Advanced Glycation End Products Inhibit Tubulogenesis and Migration of Kidney Epithelial Cells in an Ezrin-Dependent Manner Marisa A Gallicchio,* E. Anne McRobert,* Anjali Tikoo,* Mark E. Cooper, † and Leon A. Bach* ‡ *Monash University, Department of Medicine and ‡ Department of Endocrinology and Diabetes, Alfred Hospital, and † Baker Medical Research Institute, Victoria, Australia Nonenzymatic glycation of proteins to form advanced glycation end products (AGE) is implicated in diabetic complications, including nephropathy. It was shown recently that AGE bind to the ERM (ezrin, radixin, and moesin) family of membrane- cytoskeletal linker proteins in renal homogenates. Herein is reported the effects of AGE-BSA on ezrin-dependent LLC-PK1 kidney epithelial cellular functions: migration and hepatocyte growth factor (HGF)-induced tubulogenesis. LLC-PK1 cells were stably transfected with cDNA for ezrin sense, ezrin antisense, and N-ezrin. Transfection of LLC-PK1 cells with ezrin antisense and dominant negative N-ezrin decreased basal tubulogenesis and migration relative to vector-only transfection, establishing the ezrin dependency of these processes. AGE-BSA (20 or 40 M) significantly decreased HGF-induced tubulo- genesis and basal migration in two vector control lines relative to BSA-treated cells. However, AGE-BSA inhibition of both HGF-induced tubulogenesis and migration was overcome by overexpressing ezrin. These results demonstrate that the AGE– ezrin interaction significantly alters cellular function. These changes may be relevant to detrimental renal consequences as a result of diabetes. J Am Soc Nephrol 17: 414 – 421, 2006. doi: 10.1681/ASN.2005010051 N onenzymatic glycation of proteins, lipids, and nu- cleic acids resulting in the accumulation of advanced glycation end products (AGE) is a prominent feature of diabetes. AGE levels correlate with the development of chronic diabetic complications such as retinopathy, nephropa- thy, neuropathy, and vasculopathy (1–3). In the normal rat, injection of AGE leads to renal changes similar to those of diabetic nephropathy, whereas interference with AGE forma- tion reduces diabetic complications in various animal models (4 – 6). The importance of AGE as a pathogenic mechanism in diabetic nephropathy is suggested further by the finding that AGE formation/accumulation precedes diabetic renal disease (2). Together with a cellular receptor RAGE, AGE accumulate in diabetic renal tissue (2,7,8). However, the precise cellular mechanism(s) whereby AGE promote the development of dia- betic complications remains unknown. We recently described the novel binding of AGE to the ERM (ezrin, radixin, and moesin) family of proteins (9). ERM pro- teins are critical regulators of interactions between the cell membrane and the cytoskeleton (10). As members of the eryth- rocyte protein 4.1 superfamily, ERM proteins are characterized by a conserved N-domain (FERM domain), which associates with the cytoplasmic domain of specific membrane-associated proteins such as the hyaluronan receptor CD44, CD43, and CD95 and the intercellular adhesion molecules-1, -2, and -3. The C-terminal domain of ERM binds to F-actin in vitro and in vivo (11,12). However, the F-actin binding site and binding sites for membrane proteins are masked by intramolecular association between the N- and C-domains in the inactive cytoplasmic molecule (13). Tyrosine phosphorylation by receptors for EGF and hepatocyte growth factor (HGF) activate ezrin by unmask- ing the F-actin binding site (14,15). In LLC-PK1, a proximal tubule cell line, tyrosine phosphorylation of ezrin increases cell survival (16). Tubular injury features prominently in the development of renal dysfunction in diabetes (17,18). In vivo, epithelial tubulo- genesis requires the migration and repopulation of the tubular conduit with proliferating cells of the renal proximal tubular epithelium during kidney development or after renal injury. In vitro, ezrin is a mediator of HGF-induced LLC-PK1 tubulogen- esis because dominant negative N-ezrin expression and ezrin overexpression inhibit and enhance this process, respectively (15). In this model, tyrosine phosphorylation of ezrin is essen- tial, and this is also the case with respect to HGF-induced migration of these cells. As with tubulogenesis, ezrin overex- pression in LLC-PK1 cells enhances cell migration, whereas N-ezrin expression impairs HGF-induced migration (15). We recently showed that AGE-BSA inhibited LLC-PK1 tubulogen- esis and ezrin tyrosine phosphorylation (9). In this article, we describe the effect of AGE-BSA on LLC-PK1 Received January 13, 2005. Accepted November 4, 2005. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Leon A. Bach, Monash University, Department of Medicine, Alfred Hospital, Commercial Road, Prahran, Victoria 3004, Austra- lia. Phone: +61-3-9276-2387; Fax: +61-3-9276-3782; E-mail: leon.bach@ med.monash.edu.au Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1702-0414