Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-kappaB and free radical in proximal tubular cells.
BACKGROUND
Uremic toxins have been suggested to promote progression of chronic renal failure. We have shown that organic anion transporter-mediated uptake of uremic toxins induces oxidative stress in opossum kidney renal tubular cells overexpressing the transporter. Plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-kappa B (NF-kappaB) are major factors known to promote tubulointerstitial fibrosis. The present study examined the signaling pathway that is activated by uremic toxins to induce PAI-1 and activate NF-kappaB in human renal proximal tubular cells (HK-2).


METHODS
Uremic toxins in the form of organic anion were examined their ability to induce oxidative stress, PAI-1 gene expression, and NF-kappaB activation in HK-2. PAI-1 expression was measured by enzyme-linked immunosorbent assay (ELISA) and the Northern blotting. Human PAI-1 promoter activity was estimated by luciferase reporter gene (NKkappaB-luc) assay. NF-kappaB activation was measured by the pNFkappaB-luc reporter gene and electrophretic gel mobility shift assay.


RESULTS
Among organic anion species tested, indoxyl sulfate and indoleacetic acid induced free radical production in HK-2. A nonspecific transporter inhibitor (probenecid) suppressed the IS-stimulated radical production. Indoxyl sulfate and indoleacetic acid dose dependently increased the expressions of PAI-1 mRNA and protein in these cells. The luciferase reporter gene assay revealed that indoxyl sulfate and indoleacetic acid dose dependently activated NF-kappaB and PAI-1 promoter. Activation of NF-kappaB was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-kappaB inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate.


CONCLUSIONS
Uremic toxins induce free radical production by renal tubular cells and activate NF-kappaB which, in turn, up-regulates PAI-1 expression. Thus, progression of chronic renal failure may be promoted by PAI-1 up-regulation induced by uremic toxins.
Motojima
Masaru
M
Department of Pharmacology, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan.
Hosokawa
Atsuko
A
Yamato
Hideyuki
H
Muraki
Takamura
T
Yoshioka
Toshimasa
T
D000838:Anions / Q000493:pharmacokinetics; D002478:Cells, Cultured; D005609:Free Radicals / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007200:Indican / Q000493:pharmacokinetics*; D007210:Indoleacetic Acids / Q000493:pharmacokinetics*; D007676:Kidney Failure, Chronic / Q000503:physiopathology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D014118:Toxins, Biological / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D014511:Uremia / Q000503:physiopathology
D000838:Anions; D005609:Free Radicals; D007210:Indoleacetic Acids; D016328:NF-kappa B; D017395:Plasminogen Activator Inhibitor 1; D014118:Toxins, Biological; C030737:indoleacetic acid; D007200:Indican