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Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis.

Biochimica et biophysica acta (2013-06-12)
Marisa A Gallicchio, Leon A Bach
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.

MATERIALS
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