title: Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis.
abstract: Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE-BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran-TRITC uptake, significantly increased AGE-BSA-FITC uptake (167±20% vs BSA control, p<0.01) and was ezrin-dependent. AGE-BSA-FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE-BSA-FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE-BSA increased PAK1 kinase activity (212±41% vs control, p<0.05) and protein levels of Tiam1, a Rac1 activator. AGE-BSA significantly increased TGF-β1 protein levels (160±6%, p<0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82±19%, p<0.001 vs AGE) and actin polymerisation (107±11%, p<0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE-BSA-FITC uptake (81±6% and 64±7%, respectively, p<0.05 vs control for both), and AGE-stimulated TGF-β1 protein release (99±15% and 49±8% of control, p<0.05 and p<0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na(+)K(+)ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.
authors.affiliation: Monash University, Department of Medicine, Alfred Hospital, Commercial Rd., Prahran, Victoria 3004, Australia.
authors.forename: Marisa A
authors.initials: MA
authors.lastname: Gallicchio
authors.affiliation: Monash University, Department of Medicine, Alfred Hospital, Commercial Rd., Prahran, Victoria 3004, Australia; Department of Endocrinology and Diabetes, Alfred Hospital, Commercial Rd., Prahran, Victoria 3004, Australia. Electronic address: leon.bach@monash.edu.
authors.forename: Leon A
authors.initials: LA
authors.lastname: Bach
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D018374:LLC-PK1 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010873:Pinocytosis / Q000187:drug effects; D058105:Polymerization / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D000099149:Ezrin
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000199:Actins; D003598:Cytoskeletal Proteins; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate; D017127:Glycation End Products, Advanced; D019269:Phosphatidylinositol 4,5-Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D034741:RNA, Small Interfering; D012235:Rhodamines; D012710:Serum Albumin, Bovine; D000254:Sodium-Potassium-Exchanging ATPase; D053773:Transforming Growth Factor beta1; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein; D000099149:Ezrin; C081628:advanced glycation end products-bovine serum albumin; C039217:fluorescein isothiocyanate bovine serum albumin; C009434:tetramethylrhodamine isothiocyanate; C020077:1-phosphatidylinositol-4-phosphate 5-kinase; C000715348:ARF6 protein, human
keywords: AGEs; Arf6; BSA; Diabetes; GM; Glycation; HK2; LLC-PK1; Macropinocytosis; Na(+)K(+)ATPase; Nephropathy; PAK1; PIP5Kγ; SFM; TGF-β; adenosine diphosphate-ribosylation factor 6; advanced glycation end products; bovine serum albumin; growth medium; human kidney proximal tubule epithelial cells; p21-activated kinase; phosphatidylinositol 4-phosphate 5-kinase γ; porcine kidney proximal tubule epithelial cells; serum-free medium