Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells.
Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.
Berrou
J
J
INSERM U702, Hôpital Tenon AP-HP, Paris, France.
Tostivint
I
I
Verrecchia
F
F
Berthier
C
C
Boulanger
E
E
Mauviel
A
A
Marti
H P
HP
Wautier
M P
MP
Wautier
J L
JL
Rondeau
E
E
Hertig
A
A
D000906:Antibodies / Q000494:pharmacology; D015152:Blotting, Northern; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009646:Norleucine / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D016326:Extracellular Matrix Proteins; D005419:Flavonoids; D017127:Glycation End Products, Advanced; D017395:Plasminogen Activator Inhibitor 1; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D000511:alpha-Macroglobulins; C048496:N(6)-carboxymethyllysine; D009646:Norleucine; D010959:Tissue Plasminogen Activator; D020778:Matrix Metalloproteinase 2; D008239:Lysine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one