Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone

doi:10.1016/j.kint.2021.03.019

Kidney International

Volume 100, Issue 1, July 2021, Pages 79-89

https://doi.org/10.1016/j.kint.2021.03.019 Get rights and content

Vadadustat is an investigational hypoxia-inducible factor prolyl hydroxylase inhibitor that increases endogenous erythropoietin production and has been shown to decrease hepcidin levels, ameliorate iron restriction, and increase hemoglobin concentrations in anemic patients with chronic kidney disease (CKD). In studies of physiological responses to other erythropoietic stimuli, erythropoietin induced erythroblast secretion of erythroferrone (ERFE), which acts on the liver to suppress hepcidin production and mobilize iron for erythropoiesis. We therefore investigated whether vadadustat effects on erythropoiesis and iron metabolism are dependent on ERFE. Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.

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Section snippets

Methods

Full methods are detailed in the Supplementary Material. Six-week-old male EKO mice and WT littermates were placed on 8-week diets that did or did not contain 0.2% adenine, which induces CKD. To assess the treatment effects of vadadustat in the setting of CKD, for the last 3 weeks of the diets, the mice were treated daily with vadadustat solution, dosed at 75 mg/kg/d via oral gavage, or vehicle solution. Mice were killed at 14 weeks of age. We obtained whole blood, serum, plasma, liver, spleen,

Effects of vadadustat on erythropoietic parameters in WT and EKO mice with and without CKD

To assess the efficacy of vadadustat in the presence or absence of ERFE and CKD, we evaluated the effects of 3 weeks of daily vadadustat treatment in WT and EKO mice with and without adenine diet-induced CKD. In the non-CKD model, in which the mice were not anemic, vadadustat induced similar increases in hemoglobin concentrations from pretreatment values in the WT and EKO groups (Supplementary Figure S1). In the CKD model, as expected, both WT and EKO groups developed moderate anemia (Figure 1a

Discussion

Vadadustat is an HIF-PHI in development for the treatment of CKD-associated anemia. In the present study, we tested the hypothesis that ERFE, an EPO-induced, hepcidin-suppressive hormone,¹² is involved in vadadustat’s mechanism of action. We used an adenine nephropathy CKD model, treating WT and EKO mice with vadadustat or vehicle. In this CKD model, vadadustat was as effective in increasing hemoglobin concentrations in EKO mice as it was in WT mice, suggesting that ERFE-mediated hepcidin

Disclosure

MRH and TG receive research support from Akebia Therapeutics Inc. All the other authors declared no competing interests.

Acknowledgments

This research study was funded by an Akebia Therapeutics Inc. grant to TG. This work was presented at the American Society of Nephrology 2020 Kidney Week (PO0291).

