An intravital model to study mechanisms of leukocyte recruitment in experimental crescentic glomerulonephritis (CrGN)

肾小球肾炎（GN）是指肾小球的炎症，肾小球是肾脏的过滤单位，由一簇微小血管（“毛细血管”）组成。在这种疾病中，白色血细胞是免疫系统的一部分，通常保护身体免受感染，在肾小球中积聚，在那里它们“粘”在毛细血管的壁上，并被不适当地激活，释放化学物质，引起炎症和损害这些过滤单元，导致肾功能障碍。GN是透析依赖性肾衰竭的常见原因，与长期健康问题和死亡率增加有关。新月体肾炎（CrGN）是肾小球中一种特别严重的炎症，可能危及生命，如果不治疗，会导致快速进行性肾衰竭。目前治疗GN的方法是使用抑制整个免疫系统的强效药物，而不是专门针对疾病本身的治疗，因为循环的白色血细胞被募集并保留在肾小球中的机制还不清楚。这些药物并不总是有效的，可能会导致严重的副作用，因为免疫系统需要保护身体免受感染和癌症。这项研究的目标是了解循环白色血细胞被招募到肾小球引起炎症和肾衰竭的机制，以便更有针对性地，我将研究白色血细胞“粘附”在肾小球毛细血管壁上并引起炎症和损伤的机制，使用一种称为活体显微镜的技术，（使用高倍显微镜）它们在CrGN大鼠模型中穿过肾小球毛细血管时的运输和行为。我将检验这一假设，即在疾病过程中，不同的白色血细胞亚群被募集到肾小球的特定区域，这在引起肾小球损伤导致进行性肾衰竭中很重要。我将研究将这些细胞募集到肾小球并使其保留在那里的机制，重要的是，我将研究它们的命运，以确定它们是否可以在尿液中检测到，这可能是一个有用的临床工具，以评估疾病活动对治疗的反应。重要的是，我将使用一个比以前描述的更具临床相关性的CrGN模型来进行这项工作。大鼠在生物学上与人类相似，WKY大鼠品系中的实验CrGN产生的肾脏变化与人类CrGN中观察到的变化惊人相似。这是一个已建立的CrGN模型，我们的研究小组已经成功地使用了几十年来研究这种疾病。我将使用更新但日益流行的“活体显微镜”技术，研究白色血细胞在“真实的时间”内通过大鼠肾小球的运输，这些大鼠是活着的，但处于全身麻醉状态，因此没有任何痛苦。这更接近于人类肾脏的环境，因此比研究体外细胞培养系统中白色血细胞的行为和功能的其他实验技术（“体外”研究）更容易转化为人类疾病。我将在帝国理工学院（IC）进行拟议的研究，那里的肾脏部门的科学家在GN领域进行高质量的研究有着良好的记录。通过与艾特曼教授在IC的遗传学小组的合作，我们的研究小组已经确定了一些负责CrGN易感性的基因，并开发了缺乏这些负责基因的大鼠品系。这将为我提供研究白色血细胞募集和保留在肾小球中的机制所需的工具，比以前在CrGN大鼠模型中更详细。

Glomerulonephritis (GN) means inflammation of the glomeruli, which are the filtering units of the kidney made up of clusters of tiny blood vessels ("capillaries"). In this disease, white blood cells, which are part of the immune system and usually defend the body against infection, accumulate in the glomeruli where they "stick" to the walls of the capillaries and become inappropriately activated, releasing chemicals which cause inflammation and damage to these filtering units, causing kidney dysfunction. GN is a common cause of dialysis-dependent kidney failure, which is associated with long-term health problems and increased mortality. Crescentic GN (CrGN) is a particularly severe form of inflammation in the glomeruli which is potentially life-threatening and, untreated, leads to rapidly progressive kidney failure.GN is currently treated using powerful drugs that suppress the entire immune system, rather than therapy targeted specifically at the disease itself, because the mechanisms by which circulating white blood cells are recruited to and retained in the glomeruli are not well understood. These drugs are not always effective and can cause severe side effects because the immune system is needed to protect the body against infection and cancer. The goal of this research is to understand the mechanisms by which circulating white blood cells are recruited to the glomerulus to cause inflammation and kidney failure so that more targeted, safer therapy can be developed.I will study the mechanisms by which white blood cells "stick" to the glomerular capillary walls and cause inflammation and damage using a technique called intravital microscopy to directly visualise (using a high-powered microscope) their trafficking and behaviour as they travel through the glomerular capillaries in a rat model of CrGN. I will examine the hypothesis that different subpopulations of white blood cells are recruited to particular areas of the glomerulus during the disease process and that this is important in causing the glomerular damage that leads to progressive kidney failure. I will study the mechanisms that recruit these cells to the glomerulus and cause them to be retained there and, importantly, I will study their fate to determine whether they can be detected in urine, which may represent a useful clinical tool to assess disease activity in response to treatment. Importantly, I will use a more clinically relevant model of CrGN than has been previously described to carry out this work. The rat is biologically similar to humans, and experimental CrGN in the WKY rat strain produces changes in the kidney that look strikingly similar to those seen in human CrGN. This is an established model of CrGN which has been used successfully by our research group for decades to study the disease. I will be using the newer but increasingly popular technique of "intravital microscopy" to study the trafficking of white blood cells in "real time" as they travel through the glomeruli of rats that are alive but under general anaesthetic and therefore not in any distress. This much more closely resembles the environment of the human kidney and is therefore more translatable to human disease than other experimental techniques studying the behaviour and function of white blood cells in cell culture systems outside the body ("in vitro" studies). I will undertake the proposed research at Imperial College (IC) where scientists in the kidney unit have an excellent track-record conducting high quality research in the field of GN. Through collaborative work with Prof Aitman's genetics group at IC, our research group has identified a number of genes responsible for susceptibility to CrGN and have developed strains of rats which lack these responsible genes. This will provide me with the tools needed to study mechanisms of white blood cell recruitment and retention in the glomerulus in more detail than has been previously possible in rat models of CrGN.