Hypoxia‐inducible factor‐2 stabilization is not sufficient to induce erythropoietin production in deeper medullary fibroblasts

Under hypoxaemic conditions, cortical fibroblasts primarily produce erythropoietin (EPO). However, we have previously shown that most interstitial fibroblasts positive for platelet-derived growth factor receptor β (PDGFR-β) in all kidney zones are also able to produce EPO. Therefore, we wondered if either the physiological stimuli might not be sufficient to stabilize the hypoxia-inducible factor (HIF)-2 in medullary fibroblasts or if different expression patterns or functions of the HIF-regulating prolyl-4-hydroxylases (PHD) 2 and 3 might explain the restrictive EPO cell recruitment. This study shows that although HIF-2 can be clearly stabilized in deeper medullary fibroblasts by pharmacological PHD-inhibition, this is not sufficient to induce EPO in these cells. In contrast, genetic stabilization of HIF-2 by cell-specific deletion of either PHD2 or PHD2 and PHD3 in mice resulted in EPO production in all kidney zones. EPO induction in PHD2/3-KO mice was twice as high as in PHD2-KOs. PHD3 deletion slightly increased basal EPO expression. Accordingly, in contrast to PHD2, PHD3 expression was only detected in a subset of interstitial fibroblasts, without zonal accumulation or hypoxaemic upregulation. Exposure of PHD3-deficient mice to a hypoxaemic stimulus resulted in significantly higher EPO levels compared to controls, with EPO induction restricted to the cortex. Overall, our data suggest the existence of additional regulatory mechanisms beyond the HIF-2 signalling pathway that control EPO expression in deeper medullary fibroblasts. Furthermore, they identify PHD3 as an attenuating factor that delays EPO induction in a subset of cortical PDGFR-β⁺ cells, but its expression pattern is not the determining factor responsible for the cortical restriction of EPO. KEY POINTS: Pharmacological prolyl-4-hydroxylase (PHD) inhibition activates hypoxia-inducible factor (HIF)-2 signalling in interstitial fibroblasts from all renal zones. HIF-2 stabilization is not sufficient to induce erythropoietin (EPO) expression in deeper medullary fibroblasts, although they are in principle capable of producing EPO. There are two subsets of interstitial fibroblasts, PHD2⁺ and PHD2/PHD3⁺ fibroblasts, that are evenly distributed throughout the kidneys, thus also not determining the restrictive cortical induction of EPO under hypoxaemic and pharmacological conditions. Solely PHD2⁺ fibroblasts are the predominant EPO producers in the renal cortex, while PHD3 is an attenuating factor that delays EPO induction in PHD2/PHD3⁺ fibroblasts. HIF2-induced upregulation of PHD isoforms is not detectable in interstitial fibroblasts. Overall, our data suggest the existence of additional regulatory mechanisms, in addition to the HIF-2 signalling pathway, that control EPO expression in deeper medullary fibroblasts.