Academic Renal Unit, Learning and Research Bldg., Clinical Sciences North Bristol, University of Bristol, Southmead Hospital, Bristol BS10 5NB, UK.
Am J Physiol Renal Physiol. 2011 Jan;300(1):F40-8. doi: 10.1152/ajprenal.00103.2010. Epub 2010 Oct 27.
The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.
内皮糖萼是覆盖血管腔内侧的凝胶状层。肾小球内皮细胞 (GEnC) 糖萼由蛋白聚糖核心蛋白、糖胺聚糖 (GAG) 链和唾液酸化糖蛋白组成，已被证明有助于肾小球毛细血管壁的选择性筛滤作用。最近的研究表明，系统性内皮糖萼的损伤与糖尿病患者白蛋白尿的发生有关。在这项研究中，我们分析了高血糖对 GEnC 糖萼生化结构的影响，并定量分析了其蛋白质限制作用的功能变化。我们使用条件永生化的人 GEnC。通过 Western blot 和间接免疫荧光分析蛋白聚糖。通过放射性标记和阴离子交换色谱定量分析 GAG 的生物合成。使用 FITC-白蛋白分析 FITC-白蛋白穿过 GEnC 单层的大分子通透性，使用已建立的体外模型。我们观察到高糖条件下 GEnC 的 GAG 生物合成明显减少。进一步的分析证实了肝素硫酸盐 GAG 的特异性减少。蛋白聚糖核心蛋白的表达保持不变。在高糖条件下，白蛋白穿过 GEnC 单层的通透性也显著增加，而不影响内皮细胞间连接。这些结果再现了 GEnC 屏障特性的变化，这些变化是由 GEnC 糖萼中肝素硫酸盐的酶促去除引起的。它们提供了高血糖诱导的 GEnC 糖萼改变的直接证据，并证明了其作为蛋白质限制层的功能改变，从而暗示糖萼损伤参与了糖尿病蛋白尿的发病机制。
