SECRETION OF INSULIN-LIKE GROWTH FACTOR-I AND ITS BINDING-PROTEINS BY COLLECTING DUCT CELLS

Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30°C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I-IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity. In addition, its size was not affected by reduction of disulfide bonds. We conclude that cells cultured from rabbit collecting duct secrete: (1) IGF-I; (2) a high molecular weight IGF-1-containing species; and (3) IGF-I binding proteins. Since proximal tubular cells and mesangial cells possess specific IGF-I receptors, these data suggest that IGF-I may act as paracrine factor that regulates renal growth and/or function.