Heme-mediated Mitochondrial Injury, Senescence, Acute Kidney Injury and Chronic Kidney Disease

项目总结 衰老细胞(SCs)引起衰老，是急性肾损伤(AKI)的主要危险因素。SCS是细胞 周期停滞(由于上调的细胞周期抑制物p16INK4a和p21Cip1)，并表现出衰老- 相关分泌表型(SASP)，它是促炎和促凋亡的。感光剂，杀伤剂 SCS，目前正在进行临床试验。我们在血红素蛋白介导的AKI模型(HP-AKI)中证明了衰老 如多个指数所示。这种变化的意义--无论是有害的还是保护性的--都是未知的 致敬AKI。AKI领域认识到线粒体损伤驱动AKI，而衰老领域 认识到线粒体损伤会导致衰老；这一应用将这两个概念独特地结合在一起。 在HP-AKI早期，我们证明线粒体受损，其血红素含量增加；正常 暴露在这种血红素含量下的线粒体就会停止功能。血红素是一种促氧化剂四吡咯，存在于 当血红素蛋白由于细胞压力而不稳定时，无处不在的血红素蛋白家族被释放。 我们还证明了血红素诱导p16INK4a/p21Cip1和SASP，并抑制pGC-1α。在探索中 血红素诱导的线粒体损伤和p16INK4a/p21Cip1的诱导，我们主要关注两个转录因子 两者都受到线粒体损伤的上调，一个是p16INK4a的诱导者ETS1，另一个是诱导者ATF4 P21Cip1。我们的初步数据表明，这4个主要分子(ETS1、ATF4、p16INK4a、p21Cip1) 均可在HP-AKI、血红素暴露的体外肾小管上皮细胞和肾脏中诱导 缺血再灌注损伤(IRI)。我们的假设是AKI是由血红素介导的线粒体损伤引起的 以及随之而来的衰老，这是一篇需要在三个目标上进行测试的论文。目的一：确定血红素介导的作用 线粒体损伤主要表现在ETS1和ATF4的表达、衰老和AKI。利用体内的互补作用 在体外方法中，我们将顺序检查血红素介导的线粒体损伤的作用； ETS1、ATF4和ETS1/ATF4非依赖途径的贡献以及衰老在 阿琪。目的II：明确p21Cip1和p16INK4a在AKI：遗传策略中的作用。P21Cip1在AKI中的作用 通过诱导高表达p21Cip1的细胞和近端小管特异的p21KO进行检测 老鼠。P16INK4a的作用将通过诱导高表达p16INK4a的细胞和通过 可诱导近端小管特异性缺失高表达p16INK4a的细胞。目标三：界定 AKI中的感光剂。SCs存活是因为上调的抗凋亡途径，而感觉剂杀死SCs但 不是非SCs。这一目的是为了检验感觉神经降解剂在AKI中的有效性，以及在降低老龄小鼠的敏感性方面的效果。 致AKI。总之，重新提交的R01检查了AKI的衰老，顺序地将其与血红素介导的衰老联系起来 线粒体损伤，转录因子ETS1和ATF4，p16INK4a和p21Cip1。这款R01提供了新颖的 关于衰老在AKI发病机制中的作用以及增龄剂的治疗作用的见解。

PROJECT SUMMARY Senescent cells (SCs) cause senescence, a dominant risk factor for acute kidney injury (AKI). SCs are cell cycle-arrested (due to upregulated cell cycle inhibitors p16Ink4a and p21Cip1) and display a senescence- associated secretory phenotype (SASP) which is proinflammatory and proapoptotic. Senolytics, agents that kill SCs, are now in clinical trials. We demonstrate senescence in the heme protein-mediated AKI model (HP-AKI) as indicated by multiple indices. The significance of such changes – injurious or protective – is unknown as regards AKI. The AKI field recognizes that mitochondrial injury drives AKI, while the senescence field recognizes that mitochondrial injury elicits senescence; this application uniquely unites these two concepts. Early in HP-AKI, we demonstrate that mitochondria are injured and their heme content increased; normal mitochondria, exposed to such heme content, cease functioning. Heme, a prooxidant tetrapyrrole, present in the ubiquitous family of heme proteins, is freed when heme proteins are destabilized because of cellular stress. We also demonstrate that heme induces p16Ink4a/p21Cip1 and a SASP, and suppresses PGC-1α. In exploring heme-induced mitochondrial injury and induction of p16Ink4a/p21Cip1, we focused on two transcription factors both upregulated by mitochondrial injury, one, ETS1, being an inducer of p16Ink4a, the other, ATF4, an inducer of p21Cip1. Our preliminary data demonstrate that these 4 principal molecules (ETS1, ATF4, p16Ink4a, p21Cip1) are all induced in HP-AKI; in heme-exposed renal proximal tubular epithelial cells in vitro; and in renal ischemia-reperfusion injury (IRI). Our hypothesis is that AKI results from heme-mediated mitochondrial injury and ensuing senescence, a thesis to be tested in three aims. Aim I: Define the role of heme-mediated mitochondrial injury in ETS1 and ATF4 expression, senescence, and AKI. Using complementary in vivo and in vitro approaches, we will sequentially examine the role of heme-mediated mitochondrial injury; the contribution of ETS1, ATF4, and ETS1/ATF4-independent pathways; and the involvement of senescence in AKI. Aim II: Define the roles of p21Cip1 and p16Ink4a in AKI: Genetic strategies. The role of p21Cip1 in AKI will be examined by inducible deletion of high p21Cip1-expressing cells and with proximal tubule-specific p21 KO mice. The role of p16Ink4a will be examined by inducible deletion of high p16Ink4a-expressing cells and by inducible proximal tubule-specific deletion of high p16Ink4a-expressing cells. Aim III: Define the effect of senolytics in AKI. SCs survive because of upregulated anti-apoptotic pathways, while senolytics kill SCs but not non-SCs. This aim examines the efficacy of senolytics in AKI, and in reducing the sensitivity of aged mice to AKI. In sum, this resubmitted R01 examines senescence in AKI, linking it sequentially to heme-mediated mitochondrial injury, the transcription factors ETS1 and ATF4, and p16Ink4a and p21Cip1. This R01 offers novel insights regarding the role of senescence in the pathogenesis of AKI and the therapeutic utility of senolytics.