Activation of Intestinal HIF2α Ameliorates Iron‐Refractory Anemia

Abstract In clinics, hepcidin levels are elevated in various anemia‐related conditions, particularly in iron‐refractory anemia and in high inflammatory states that suppress iron absorption, which remains an urgent unmet medical need. To identify effective treatment options for various types of iron‐refractory anemia, the potential effect of hypoxia and pharmacologically‐mimetic drug FG‐4592 (Roxadustat) are evaluated, a hypoxia‐inducible factor (HIF)‐prolyl hydroxylase (PHD) inhibitor, on mouse models of iron‐refractory iron‐deficiency anemia (IRIDA), anemia of inflammation and 5‐fluorouracil‐induced chemotherapy‐related anemia. The potent protective effects of both hypoxia and FG‐4592 on IRIDA as well as other 2 tested mouse cohorts are found. Mechanistically, it is demonstrated that hypoxia or FG‐4592 could stabilize duodenal Hif2α, leading to the activation of Fpn transcription regardless of hepcidin levels, which in turn results in increased intestinal iron absorption and the amelioration of hepcidin‐activated anemias. Moreover, duodenal Hif2α overexpression fully rescues phenotypes of Tmprss6 knockout mice, and Hif2α knockout in the gut significantly delays the recovery from 5‐fluorouracil‐induced anemia, which can not be rescued by FG‐4592 treatment. Taken together, the findings of this study provide compelling evidence that targeting intestinal hypoxia‐related pathways can serve as a potential therapeutic strategy for treating a broad spectrum of anemia, especially iron refractory anemia.


Introduction
Nearly a quarter of the world's population suffers from anemia. [1,2]Anemia of inflammation (AI) and iron-deficiency-induced anemia are the two most common forms worldwide.And they often coexist, causing a severe additional burden on the recovery from their primary underlying diseases in people with a high prevalence of nutritional deficiencies, chronic infections, or chronic systemic inflammation. [3,4]echanisms causing iron deficiency with inflammation are centered on increased hepatic hepcidin (encoded by HAMP) and decreased iron exporter ferroportin (FPN, also known as SLC40A1).This dynamic interplay hinders iron absorption from duodenal enterocytes and iron mobilization by the reticuloendothelial system, posing a significant challenge to effective oral iron supplementation.[7] When serum transferrin is saturated, the homeostatic the key scientific question of the study, which is to investigate the potential effect of hypoxia on a mouse model of hepcidin-activated anemia.B) Representative images of 8-week-old control and Tmprss6 −/− mice before and after placement in a hypoxia chamber (O 2 :10%) for 4 weeks.C-Q) RBC C), HGB D) HCT E), MCV F), RET G), renal Epo mRNA H), serum Epo I), Erfe mRNA normalized to the erythroid marker GypA in the bone marrow J) and spleen K), hepatic Hamp mRNA L), serum hepcidin M), splenic iron N), serum iron O), hepatic iron P) and duodenal iron Q) levels were detected in normoxia-and hypoxia-exposed control and Tmprss6 −/− mice.R-T) Representative duodenum images of Hif2, Hif1 and Fpn immunohistochemistry R), duodenal Fpn mRNA S), representative western blot results of duodenal Hif2, Fpn and Dmt1 T) were shown in normoxia-and hypoxia-exposed control and Tmprss6 −/− mice.Scale bars, 100 μm.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons), n = 4-9 mice per group.
To address this clinically relevant question, we examined and demonstrated either hypoxia exposure or treatment with FG-4592, an oral HIF-prolyl hydroxylase (PHD) inhibitor, significantly accelerated the recovery from various forms of hepcidinactivated anemias.Mechanistically, this benefit resulted from stabilized Hif2 and upregulated duodenal Fpn expression that facilitated increased duodenal iron absorption independent of hepatic Hamp expression.These findings suggest duodenal Hif2-Fpn axis could serve as a promising therapeutic target to treat anemias.
Next, we found the staining of both Hif2 and Hif1 in the duodenum, two well-known master transcription factors that regulate cellular responses to hypoxia, are significantly stronger in hypoxia-treated mice compared to normoxia-exposed mice.And Hif2 appears to be present in the villi but Hif1 in the lamina propria (Figure 1R).Duodenal nuclear receptor coactivator 4 (Ncoa4), [28] Fpn, [29] and divalent metal transporter 1 (Dmt1) [21] were reported previously as targets of HIFs and responsible for iron transport.Hypoxia treatment failed to trigger Ncoa4 expressions among control and Tmprss6 −/− mice (Figure S1J, Supporting Information), suggesting Ncoa4-mediated intestinal ferritinophagy could not be required for erythropoiesis under hypoxia treatment.Duodenal Dmt1 levels were unchanged (Figure S1K, Supporting Information), but Fpn levels were increased significantly in Tmprss6 −/− mice upon hypoxia treatment (Figure 1S).Consistently, although hepcidin levels in hypoxiatreated-Tmprss6 −/− mice remained unchanged, duodenal Fpn protein levels were significantly higher compared to normoxiatreated-Tmprss6 −/− mice (Figure 1R-T; Figure S1L, Supporting Information).All these observations suggest hypoxia promotes iron absorption and erythropoiesis recovery of IRIDA by a regulatory mechanism independent of the canonical hepatic Hampduodenum Fpn axis.
To further confirm whether hypoxia treatment could improve IRIDA through hepatic Hamp-duodenum Fpn axis, we constructed a hepatocyte-specific Tmprss6 knockout mouse, Tmprss6 flox/flox ; Alb-Cre (Tmprss6-LKO).Decreased hepatic Tm-prss6 and increased hepatic Hamp expressions were confirmed by RT-qPCR (Figure S2A,N, Supporting Information).Similar to Tmprss6 −/− mice, Tmprss6-LKO mice exposed to hypoxia for 4 weeks exhibited significantly improved hematological alterations, unchanged hepatic Hamp and serum hepcidin levels, but significantly higher serum iron levels, less duodenum iron retention with higher duodenal Fpn expressions (Figure S2, Supporting Information) compared to normoxiatreated-Tmprss6-LKO mice.Together, these data suggest that hypoxia-induced recovery of anemic phenotypes in these IRIDA mice mainly results from increased duodenal Fpn expression.

