High concentration of glucose inhibits glomerular endothelial eNOS through a PKC mechanism.
Kidney glomeruli are important targets of diabetic nephropathy. We hypothesized a high concentration of glucose could suppress glomerular endothelial nitric oxide synthase (eNOS) by a protein kinase C (PKC) mechanism, as has been found in other tissues. Mouse kidney slices (150-200 microm) were bathed in Hanks' solution with 100 microM L-arginine and exposed to either 5 or 20-30 mM D-glucose. Immunofluorescence identified only eNOS in normal mouse glomeruli. Measurements of glomerular NO concentration with NO-sensitive fluorescent dye (4,5-diaminofluorescein diacetate) using confocal microscopy and NO-sensitive microelectrodes verified that resting glomeruli had active production of NO that was inhibited by N(G)-nitro-L-arginine methyl ester. High-concentration (20-30 mM) D-glucose inhibited 60-70% of the NO production within 15-30 min; L-glucose at the same concentration did not have any effect. Inhibition of PKC-beta with 100 nM ruboxistaurin prevented eNOS suppression in high-glucose media. Activation of PKC with 100 nM phorbol ester also suppressed the glomerular NO concentration. We concluded that eNOS in the renal glomerular capillary endothelial cells is suppressed by activity of PKC at high-glucose concentrations comparable to those in diabetic animals and humans. The consequence is a rapid decline in the generation of NO in the glomerular endothelial cells in the presence of a high concentration of glucose.
Chu
Shaoyou
S
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA. schu@hsc.unt.edu
Bohlen
H Glenn
HG
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D005947:Glucose / Q000494:pharmacology*; D006943:Hyperglycemia / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007678:Kidney Glomerulus / Q000201:enzymology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008839:Microelectrodes; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta
C111205:4,5-diaminofluorescein; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005947:Glucose; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester