Alteration of the DNA Methylation Signature of Renal Erythropoietin-Producing Cells Governs the Sensitivity to Drugs Targeting the Hypoxia-Response Pathway in Kidney Disease Progression

Abstract

Chronic kidney disease (CKD) affects more than 10% of the population worldwide and burdens citizens with heavy medical expenses in many countries. Because a vital erythroid growth factor, erythropoietin (EPO), is secreted from renal interstitial fibroblasts [renal EPO-producing (REP) cells], anemia arises as a major complication of CKD. We determined that hypoxia-inducible factor 2α (HIF2α), which is inactivated by HIF-prolyl hydroxylase domain-containing proteins (PHDs) in an oxygen-dependent manner, tightly regulates EPO production in REP cells at the gene transcription level to maintain oxygen homeostasis. HIF2α-mediated disassembly of the nucleosome in the EPO gene is also involved in hypoxia-inducible EPO production. In renal anemia patients, anemic and pathological hypoxia is ineffective toward EPO induction due to the inappropriate over-activation of PHDs in REP cells transformed into myofibroblasts (MF-REP cells) due to kidney damage. Accordingly, PHD inhibitory compounds are being developed for the treatment of renal anemia. However, our studies have demonstrated that the promoter regions of the genes encoding EPO and HIF2α are highly methylated in MF-REP cells, and the expression of these genes is epigenetically silenced with CKD progression. This finding notably indicates that the efficacy of PHD inhibitors depends on the CKD stage of each patient. In addition, a strategy for harvesting renal cells, including REP cells from the urine of patients, is proposed to identify plausible biomarkers for CKD and to develop personalized precision medicine against CKD by a non-invasive strategy.

Efficient isolation of interstitial fibroblasts directly from mouse kidneys or indirectly after ex vivo expansion

“…This protocol for the isolation of fibroblasts from healthy kidneys is applicable to the isolation of myofibroblasts from fibrotic kidneys, which originate from REP cells in injured kidneys ( Souma et al., 2013 ; Sato et al., 2019a , 2019b ). Because myofibroblasts occupy a large portion of fibrotic kidneys in mice subjected to unilateral ureteral obstruction, sufficient numbers of myofibroblasts are isolated from the mice for molecular and cellular analyses ( Souma et al., 2013 ).…”

Heterogeneity of Fibroblasts in Healthy and Diseased Kidneys

“…EPO production is stimulated by hypoxia and regulated by hypoxia-inducible factors (HIFs). In normoxic conditions, HIFs are hydroxylated by HIF-prolyl hydroxylase domain-containing proteins (PHDs), and hydroxylated HIFs are degraded by the ubiquitin-proteasome system [36,37]. In hypoxic conditions, the hydroxylation and degradation of HIFs is inhibited, resulting in the transcriptional activation of HIF-inducible genes, including EPO.…”

DNA Methylation Dysfunction in Chronic Kidney Disease

“…HIF-1α and HIF-2α may play different roles in various anemia conditions; HIF-2α, for example, mediates nucleosome disassembly, in the mechanism of hypoxia-dependent erythropoietin induction. Animal studies have clarified that, when oxygen is available, HIF is inactivated by post biosynthetic modifications of basic amino acid residues in the α subunits [17], an oxygen-dependent function which is accomplished by proteins containing a HIF-prolyl hydroxylase domain (PHDs) [7]. Sato and coworkers recently showed that, in CKD related anemia, there is a disruption of the mechanism linking hypoxia with the consequent EPO induction.…”

“…This is due to (I) metaplasia of renal EPO producing cells (REPs) into myofibroblasts (MF-REPs); (II) the pathological hyperactivation of PHDs as the result of kidney damage [7]. Although PHD inhibitors (e.g., roxaduxat) have been considered in the treatment of CKD-related anemia, their action may be partly neutralized, however, as CKD progression proceeds, since the HIF-2α and EPO encoding gene promoter regions are hypermethylated and therefore epigenetically silenced in MF-REPs, thus hampering the expression of these genes [7]. This is indeed a field of active research for personalized treatment applications in CKD patients, provided that a robust assessment of disease stage is accomplished.…”

“…Sato and coworkers recently showed that, in CKD related anemia, there is a disruption of the mechanism linking hypoxia with the consequent EPO induction. This is due to (I) metaplasia of renal EPO producing cells (REPs) into myofibroblasts (MF-REPs); (II) the pathological hyperactivation of PHDs as the result of kidney damage [ 7 ]. Although PHD inhibitors (e.g., roxaduxat) have been considered in the treatment of CKD-related anemia, their action may be partly neutralized, however, as CKD progression proceeds, since the HIF-2α and EPO encoding gene promoter regions are hypermethylated and therefore epigenetically silenced in MF-REPs, thus hampering the expression of these genes [ 7 ].…”