Expression of the multiligand receptor megalin in liver

Abstract

Confocal microscopy has indicated that hepatic stellate cells (HSCs) might express megalin [1]. Since megalin is the receptor of the retinol-retinol binding protein-complex in kidney and is responsible for the resorption of vitamin A and other metabolites from urine [2], we assumed participation of this endocytotic multiligand receptor in the retinol storage system of HSCs. Based on the observation that HSCs lose their ability to store retinol during fibrogenesis, we compared megalin expression in healthy and fibrotic livers after CCl4 challenge. A megalin-specific rabbit antiserum was generated by immunizing rabbits with the recombinantly expressed cytoplasmic tail of megalin. Control immunoblots of these antibodies on kidney samples revealed reactivity with both a high and a low molecular mass band. The high molecular mass band (>500 kD) presumably corresponds to the whole transmembrane protein megalin, consisting of an ectodomain, a transmembrane domain and a cytoplasmic tail (CT). In contrast, the low molecular mass band (30–35 kD) represents the megalin C-terminal fragment (MCTF) as a result of ectodomain shedding and consists of the transmembrane domain and the CT [3]. In experiments using liver homogenates, only the low molecular mass band of the MCTF was detected by the anti-megalin antibodies. No evidence was obtained for the expression of whole megalin in liver. Furthermore, there was no expression difference between liver homogenates from CCl4-challenged mice and controls. Immunohistochemistry using anti-megalin antibodies and collagen staining of liver sections revealed reactivity with non-parenchymal cells in centrilobular areas with intense fibrosis but not with HSCs. The lack of megalin expression in HSCs as demonstrated by our immunostaining data, contradicts previous reports [1] and does not support a role of megalin in hepatic retinol uptake.