Biophysical Properties of Renal Glomeruli and Podocytes

描述（由申请人提供）：组织和细胞的机械性能（弹性模量、变形性或刚度）对于确定组织的正常结构和功能至关重要。足细胞应具有特征性的机械性质，使其能够在毛细血管血流动力学力的存在下维持肾小球毛细血管壁的结构和完整性。形成狭缝隔膜的足突和产生它们的次级突含有肌动蛋白束和肌动蛋白交联和成束蛋白（1-辅肌动蛋白和细丝蛋白），这些蛋白决定了它们的机械特性。人类肾小球疾病和许多导致局灶性硬化的疾病模型是由足细胞细胞骨架或粘附蛋白的突变或异常引起的，这些突变或异常应该影响它们的机械特性。我们的初步数据显示，在四个模型中，肾小球和足细胞比正常软。我们的假设是，足细胞具有特定的机械特性，由其细胞骨架的结构和组成以及允许它们支持肾小球毛细血管结构和功能的机械环境决定。我们专注于三种小鼠模型，Col 4a 3-/-（Alport模型，异常GBM），条件性足细胞整合素21-/-（异常细胞间隙-GBM连接）和Actn 4-/-（异常肌动蛋白交联），基于其不同的分子病理学，将定义特定的机械传感和响应途径，导致肾小球损伤，由于未能感知或响应适当的机械信号。这三个具体目标是：目标1。定义WT、Col 4a 3-/-、21整联蛋白-/-和Actn 4-/-小鼠在疾病前状态和整个疾病过程中的肾小球和肾小球毛细血管壁的机械特性（变形性）。目标2.定义来自WT、Col 4a 3-/-、21整合素-/-和Actn 4-/-小鼠的小鼠足细胞的机械特性，以及不同机械环境对它们的影响。目标3。定义21整合素和细胞骨架交联剂1-辅肌动蛋白-4和细丝蛋白的机制，确定足细胞对基质产生的机械信号的结构和生物物理反应，以测试抑制疾病模型异常机械表型的蛋白质或构建体。从这个新的生物物理学角度接近肾小球疾病，并定义肾小球和足细胞的机械特性的决定因素，将允许开发新的调查路线和新的干预措施在肾小球疾病。 公共卫生相关性：过滤血液（肾小球）并暴露于血压和流量的肾脏部分在许多肾脏疾病中受损。在某些情况下，损坏是由于过滤器的缺陷而发生的，而在其他情况下，损坏是由于血压和流量的过度作用力而发生的。在任何一种情况下，过滤器的机械性能都很重要。肾脏疾病领域的大多数工作都集中在遗传或生化原因上。我们认为，与其他组织一样，组织和环境中的机械因素是正常功能和疾病的重要组成部分。在本提案中，我们将评估过滤器的机械性能和三种不同遗传疾病模型中的一种重要细胞类型。每个疾病模型都有一个原因，通过对疾病模型的机械特性进行分析，将告诉我们很多关于我们感兴趣的过滤器和细胞如何感知和响应机械力的信息。这种治疗肾脏疾病的新方法将为我们提供信息，帮助我们了解某些肾脏疾病的原因，并可能通过了解如何加强过滤器来确定治疗它们的新方法。

DESCRIPTION (provided by applicant): The mechanical properties of tissues and cells (elastic modulus, deformability, or stiffness) are essential for determining the normal structure and function of tissues. Podocytes should have characteristic mechanical properties that permit them to maintain the structure and the integrity of glomerular capillary walls in the presence of capillary hemodynamic forces. The foot processes that form the slit diaphragms and the secondary processes that give rise to them contain actin bundles and actin cross-linking and bundling proteins (1-actinin and filamin) that determine their mechanical properties. Human glomerular diseases and a number of disease models that result in focal sclerosis result from mutations or abnormalities in podocyte cytoskeletal or adhesion proteins that should affect their mechanical properties. Our preliminary data show that in four models, glomeruli and podocytes are softer than normal. Our hypothesis is that podocytes have specific mechanical characteristics determined by the structure and composition of their cytoskeletons and their mechanical environment that permit them to support glomerular capillary structure and function. We focus on three mouse models, Col4a3-/- (Alport model, abnormal GBM), conditional podocyte integrin 21-/- (abnormal cytoskeletal-GBM connection), and Actn4-/- (abnormal actin cross-linking), that based on their distinct molecular pathology, will define specific mechanosensing and response pathways that lead to glomerular injury due to failure to sense or respond appropriately to mechanical signals. The three specific aims are: Aim 1. Define the mechanical properties (deformability) of mouse glomeruli and glomerular capillary walls from WT, Col4a3-/-, 21 integrin-/-, and Actn4-/- mice at a pre-disease state, and through the course of disease. Aim 2. Define the mechanical properties of mouse podocytes from WT, Col4a3-/-, 21 integrin-/-, and Actn4-/- mice, and the effects of different mechanical environments on them. Aim 3. Define the mechanisms by which 21 integrin and the cytoskeletal cross-linkers 1-actinin-4 and filamin, determine structural and biophysical responses to matrix-generated mechanical signals in podocytes to test for proteins or constructs that suppress the abnormal mechanical phenotype of the disease models. Approaching glomerular disease from this novel biophysical perspective, and defining the determinants of the mechanical properties of glomeruli and podocytes, will permit development of new lines of investigation and new interventions in glomerular diseases. PUBLIC HEALTH RELEVANCE: The part of the kidney that filters the blood (glomerulus) and is exposed to blood pressure and flow is damaged in many kidney diseases. In some cases the damage occurs due to defects in filter, and in others the damage occurs due excessive force from blood pressure and flow. In either situation, the mechanical properties of the filters are important. Most work in the field of kidney disease has focused on genetic or biochemical causes. We believe that as is the case for other tissues, mechanical factors in the tissue and environment are important components of normal function and disease. In this proposal, we will assess the mechanical properties of the filters and one important cell type in three different genetic disease models. Each disease model has a cause, the analysis of which with the mechanical properties of the disease model, will tell us a great deal about how the filters and cells we are interested in sense and respond to mechanical force. This new approach to kidney disease will give us information that will help us understand the causes of some kidney diseases, and may identify new approaches to their treatment, possibly by understanding how to strengthen the filters.