FG-4592 Therapeutically Ameliorates IRIDA in Mice
FG-4592 is an orally reversible inhibitor of HIF-PHD, which stabilizes HIFs and mimics natural hypoxia responses. [30,31]Therefore, we examined the effect of FG-4592 in the IRIDA mouse models.After 4 weeks treatment of oral FG-4592, Tmprss6 −/− mice showed near complete hair growth and improved RBCs, HGB, HCT, and RET levels (Figure 2A-F), and improved extramedullary hematopoiesis stress shown by decreased spleen mass and spleen H&E staining (Figure 2G; Figure S3, Supporting Information).Additionally, FG-4592 decreased the levels of renal Epo and Erfe in BM and spleen, but didn't affect serum Epo, hepatic Hamp, serum hepcidin, or splenic non-heme iron levels (Figure 2H-N) in Tmprss6 −/-mice.FG-4592-treated-Tmprss6 −/− mice exhibited elevated serum iron and hepatic non-heme iron levels, decreased duodenal iron levels (Figure 2O-Q), increased duodenal protein expressions of Hif2 and Hif1, unchanged Dmt1 levels and increased Fpn mRNA and protein levels by IHC and western blot (Figure 2R-U) compared to vehicle-treated-Tmprss6 −/− mice.
Similarly, FG-4592-fed Tmprss6-LKO mice presented significant improvement in anemia-and iron-related parameters without the change of hepatic Hamp levels compared to vehicletreated-Tmprss6-LKO mice (Figure S4, Supporting Information).Taken together, these data indicate FG-4592 has potent activity for improving IRIDA independent of the hepatic Hamp-duodenal Fpn axis.

