Autophagy in Polycystic Kidney Disease (PKD)

自噬是在所有真核细胞中发生的一个过程，使细胞在压力条件下保持存活。在……里面 自噬：将受损的细胞器隔离到双膜自噬小体中， 随后与溶酶体融合，它们的货物被运送到那里进行降解和回收。健康的人 在肾脏中，自噬对肾小管上皮细胞的动态平衡和活性起着重要的作用。的确有 PKD是一种被抑制的自噬的间接证据：1)许多保护PKD的药物是 自噬诱导剂。2)初步/已发表的数据显示PKD1-/-小鼠的自噬通量减少 肾脏和PC1-/-细胞。3)自噬和细胞凋亡之间存在着相互作用，而细胞凋亡是包囊生长的一个关键过程。 总体假设是自噬通量(自噬小体-溶酶体融合和降解)减少。 在多囊肾早期，阻断自噬的干预将使PKD恶化，反之亦然 自噬诱导剂，如热量限制，2-脱氧葡萄糖或海藻糖将增加自噬，减少 以自噬依赖的方式诱导细胞凋亡，改善PKD。自噬的存在(自噬通量， 自噬小体和自溶小体的存在)、有丝分裂吞噬(含有线粒体的自噬小体)和 自噬抑制(使用基因技术)、自噬诱导(使用自噬诱导剂 已知可预防PKD)对囊泡生长和细胞凋亡的影响将在PKD-/-小鼠和PKD-/-小鼠中确定 细胞。更好地理解诱导自噬可以预防PKD的机制可能会导致 为诊断和治疗PKD寻找新的靶点。在目标1中，自噬的影响 对PKD1-/-小鼠肾脏进行抑制或诱导实验。自噬通量和细胞凋亡将被确定 在PKD_1-/-小鼠肾脏中的时间过程中。将确定肾脏中的自噬敲除是否- 特异性双基因敲除ATG7、PKD1-/-小鼠的细胞凋亡、增殖、PKD和肾功能恶化。这个 自噬诱导剂(热量限制、2-脱氧葡萄糖或海藻糖)对自噬、细胞凋亡和细胞周期的影响 检测PKD-/-小鼠和ATG7、PKD双基因敲除小鼠的增殖、PKD和肾功能。 一种新的自噬诱导剂(海藻糖)对PKD的影响将被确定。在目标2中，自噬途径 在PKD_1-/-肾小管上皮细胞将被确定。这些实验将使用更复杂的检测技术 自噬通量和有丝分裂吞噬以及自噬诱导和遗传的药理学和遗传学技术 不能在体内进行的抑制。不同机械方法诱导自噬的效果 (葡萄糖剥夺、海藻糖、Beclin1肽)和自噬抑制(3甲基腺嘌呤、氯喹、 显性阴性ATG4)在自噬途径和通量、有丝分裂吞噬、细胞凋亡、增殖、囊变上的表达 规模/数量将另行确定。我们的结果将为PKD代表一种 1例自噬减少。我们的结果将提供对热量作用机制的机械论见解。 限制或化学热量限制，防止PKD，也是自噬诱导者。我们的 这一结果将为未来自噬诱导剂在PKD中的潜在应用提供治疗方面的见解。

Autophagy is a process that takes place in all eukaryotic cells that keeps cells alive under stressful conditions. In autophagy there is the sequestration of damaged organelles into double-membraned autophagosomes that subsequently fuse with lysosomes where their cargoes are delivered for degradation and recycling. In the healthy kidney, autophagy plays an important role in the homeostasis and viability of renal tubular epithelial cells. There is indirect evidence that PKD is a case of suppressed autophagy: 1) Many of the agents that protect against PKD are autophagy inducers. 2) Preliminary/published data demonstrate decreased autophagic flux in Pkd1 -/- mouse kidneys and PC1 -/- cells. 3) There is cross-talk between autophagy and apoptosis, a key process in cyst growth. The overall hypothesis is that autophagic flux (autophagosome-lysosome fusion and degradation) is decreased early in the polycystic kidney and that interventions that block autophagy will worsen PKD and conversely autophagy inducers like caloric restriction, 2-deoxy-glucose or trehalose will increase autophagy, decrease apoptosis and improve PKD in an autophagy–dependent manner. The presence of autophagy (autophagic flux, presence of autophagosomes and autolysosomes), mitophagy (autophagosomes containing mitochondria) and the effect of autophagy inhibition (using genetic techniques), autophagy induction (using autophagy-inducers that are known to protect against PKD) on cyst growth and apoptosis will be determined in Pkd1 -/- mice and PKD1 -/- cells. An improved understanding of the mechanism by which induction of autophagy can prevent PKD may lead to the identification of new targets for both diagnosis and therapy of PKD. In Aim 1, the effect of autophagy inhibition or induction in will be tested Pkd1 -/- mouse kidneys. Autophagic flux and apoptosis will be determined during a time course in Pkd1 -/- mouse kidneys. It will be determined whether autophagy knockout in kidney- specific double knockout ATG7, Pkd1 -/- mice worsens apoptosis, proliferation, PKD and kidney function. The effect of autophagy inducers (caloric restriction, 2-deoxy-glucose or trehalose) on autophagy, apoptosis and proliferation, PKD and renal function will be determined in Pkd1 -/- mice and ATG7, Pkd1 double knockout mice. The effect of a novel autophagy inducer (trehalose) on PKD will be determined. In Aim 2, pathways of autophagy in PKD1 -/- tubular cells will be determined. These experiments will use more sophisticated techniques of detection of autophagic flux and mitophagy and pharmacological and genetic techniques of autophagy induction and inhibition that cannot be performed in vivo. The effect of autophagy induction by mechanistically distinct methods (glucose deprivation, trehalose, Beclin1 peptide) and autophagy inhibition (3 methyladenine, chloroquine, expression of dominant negative ATG4) on autophagy pathways and flux, mitophagy, apoptosis, proliferation, cyst size/number will be determined. Our results will provide a definitive test of the hypothesis that PKD represents a case of decreased autophagy. Our results will offer mechanistic insights into the mechanism of action of caloric restriction or chemical caloric restriction that protect against PKD and that are also autophagy inducers. Our results will provide therapeutic insights into the potential future use of autophagy inducers in PKD.