title: Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis.
abstract: CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent pathway.
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584, CX, Utrecht, The Netherlands.|Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: K G F
authors.initials: KG
authors.lastname: Gerritsen
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584, CX, Utrecht, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Bovenschen
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584, CX, Utrecht, The Netherlands. T.Q.Nguyen@umcutrecht.nl.
authors.forename: T Q
authors.initials: TQ
authors.lastname: Nguyen
authors.affiliation: Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Sprengers
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M P
authors.initials: MP
authors.lastname: Koeners
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: A N
authors.initials: AN
authors.lastname: van Koppen
authors.affiliation: Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Joles
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584, CX, Utrecht, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Goldschmeding
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kok
keywords: Biomarker; CCN-2; CTGF; Hepatic clearance; LRP1