的作用。因此，ADPKD肾脏中可能存在许多活跃的激酶 在尚未发现的、可能是很好的治疗靶点的囊肿生长过程中起着重要作用。 在坦普尔大学的詹姆斯·邓肯的合作下，我们现在已经采用了一种新的方法来 以一种公正的方式广泛筛查PKD肾脏，以寻找在 PKD肾脏与野生型肾脏裂解物的比较。在本实验中，活性激酶具有亲和力。 通过将裂解物传递到含有激酶抑制剂鸡尾酒的多重抑制剂珠子(MIB)上而捕获。 然后用LC分离和定量串联质谱仪(LC-MS)鉴定结合的激酶。 MS/MS)。使用这种方法，我们现在已经确定了一些已知和未知的激酶 特别是在PKD肾脏中上调和下调。 这项提案的主要目标是评估到目前为止已确定的几种其活性的激酶的作用。 在PKD肾脏中增加，并确定抑制或激活任何已识别的激酶是否减慢 在ADPKD动物模型中，可抑制间质纤维化，保护肾功能。到时候我们会的 采取有偏见和无偏见的方法来确定由这些激酶调节的信号通路 是发病机制的关键，其目标是形成对相关信号中枢的更完整的图景 以及在PKD肾脏中异常激活的网络。此外，我们将使用这项技术来广泛地 从不同早期和晚期的ADPKD小鼠模型中筛选PKD肾脏 在人类ADPKD患者的肾脏中无偏见地识别其他 PKD肾脏中被激活和被抑制的激酶以确定两者之间的异同 小鼠模型，哪些激酶可能与人类疾病最相关，以及不同的激酶组是否 在囊肿诱导期后早期被激活，是囊肿长出的早期“驱动力”。最终，我们希望这是 新的信息将确定新的安全药物靶点和合理的联合治疗方法来减缓 然后可以转化为临床试验的包囊生长。

Autosomal-dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide and is a common cause of end-stage kidney disease (ESKD). ADPKD is caused by mutations in one of two genes, PKD1 or PKD2, which encodes polycystin 1 (PC) and PC2 respectively. Loss of PC1 or PC2 results in activation of numerous kinases and downstream signaling pathways, which is central to the pathogenesis of cyst growth in ADPKD. In addition, pharmacologically inhibiting a number of different kinases up-regulated in PKD kidneys has been shown to slow cyst growth in animal models of PKD making kinase inhibitors among the most promising class of drug candidates to treat patients with ADPKD. However, while the human kinome consists of more than 500 kinases, only a fraction of these kinases have been tested to determine if they play a role in ADPKD pathogenesis. As a result, there are likely many kinases that are active in ADPKD kidneys that play prominent roles in cyst growth that are yet-to-be discovered and may be good therapeutic targets. In collaboration with James Duncan at Temple University, we have now adapted a novel approach to broadly screen PKD kidneys in an unbiased manner for kinases that are more active in lysates from PKD kidneys compared with lysates from wild type kidneys. In this assay, active kinases are affinity captured by passing lysates over multiplex inhibitor beads (MIB) containing a cocktail of kinase inhibitors. Bound kinases are then identified by LC separation followed by quantitative tandem mass spectrometry (LC- MS/MS). Using this approach, we have now identified a number of both known and unknown kinases specifically up-regulated and down-regulated in PKD kidneys. The major goal of this proposal is to assess the role of several of the kinases identified thus far whose activity is increased in PKD kidneys and determine whether inhibiting or activating any of the kinases identified slows cyst growth, inhibits interstitial fibrosis, and preserves renal function in animal models of ADPKD. We will then take both biased and unbiased approaches to identify the signaling pathways regulated by these kinases that are critical to pathogenesis with the goal of developing a more complete picture of the relevant signaling hubs and networks that are aberrantly activated in PKD kidneys. In addition, we will use this technology to broadly screen PKD kidneys from a variety of different “early” and “late” mouse models of ADPKD at different stages in cyst formation and in kidneys from human patients with ADPKD to identify in an unbiased manner additional kinases that are activated and inhibited in PKD kidneys to determine the similarities and differences between mouse models, which kinases may be most relevant to human disease, and whether distinct sets of kinases are activated early post cyst induction and function as early “drivers” of cyst growth. Ultimately, we hope this new information will identify new safe drug targets and rational approaches to combination therapies to slow cyst growth that can then be translated into clinical trials.