High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1.
BACKGROUND
High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44).


METHODS
Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation.


RESULTS
In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1.


CONCLUSIONS
In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.
Glogowski
E A
EA
Department of Medicine, University of Toronto, Ontario, Canada.
Tsiani
E
E
Zhou
X
X
Fantus
I G
IG
Whiteside
C
C
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology