Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.
Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.
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DOI：
10.1053/ajkd.2001.20593
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
S. Tang;K. Lai;T. Chan;H. Lan;S. Ho;S. Sacks
中科院分区：
文献类型：
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作者：
S. Tang;K. Lai;T. Chan;H. Lan;S. Ho;S. Sacks
补体越来越多地参与持续性蛋白尿引起的进行性肾脏疾病的发病机制。我们以前已经表明，顶端血清蛋白刺激C3在培养的人近端肾小管上皮细胞（PTECs），和刺激剂是一种非白蛋白化合物的30至100 kD。我们在这项研究中假设，转铁蛋白和脱铁转铁蛋白，也是蛋白尿的重要组成部分，在这个分子量范围内，可能是罪魁祸首。通过从肿瘤肾切除术标本的正常极鉴别筛选肾皮质组织获得人PTEC，其特征在于主要是近端肾小管起源。补体C3信使RNA（mRNA）的表达进行了分析，在汇合生长停滞的PTEC单层培养基中含有不同浓度（2.5至20毫克/毫升）的转铁蛋白通过逆转录和聚合酶链反应。使用纯人白蛋白作为对照蛋白。在不同的时间点后，通过夹心酶联免疫吸附试验对细胞培养上清液进行检测和定量C3蛋白分泌。转铁蛋白以剂量依赖性方式增强C3分泌速率，在10 mg/mL的剂量下达到最大刺激。使用Transwell技术的选定实验表明，C3释放主要是在静息状态下的顶端。在顶侧而不是基底侧添加10 mg/mL转铁蛋白刺激顶侧和基底侧C3分泌，并将C3分泌的基底侧-顶侧比率从0.45 +/- 0.16增加到0.93 +/- 0.24（P：< 0.02）。转铁蛋白以时间和剂量依赖性方式上调组成型C3 mRNA表达，24小时后达到峰值。类似程度的C3上调时，铁贫转铁蛋白，脱铁转铁蛋白，被用来代替。这些结果表明，C3的合成在PTECs上调转铁蛋白，蛋白质，而不是铁部分可以解释所观察到的效果。这些发现提供了蛋白尿与肾小管补体过度表达相关的证据。
Complement is increasingly implicated in the pathogenesis of progressive renal disease resulting from persistent proteinuria. We have previously shown that apical serum proteins stimulate C3 in cultured human proximal tubular epithelial cells (PTECs), and that the stimulant is a nonalbumin compound of 30 to 100 kd. We postulated in this study that transferrin and apotransferrin, also important components of proteinuric urine in this molecular-weight range, might be the culprit. Human PTECs were obtained by differential sieving of renal cortical tissue from the normal pole of tumor nephrectomy specimens and characterized to be predominantly of proximal tubular origin. Complement C3 messenger RNA (mRNA) expression was analyzed in confluent growth-arrested PTEC monolayers in media containing different concentrations (2.5 to 20 mg/mL) of transferrin by reverse transcription and polymerase chain reaction. Pure human albumin was used as a control protein. C3 protein secretion was detected and quantified by a sandwich enzyme-linked immunosorbent assay on cell culture supernatants after distinct time points. Transferrin enhanced the rate of C3 secretion in a dose-dependent manner, reaching maximal stimulation at doses of 10 mg/mL. Selected experiments using the Transwell technique showed that C3 release was predominantly apical in the resting state. The addition of 10 mg/mL of transferrin apically but not basolaterally stimulated both apical and basolateral C3 secretion and increased the basolateral-apical ratio of C3 secretion from 0.45 +/- 0.16 to 0.93 +/- 0.24 (P: < 0.02). Constitutive C3 mRNA expression was upregulated by transferrin in a time- and dose-dependent fashion, reaching a peak after 24 hours. A similar degree of C3 upregulation was reproduced when iron-poor transferrin, apotransferrin, was used instead. These results indicate that C3 synthesis in PTECs is upregulated by transferrin, for which protein rather than iron moiety may account for the observed effects. These findings provide evidence linking proteinuria with overexpression of tubular complement.