The drug-specific properties of hypoxia-inducible factor-prolyl hydroxylase inhibitors in mice reveal a significant contribution of the kidney compared to the liver to erythropoietin induction.

AIMS: Kidney disease often leads to anemia due to a defect in the renal production of the erythroid growth factor erythropoietin (EPO), which is produced under the positive regulation of hypoxia-inducible transcription factors (HIFs). Chemical compounds that inhibit HIF-prolyl hydroxylases (HIF-PHs), which suppress HIFs, have been developed to reactivate renal EPO production in renal anemia patients. Currently, multiple HIF-PH inhibitors, in addition to conventional recombinant EPO reagents, are used for renal anemia treatment. This study aimed to elucidate the therapeutic mechanisms and drug-specific properties of HIF-PH inhibitors. METHODS AND KEY FINDINGS: Gene expression analyses and mass spectrometry revealed that HIF-PH inhibitors (daprodustat, enarodustat, molidustat, and vadadustat) alter Epo gene expression levels in the kidney and liver in a drug-specific manner, with different pharmacokinetics in the plasma and urine after oral administration to mice. The drug specificity revealed the dominant contribution of EPO induction in the kidneys rather than in the liver to plasma EPO levels after HIF-PH inhibitor administration. We also found that several HIF-PH inhibitors directly induce duodenal gene expression related to iron intake, while these drugs indirectly suppress hepatic hepcidin expression to mobilize stored iron for hemoglobin synthesis through induction of the EPO-erythroferrone axis. SIGNIFICANCE: Renal EPO induction is the major target of HIF-PH inhibitors for their therapeutic effects on erythropoiesis. Additionally, the drug-specific properties of HIF-PH inhibitors in EPO induction and iron metabolism have been shown in mice, providing useful information for selecting the proper HIF-PH inhibitor for each renal anemia patient.

Drugs activating hypoxia-inducible factors correct erythropoiesis and hepcidin levels via renal EPO induction in mice.

The erythroid growth factor erythropoietin (EPO) is mainly produced by the kidneys in adult mammals and induces expansion of erythroid cells and iron use for hemoglobin synthesis. The liver also produces EPO at a lower level than the kidneys. Renal and hepatic EPO production is fundamentally regulated by hypoxia-inducible transcription factors (HIFs) in a hypoxia/anemia-inducible manner. Recently, small compounds that activate HIFs and EPO production in the kidneys by inhibiting HIF-prolyl hydroxylases (HIF-PHIs) have been launched to treat EPO-deficiency anemia in patients with kidney disease. However, the roles of the liver in the HIF-PHI-mediated induction of erythropoiesis and iron mobilization remain controversial. Here, to elucidate the liver contributions to the therapeutic effects of HIF-PHIs, genetically modified mouse lines lacking renal EPO-production ability were analyzed. In the mutant mice, HIF-PHI administration marginally increased plasma EPO concentrations and peripheral erythrocytes by inducing hepatic EPO production. The effects of HIF-PHIs on the mobilization of stored iron and on the suppression of hepatic hepcidin, an inhibitory molecule for iron release from iron-storage cells, were not observed in the mutant mice. These findings demonstrate that adequate induction of EPO mainly in the kidney is essential for achieving the full therapeutic effects of HIF-PHIs, which include hepcidin suppression. The data also show that HIF-PHIs directly induce the expression of duodenal genes related to dietary iron intake. Furthermore, hepatic EPO induction is considered to partially contribute to the erythropoietic effects of HIF-PHIs but to be insufficient to compensate for the abundant EPO induction by the kidneys.

Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.

Gene expression changes related to bone mineralization, blood pressure and lipid metabolism in mouse kidneys after space travel.

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

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

Renal interstitial fibroblasts are responsible for producing the erythroid growth factor Epo and the vasopressor renin in addition to kidney fibrosis, in which they are transformed into myofibroblasts. Therefore, analyses of fibroblasts may elucidate the complex mechanisms of kidney diseases. However, the fragility of these cells makes their isolation for in vitro analyses and ex vivo cultivation difficult. We have overcome these difficulties by mildly dissociating mouse kidneys and coculturing fibroblasts with other kidney cells in semisolid medium. For complete details on the use and execution of this protocol, please refer to Sato et al. (2019a) and Miyauchi et al. (2021).

Renal interstitial fibroblasts coproduce erythropoietin and renin under anaemic conditions.

BACKGROUND: Erythrocyte mass contributes to maintaining systemic oxygen delivery and blood viscosity, with the latter being one of the determinants of blood pressure. However, the physiological response to blood pressure changes under anaemic conditions remain unknown. METHODS AND FINDINGS: We show that anaemia decreases blood pressure in human patients and mouse models. Analyses of pathways related to blood pressure regulation demonstrate that anaemia enhances the expression of the gene encoding the vasopressor substance renin in kidneys. Although kidney juxtaglomerular cells are known to continuously produce renin, renal interstitial fibroblasts are identified in the present study as a novel site of renin induction under anaemic hypotensive conditions in mice and rats. Notably, some renal interstitial fibroblasts are found to simultaneously express renin and the erythroid growth factor erythropoietin in the anaemic mouse kidney. Antihypertensive agents but not hypoxic stimuli induced interstitial renin expression, suggesting that blood pressure reduction triggers interstitial renin induction in anaemic mice. The interstitial renin expression was also detected in injured fibrotic kidneys of the mouse and human, and the renin-expressing interstitial cells in murine fibrotic kidneys were identified as myofibroblasts originating from renal interstitial fibroblasts. Since the elevated expression levels of renin in fibrotic kidneys along with progression of renal fibrosis were well correlated to the systemic blood pressure increase, the renal interstitial renin production seemed to affect systemic blood pressure. INTERPRETATION: Renal interstitial fibroblasts function as central controllers of systemic oxygen delivery by producing both renin and erythropoietin. FUNDING: Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) KAKENHI (17K19680, 15H04691, and 26111002) and the Takeda Science Foundation.

An immortalized cell line derived from renal erythropoietin-producing (REP) cells demonstrates their potential to transform into myofibroblasts.

The erythroid growth factor erythropoietin (Epo) is produced by renal interstitial fibroblasts, called REP (renal Epo-producing) cells, in a hypoxia-inducible manner. In chronic kidney disease (CKD), REP cells lose their Epo-production ability, leading to renal anaemia. Concurrently, REP cells are suggested to be transformed into myofibroblasts, which are the major player of renal fibrosis. Although establishment of cultured cell lines derived from REP cells has been a long-term challenge, we here successfully established a REP-cell-derived immortalized and cultivable cell line (Replic cells) by using a genetically modified mouse line. Replic cells exhibited myofibroblastic phenotypes and lost their Epo-production ability, reflecting the situation in renal fibrosis. Additionally, we found that cell-autonomous TGFß signalling contributes to maintenance of the myofibroblastic features of Replic cells. Furthermore, the promoters of genes for Epo and HIF2α, a major activator of Epo gene expression, were highly methylated in Replic cells. Thus, these results strongly support our contention that REP cells are the origin of myofibroblasts in fibrotic kidneys and demonstrate that cell-autonomous TGFß signalling and epigenetic silencing are involved in renal fibrosis and renal anaemia, respectively, in CKD. The Replic cell line is a useful tool to further investigate the molecular mechanisms underlying renal fibrosis.