Hilliard Lucinda M, Osicka Tanya M, Clavant Steven P, Robinson Phillip J, Nikolic-Paterson David J, Comper Wayne D
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
J Lab Clin Med. 2006 Jan;147(1):36-44. doi: 10.1016/j.lab.2005.08.008.
This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.
本研究使用离体灌注大鼠肾（IPK）模型，探讨尿白蛋白降解途径的存在以及受体介导的内吞作用在此过程中所提出的作用。通过放射性以及在214nm处的吸光度分析IPK尿液中白蛋白衍生肽的相对大小分布，以及它们对胰蛋白酶消化的敏感性。分别用衣霉素A和肉豆蔻酰三甲基溴化铵（MTMAB）灌注肾脏，研究受体介导的内吞作用调节剂液泡型H(+) ATP酶（v-ATP酶）和发动蛋白GTP酶的抑制剂的作用。正常IPK尿液含有轻度降解（定义为约10 - 40kDa；43.0±8.3%）和重度降解（定义为<10kDa；22.6±7.7%）的白蛋白肽以及完整白蛋白（34.5±4.1%）。肽的相对大小分布通过放射性和在214nm处的吸光度相似，并且在胰蛋白酶消化后两种图谱都减少为非常小的肽。与正常IPK尿液相比，给予衣霉素A或MTMAB导致排泄的完整白蛋白比例显著增加（衣霉素A：55.8±11.6%；MTMAB：50.0±11.9%）。这与轻度（衣霉素A：27.6±9.8%；MTMAB：39.9±11.5%）和重度降解（衣霉素A：16.6±7.4%；MTMAB：10.0±2.5%）白蛋白比例的降低一致，并且与肾小球对白蛋白的通透性变化无关，因为在存在衣霉素A的情况下，Ficoll（半径范围20 - 60Å）的分数清除率没有观察到显著变化。本研究证明了IPK尿液中白蛋白肽的存在，并表明受体介导的内吞作用在尿白蛋白降解中起作用。
