Aron D C, Rosenzweig J L, Abboud H E
Department of Medicine, Veterans Administration Medical Center, Case Western Reserve University, Cleveland, Ohio 44106.
J Clin Endocrinol Metab. 1989 Mar;68(3):585-91. doi: 10.1210/jcem-68-3-585.
Insulin-like growth factor I (IGF-I) has been found in the kidney, but its precise cellular localization is not known. Since there is evidence that IGF-I is an autocrine factor in many tissues and since murine mesangial cells have IGF-I receptors, we examined whether human mesangial cells produce IGF-I. Culture medium conditioned by mesangial cells was concentrated by reverse phase chromatography and applied to a Sephadex G-100 column equilibrated in a denaturing buffer. Two major species with apparent mol wt (MW) of 7,500 and 25,000 daltons were identified by IGF-I RIA. To determine whether the high MW species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]Thr59-IGF-I for 2 h at 30 C, and applied to a Sephadex G-100 column equilibrated in a nondissociating buffer. The major peak of radioactivity was confined to a high MW region; there was no radioactivity in the fractions corresponding to 7,500 daltons. Further characterization of 7,500 dalton IGF-I immunoreactive species by reverse phase high performance liquid chromatography showed that it coeluted with synthetic human IGF-I. Isoelectric focusing revealed it to have a pI between 8.1 and 8.5, corresponding to the pI of human IGF-I of 8.25. Northern blot analyses of poly(A)+ RNA from human mesangial cells and human liver using a cDNA probe for human IGF-I showed that a 2.0-kilobase transcript predominated in the mesangial cells, whereas the liver contained 1.1- and 2.0-kilobase species. Specific binding of IGF-I to mesangial cells was demonstrated, and competition curves indicated a rank order of potency (IGF-I greater than IGF-II greater than insulin) consistent with type I IGF receptors. We conclude that human mesangial cells 1) express IGF-I mRNA transcripts, 2) secrete IGF-I and IGF-I-binding activity, and 3) possess specific IGF-I receptors. These data suggest that IGF-I may act as an autocrine or paracrine factor that regulates glomerular cell functions.
胰岛素样生长因子I（IGF-I）已在肾脏中被发现，但其精确的细胞定位尚不清楚。由于有证据表明IGF-I在许多组织中是一种自分泌因子，且小鼠系膜细胞有IGF-I受体，我们研究了人系膜细胞是否产生IGF-I。用系膜细胞条件培养基经反相色谱浓缩后，加样到用变性缓冲液平衡的Sephadex G-100柱上。通过IGF-I放射免疫分析鉴定出两种主要的表观分子量（MW）分别为7500和25000道尔顿的物质。为确定高分子量物质是否具有IGF-I结合活性，将适当的组分脱盐，于30℃与[125I]Thr59-IGF-I孵育2小时，然后加样到用非解离缓冲液平衡的Sephadex G-100柱上。放射性主峰局限于高分子量区域；对应于7500道尔顿的组分中无放射性。用反相高效液相色谱对7500道尔顿的IGF-I免疫反应性物质作进一步鉴定，结果显示它与合成的人IGF-I共洗脱。等电聚焦显示其pI在8.1至8.5之间，与人IGF-I的pI 8.25相符。用人IGF-I的cDNA探针，对人系膜细胞和人肝脏的聚腺苷酸加尾（poly(A)+）RNA进行Northern印迹分析，结果显示系膜细胞中占优势的是2.0千碱基的转录本，而肝脏中含有1.1和2.0千碱基的转录本。已证实IGF-I与人系膜细胞有特异性结合，竞争曲线表明其活性顺序为（IGF-I＞IGF-II＞胰岛素），与I型IGF受体相符。我们得出结论，人系膜细胞1）表达IGF-I mRNA转录本，2）分泌IGF-I及IGF-I结合活性物质，3）拥有特异性IGF-I受体。这些数据提示IGF-I可能作为一种自分泌或旁分泌因子调节肾小球细胞功能。
