Cell Biology – Immunology – Pathology
Kidney International (2003) 63, 1671–1680; doi:10.1046/j.1523-1755.2003.00906.x
Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-
B and free radical in proximal tubular cells
Masaru Motojima, Atsuko Hosokawa, Hideyuki Yamato, Takamura Muraki and Toshimasa Yoshioka
Department of Pharmacology and Department of Medical Education, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan; and Biomedical Research Laboratories, Kureha Chemical Industry Co., Tokyo, Japan
Correspondence: Toshimasa Yoshioka, M.D., Department of Pharmacology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: yoshioka@research.twmu.ac.jp
Received 29 May 2002; Revised 29 September 2002; Re-revised 21 November 2002; Accepted 13 November 2002.
Abstract
Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-
B 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-
B) 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-B 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-B 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 (NKB-luc) assay. NF-B activation was measured by the pNFB-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-B and PAI-1 promoter. Activation of NF-B was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-B inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate.
Conclusion
Uremic toxins induce free radical production by renal tubular cells and activate NF-B 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.
Keywords:
uremic toxins, proximal tubular, oxidative stress, NF-kB, PAI-1, renal fibrosis
