Cell Biology Immunology Pathology
Kidney International (2005) 68, 569–583; doi:10.1111/j.1523-1755.2005.00435.x
Synergistic effect of hypoxia and TNF-
on production of PAI-1 in human proximal renal tubular cells
XUAN LI, HIDEKI KIMURA, KIICHI HIROTA, KENJI KASUNO, KUNIO TORII, TOSHIHARU OKADA, HISANORI KUROOKA, YOSHIFUMI YOKOTA and HARUYOSHI YOSHIDA
Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, Fukui University, Fukui, Japan; Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, Osaka, Japan; Department of Anesthesia, The Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, >Japan; Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan; and Department of Molecular Genetics, School of Medicine, Faculty of Medical Sciences, Fukui University, Fukui, Japan
Correspondence: Hideki Kimura, M.D., Division of Nephrology, Department of General Medicine School of Medicine, Faculty of Medical Science, Fukui University, 23 Shimoaizuki, Matsuoka, Fukui 910–1193 Japan E-Mail: hkimura@fmsrsa.fukui-med.ac.jp
Received 30 April 2004; Revised 27 October 2004; Re-revised 22 February 2005; Accepted 3 March 2005.
Abstract
Synergistic effect of hypoxia and TNF-
on production of PAI-1 in human proximal renal tubular cells.
Background
Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-
(TNF-) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs).
Methods
Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O2) and/or TNF- at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O2 without TNF-). Hypoxia-inducible factor-1 (HIF-1) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay.
Results
In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1 (HIF-1) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-. Luciferase reporter gene assay showed that hypoxia, TNF-, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1 significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-
B (NF-B) caused a moderate decrease in PAI-1 production under hypoxia.
Conclusion
Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1 and partially via NF-B activation in HPTECs. TNF- can synergistically enhance this hypoxia-induced PAI-1 expression.
Keywords:
PAI-1, hypoxia, TNF-, HIF-1, synergistic effect, human proximal tubular cells, cDNA array
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