Glucose-Induced Stiffening of Extracellular Matrix in Diabetic Nephropathy

这个项目的目标是确定肾脏细胞外基质(ECM)硬度的变化是如何引起的 晚期糖基化影响糖尿病肾病的进展。我们最重要的假设是 糖基化终末产物(AGEs)形成交联物，增加肾脏细胞外基质的硬度，改变整合素 触发细胞外基质合成增加的信号。新合成的基质进一步交联化， 从而创造了一个增加了刚性和ECM合成的前馈循环。我们建议这一机制 有助于细胞外基质在肾小球和肾小管间质中堆积，这是糖尿病的特征 肾脏疾病。我们的初步研究表明，将肾小管与葡萄糖或核糖孵育显著 增加管状基底膜的硬度。本申请的目的是(1)确定 糖暴露改变肾小管和肾小球细胞外基质硬度的机制 以及(2)确定僵硬度增加改变整合素信号并导致增加的机制 ECM生产。目标1采用的方法将包括对管状基底膜的测量。 使用我们新开发的基于微悬臂梁的刚性在正常和糖化修饰的肾小管中的硬度 测量技术。肾小球硬度将使用微型压缩测试进行测量。 组织的机械特征将通过质谱学与生化分析相关联，以 评估交联剂的形成。对于目标2，我们将使用体外细胞培养模型来模拟机械 正常和糖修饰的肾小管和肾小球细胞外基质的性质。僵硬引起的细胞变化 表型、细胞增殖和细胞外基质的产生将在野生型和整合素α1和α2缺失的小管中进行评估 上皮性和肾小球系膜细胞。这些目标的成功实现将确立年龄的作用-- 介导的细胞外基质硬化是糖尿病肾病进展的潜在因素，并将提示 糖尿病和整合素缺失小鼠模型的后续研究。

The goal of this project is to determine how changes in kidney extracellular matrix (ECM) stiffness caused by advanced glycation impact progression of diabetic nephropathy. Our overarching hypothesis is that advanced glycation end products (AGEs) form crosslinks that increase ECM stiffness in the kidney and alter integrin signaling to trigger increased extracellular matrix synthesis. The newly synthesized matrix is further crosslinked, thus creating a feed forward cycle of increased stiffness and ECM synthesis. We propose that this mechanism contributes to accumulation of ECM in the glomerulus and tubulointerstitium that is characteristic of diabetic kidney disease. Our preliminary studies show that incubating renal tubules with glucose or ribose significantly increases the stiffness of the tubular basement membrane. The aims of this application are to (1) Determine the mechanism by which sugar exposure alters the stiffness of kidney tubular and glomerular extracellular matrix and (2) Determine the mechanism by which increased stiffness alters integrin signaling and leads to increased ECM production. The methods employed in aim 1 will include measurement of tubular basement membrane stiffness in normal and sugar modified kidney tubules using our newly developed microcantilever-based stiffness measurement technique. Glomerular stiffness will be measured using microscale compression testing. Mechanical characterization of the tissue will be correlated with biochemical analysis via mass spectrometry to evaluate crosslink formation. For aim 2, we will use in vitro cell culture models that mimic the mechanical properties of normal and sugar modified tubular and glomerular ECM. Stiffness induced changes in cell phenotype, cell proliferation and ECM production will be evaluated in wild type and integrin α1 and α2-null tubular epithelial and glomerular mesangial cells. Successful completion of these aims will establish the role of AGE- mediated ECM stiffening as a potential contributor to progression of diabetic kidney disease and will inform subsequent studies in diabetic and integrin-null mouse models.