Transferrin but not albumin mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells

Complement is increasingly implicated in the pathogenesis of progressive re nal 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 hd. We postulated in this study that transferrin and apotransf errin, also important components of proteinuric urine in this molecular-wei ght 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 gr owth-arrested PTEC monolayers in media containing different concentrations (2.5 to 20 mg/mL) of transferrin by reverse transcription and polymerase ch ain reaction, Pure human albumin was used as a control protein. C3 protein secretion was detected and quantified by a sandwich enzyme linked immunosor bent assay on cell culture supernatants after distinct time points. Transfe rrin enhanced the rate of C3 secretion in a dose-dependent manner, reaching maximal stimulation at doses of 10 mg/mL, Selected experiments using the T ranswell technique showed that C3 release was predominantly apical in the r esting state. The addition of 10 mg/mL of transferrin apically but not baso laterally 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 trans ferrin in a time- and dose-dependent fashion, reaching a peak after 24 hour s. A similar degree of C3 upregulation was reproduced when iron-poor transf errin, apotransferrin, was used instead, These results indicate that C3 syn thesis in PTECs is upregulated by transferrin, for which protein rather tha n iron moiety may account for the observed effects. These findings provide evidence linking proteinuria with overexpression of tubular complement, (C) 2001 by the National Kidney Foundation, Inc.