BACKGROUND: Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia. METHODS: Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury. RESULTS: Hypoxia increased mRNA expression of TF (6 h: 2.3 ± 0.05 fold, p < 0.001, 24 h: 5.6 ± 2.4 fold, p < 0.05) and suppressed TFPI (6 h: 0.54 ± 0.04 fold, p < 0.05, 24 h: 0.24 ± 0.06 fold, p < 0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA. CONCLUSION: Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.
BACKGROUND: Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia. METHODS: Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury. RESULTS:Hypoxia increased mRNA expression of TF (6 h: 2.3 ± 0.05 fold, p < 0.001, 24 h: 5.6 ± 2.4 fold, p < 0.05) and suppressed TFPI (6 h: 0.54 ± 0.04 fold, p < 0.05, 24 h: 0.24 ± 0.06 fold, p < 0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA. CONCLUSION:Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.
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