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As a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the adequate supply of iron to the bone marrow for red blood cell production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine fibrinogen-like 1 (FGL1) as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia, and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in higher hepcidin levels at baseline and after bleeding. FGL1 exerts its activity by directly binding to bone morphogenetic protein 6 (BMP6), thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription.
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Anemia is common in patients with chronic kidney disease and is associated with a high burden of morbidity and adverse clinical outcomes. In 2012, Kidney Disease: Improving Global Outcomes (KDIGO) published a guideline for the diagnosis and management of anemia in chronic kidney disease. Since then, new data from studies assessing established and emerging therapies for the treatment of anemia and iron deficiency have become available. Beginning in 2019, KDIGO planned 2 Controversies Conferences to review the new evidence and its potential impact on the management of anemia in clinical practice. Here, we report on the second of these conferences held virtually in December 2021, which focused on a new class of agents—the hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs). This report provides a review of the consensus points and controversies from this second conference and highlights areas that warrant prioritization for future research.
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This phenomenon was formerly considered to indicate improved iron utilization specific to HIF-PHIs, but it seems that the decrease in hepcidin levels is mainly mediated by erythroferrone, a hormone produced by erythroid progenitor cells in response to EPO (Liu, Davidoff, Niss, & Haase, 2012; Mastrogiannaki et al., 2012; Volke et al., 2009). However, a preclinical study demonstrated that administration of vadadustat resulted in decreased hepcidin levels even in erythroferrone knockout mice (Hanudel et al., 2021), indicating that HIF-PHIs might have a potential to suppress hepcidin levels independent of EPO production. Phase 3 clinical trials were conducted to evaluate efficacy and safety of HIF-PHIs in both NDD and DD CKD patients.
For the past 3 decades, erythropoiesis-stimulating agents (ESA) in conjunction with iron supplementation has been the mainstay of treatment for anemia in chronic kidney disease (CKD). Although ESAs are well-established and highly efficacious treatment, clinical trials demonstrated that the use of ESAs with a high hemoglobin (Hb) target was associated with increased risk of cardiovascular events. This safety concern raised considerable interest in developing an alternative therapeutic strategy. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) are such novel agents to treat anemia in CKD. They stimulate endogenous erythropoietin production via HIF activation and thereby induce erythropoiesis. At least 6 small-molecule HIF-PHIs have been developed to date. The phase 3 clinical trials demonstrated that their effects were noninferior to ESAs. HIF-PHIs may have several advantages over the conventional treatment, such as oral route of administration and their ability to raise Hb levels in patients with chronic inflammation. Although many of the phase 3 clinical trials demonstrated that HIF-PHIs were noninferior to placebo or ESAs with respect to cardiovascular safety, one of the compounds failed to meet the prespecified noninferiority criterion in non-dialysis-dependent CKD patients, and some studies of another HIF-PHI indicated potential risks for thromboembolic events. While the regulatory agencies of some countries including Japan and the European Union concluded that roxadustat, one of the HIF-PHIs, had a favorable benefit-risk profile, the U.S. Food and Drug Administration decided not to approve the drug because of safety reasons. In order to establish the optimal anemia management in CKD, further studies are needed to evaluate important aspects of HIF-PHIs, such as long-term safety, appropriate Hb target, and the types of patients who would gain benefits from these new drugs.
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There are potential renal benefits of HIF-PHIs as well. A recent study showed that vadadustat treatment decreased serum urea nitrogen and creatinine levels and decreased expression of kidney fibrosis markers in a mouse model of CKD.72 Administration to healthy rats of roxadustat or molidustat, which is another HIF-PHI that is currently in phase 3 clinical trials, increased renal blood flow and glomerular filtration rate through a nitric oxide–dependent mechanism.78
Hypoxia-inducible factors (HIFs) were discovered as activators of erythropoietin gene transcription in response to reduced oxygen (O₂) availability. O₂-dependent hydroxylation of HIFs on proline and asparagine residues regulates protein stability and transcriptional activity, respectively. Mutations in genes encoding components of the O₂-sensing pathway cause familial erythrocytosis. Several small-molecule inhibitors of HIF prolyl hydroxylases are currently in clinical trials as erythropoiesis-stimulating agents. HIFs are overexpressed in bone marrow neoplasms, and the development of HIF inhibitors may improve outcomes in these disorders.
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Basic ResearchAmelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone

Graphical abstract

Section snippets

Methods

Effects of vadadustat on erythropoietic parameters in WT and EKO mice with and without CKD

Discussion

Disclosure

Acknowledgments

Kidney Int

Blood Rev

Kidney Int

Kidney Int

Gastroenterology

Kidney Int

Kidney Int

Kidney Int Rep

J Biol Chem

J Biol Chem

J Biol Chem

Blood

J Biol Chem

Blood

Blood

Blood

Gastroenterology

Kidney Int

Kidney Int

Semin Nephrol

Am J Kidney Dis

Kidney Int

Kidney Int

Kidney Int

Kidney Int

Prevalence of anemia in chronic kidney disease in the United States

PLoS One

Effect of anaemia on mortality, cardiovascular hospitalizations and end-stage renal disease among patients with chronic kidney disease

Nephrology (Carlton)

Anemia and incident end-stage renal disease

Kidney360

Mechanisms of anemia in CKD

J Am Soc Nephrol

Targeting hypoxia-inducible factors for the treatment of anemia in chronic kidney disease patients

Am J Nephrol

Clinical trial of vadadustat in patients with anemia secondary to stage 3 or 4 chronic kidney disease

Am J Nephrol

Effects of vadadustat on hemoglobin concentrations in patients receiving hemodialysis previously treated with erythropoiesis-stimulating agents

Nephrol Dial Transplant

Identification of erythroferrone as an erythroid regulator of iron metabolism

Nat Genet

Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization

Science (New York, NY)

Hepcidin—a potential novel biomarker for iron status in chronic kidney disease

Clin J Am Soc Nephrol

Reduction of serum hepcidin by hemodialysis in pediatric and adult patients

Clin J Am Soc Nephrol

Non-renal-related mechanisms of FGF23 pathophysiology

Curr Osteoporos Rep

Erythropoietin and renoprotection

Curr Opin Nephrol Hypertens

Remote conditioning or erythropoietin before surgery primes kidneys to clear ischemia-reperfusion-damaged cells: a renoprotective mechanism?

Am J Physiol Renal Physiol

Erythropoietin ameliorates ischemia/reperfusion-induced acute kidney injury via inflammasome suppression in mice

Int J Mol Sci

Erythropoiesis stimulating agents and reno-protection: a meta-analysis

BMC Nephrol

Basic Research
Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone