Effects of long-term tubular HIF-2α overexpression on progressive renal fibrosis in a chronic kidney disease model
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Renal fibrosis is the final manifestation of chronic kidney disease(CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha(HIF-2α) is an important regulator of chronic hypoxia, and thelate-stage renal tubular HIF-2α activation exerts protective effectsagainst renal fibrosis. However, its specific role in progressiverenal fibrosis remains unclear. Here, we investigated the effectsof the long-term tubular activation of HIF-2α on renal functionand fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelialcells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg)mice and wild-type (WT) controls through a 6-week adeninediet. Tg mice were maintained on doxycycline (DOX) for thediet period to induce Tg HIF-2α expression. Primary TECs isolatedfrom Tg mice were treated with DOX (5 μg/ml), transforminggrowth factor-β1 (TGF-β1) (10 ng/ml), and a combinationof both for 24, 48, and 72 hr. Blood was collected to analyzecreatinine (Cr) and blood urea nitrogen (BUN) levels. Pathologicalchanges in the kidney tissues were observed using hematoxylinand eosin, Masson’s trichrome, and Sirius Red staining. Meanwhile,the expression of fibronectin, E-cadherin and α-smoothmuscle actin (α-SMA) and the phosphorylation of p38 mitogenactivatedprotein kinase (MAPK) was observed using westernblotting. Our data showed that serum Cr and BUN levels weresignificantly lower in Tg mice than in WT mice following theadenine diet. Moreover, the protein levels of fibronectin andE-cadherin and the phosphorylation of p38 MAPK were markedlyreduced in the kidneys of adenine-fed Tg mice. These resultswere accompanied by attenuated fibrosis in Tg mice followingadenine administration. Consistent with these findings, HIF-2αoverexpression significantly decreased the expression of fibronectinin TECs, whereas an increase in α-SMA protein levels wasobserved after TGF-β1 stimulation for 72 hr. Taken together,these results indicate that long-term HIF-2α activation in CKDmay inhibit the progression of renal fibrosis and improve renalfunction, suggesting that long-term renal HIF-2α activation maybe used as a novel therapeutic strategy for the treatment of CKD.
Abstract
Renal fibrosis is the final manifestation of chronic kidney disease(CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha(HIF-2α) is an important regulator of chronic hypoxia, and thelate-stage renal tubular HIF-2α activation exerts protective effectsagainst renal fibrosis. However, its specific role in progressiverenal fibrosis remains unclear. Here, we investigated the effectsof the long-term tubular activation of HIF-2α on renal functionand fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelialcells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg)mice and wild-type (WT) controls through a 6-week adeninediet. Tg mice were maintained on doxycycline (DOX) for thediet period to induce Tg HIF-2α expression. Primary TECs isolatedfrom Tg mice were treated with DOX (5 μg/ml), transforminggrowth factor-β1 (TGF-β1) (10 ng/ml), and a combinationof both for 24, 48, and 72 hr. Blood was collected to analyzecreatinine (Cr) and blood urea nitrogen (BUN) levels. Pathologicalchanges in the kidney tissues were observed using hematoxylinand eosin, Masson’s trichrome, and Sirius Red staining. Meanwhile,the expression of fibronectin, E-cadherin and α-smoothmuscle actin (α-SMA) and the phosphorylation of p38 mitogenactivatedprotein kinase (MAPK) was observed using westernblotting. Our data showed that serum Cr and BUN levels weresignificantly lower in Tg mice than in WT mice following theadenine diet. Moreover, the protein levels of fibronectin andE-cadherin and the phosphorylation of p38 MAPK were markedlyreduced in the kidneys of adenine-fed Tg mice. These resultswere accompanied by attenuated fibrosis in Tg mice followingadenine administration. Consistent with these findings, HIF-2αoverexpression significantly decreased the expression of fibronectinin TECs, whereas an increase in α-SMA protein levels wasobserved after TGF-β1 stimulation for 72 hr. Taken together,these results indicate that long-term HIF-2α activation in CKDmay inhibit the progression of renal fibrosis and improve renalfunction, suggesting that long-term renal HIF-2α activation maybe used as a novel therapeutic strategy for the treatment of CKD.
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