Advanced glycation end products inhibit Na+ K+ ATPase in proximal tubule epithelial cells: role of cytosolic phospholipase A2alpha and phosphatidylinositol 4-phosphate 5-kinase gamma.
Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na+ K+ ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40 microM) induced 3H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A2 (cPLA2) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [86Rb+] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA2alpha inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the alpha-adaptin but not beta2- or mu2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kgamma siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kgamma to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA2alpha activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.
Gallicchio
Marisa A
MA
Monash University, Department of Medicine, Alfred Hospital, Commercial Rd., Prahran, 3004, Australia.
Bach
Leon A
LA
D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D054513:Group IV Phospholipases A2 / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013552:Swine; D050656:Transcription Factor AP-2 / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D017127:Glycation End Products, Advanced; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D050656:Transcription Factor AP-2; D016718:Arachidonic Acid; D051546:Cyclooxygenase 2; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D054513:Group IV Phospholipases A2; D000254:Sodium-Potassium-Exchanging ATPase