EPO synthesis induced by HIF-PHD inhibition is dependent on myofibroblast transdifferentiation and colocalizes with non-injured nephron segments in murine kidney fibrosis

Abstract

Aim: Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis.

Methods: We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat. Lectin absorption chromatography was used to assess liver-derived EPO. In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy.

Results: In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy.

Conclusions: Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.

Keywords: anemia; chronic kidney disease; erythropoietin; hypoxia-inducible factor; molidustat; prolyl hydroxylase domain.