Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼90 kDa and ∼70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met706 is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM) (and to a lesser extent Ang II) for 48–72 h increased ACE2 activity in the media (p血管紧张素转换酶2（Angiotensin-converting enzyme 2, ACE2）在肾脏近端小管中呈高表达，可将血管紧张素（Ang）II切割为血管紧张素-(1-7)。糖尿病患者尿液中的ACE2水平升高，提示ACE2可能从肾小管细胞脱落。本研究旨在明确ACE2是否可从近端肾小管细胞脱落、鉴定ACE2的剪切片段，并探究其脱落的相关通路。本研究采用小鼠近端肾小管细胞原代培养体系，通过合成底物检测ACE2活性，并借助免疫印迹与质谱分析法对ACE2片段进行分析。小鼠近端肾小管细胞的培养液中，ACE2活性呈时间依赖性升高，提示存在组成型ACE2脱落现象。免疫印迹检测显示，培养液中的ACE2呈现约90 kDa与约70 kDa的两条条带；与之相比，细胞裂解液或小鼠肾皮质样本中的全长ACE2条带分子量约为100 kDa。对两个去糖基化片段进行质谱分析，结果鉴定出分别匹配小鼠ACE2第18-706位与第18-577位氨基酸序列的肽段。其中18-706肽段的C端存在非胰蛋白酶切割位点，提示Met706可能为ACE2的候选剪切位点。将细胞在高浓度D-葡萄糖（25 mM）环境中孵育48~72 h，可使培养液中ACE2活性升高，血管紧张素II也可产生一定程度的该效应但程度较轻（p