FG-4592 Mediates Iron Homeostasis in Acute and Chronic Inflammatory Conditions
AI is defined as normocytic anemia with systemic inflammation and elevated hepcidin levels, [3] which is prevalent in patients with infections, malignancies, or autoimmune disorders.To investigate the effect of hypoxia or FG-4592 on AI, mice were injected with a single dose of turpentine oil (TO) to establish a widely used mouse model of acute-phase inflammation (Figure 4A). [34,35]Early after a single dose of TO injection (at the 16 th hour), serum IL-6 levels, hepatic Hamp and serum hepcidin levels were significantly higher (Figure 4B-D) compared to the vehicle-injected controls.Interestingly, hepatic Hamp and serum hepcidin levels failed to decrease after hypoxia or FG-4592 treatment for 14 h post TO-treatment compared to the untreated TO group (Figure 4C,D).Serum iron in the TO-treated group was lower than in vehicle-injected controls, whereas increased significantly upon either hypoxia or FG-4592 treatment (Figure 4E), consistent with upregulated duodenum Fpn levels (Figure S6, Supporting Information).
In addition, mice were injected weekly with TO over 3 weeks to induce AI, followed by a 2-week recovery period and treated with either hypoxia or FG-4592 for an additional 3 weeks (Figure 4F).The increased WBC counts reflected the existence of chronic inflammation (Figure 4G).Greater anemia-related blood parameters were presented after hypoxia or FG-4592 treatment compared to untreated TO-induced chronic anemia (Figure 4H-J).Mice chronically exposed to TO showed higher levels of renal Epo and serum Epo, which were decreased by hypoxia or FG-4592 treatment (Figure S7A, Supporting Information; Figure 4K).Erfe levels in BM and spleen were unchanged (Figure S7B,C, Supporting Information).Additionally, hepatic Hamp and serum hepcidin levels were significantly increased (Figure 4L,M), accompanied with increased levels of splenic iron, serum iron, and hepatic iron (Figure 4N-P), upon hypoxia or FG-4592 treatment, compared to TO-treated group.Importantly, stronger IHC staining of duodenal Hif2, Hif1, Fpn mRNA and protein levels were observed in hypoxia-exposed or FG-4592-treated mice (Figure 4Q,R).And only hypoxia-treated mice showed higher Dmt1 levels (Figure S7D, Supporting Information).These results suggest hypoxia and FG-4592 treatment can improve chronic inflammation-induced anemia mainly by increasing duodenum Fpn expression.

FG-4592 Accelerates Recovery from 5-Fluorouracil-Induced Anemia
Anemia remains a common complication of cancer therapy. [36]o evaluate whether hypoxia or FG-4592 modulates the responses to chemotherapy-related anemia (CRA), mice were subjected to the antimetabolite 5-fluorouracil (5-FU), a widely used chemotherapeutic drug to induce a severe and persistent anemia. [37]We compared anemia recovery time with or without hypoxia exposure or FG-4592 treatment on days 14 and 21 after the initiation of 5-FU treatment (Figure 5A).The recovery from anemia was accelerated in hypoxia-or FG-4592treated mice compared to untreated 5-FU-injected mice, respectively (Figure 5B-E).In addition, we found that in the BM, the cell counts of R1 were unchanged, but R2-R5 decreased significantly at day 14, while R2, R4, and R5 recovered at day 21 upon 5-FU injection (Figure 5F; Figure S8A,B, Supporting Information).Notably, either hypoxia or FG-4592 treatment for 7 days could significantly increase R1 numbers and continue the recovery of R2-R5 cell populations upon 5-FU injection at days 14 and 21 (Figures 5F; S8A,B, Supporting Information).
Interestingly, hepatic Hamp and serum hepcidin levels were increased significantly at day 7, suggesting 5-FU-induced CRA belongs to hepcidin-activated anemia (Figure S8C,D, Supporting Information).In addition, hepatic Hamp and serum hepcidin levels were decreased at day 14 and back to normal at day 21 post 5-FU injection, accompanied by higher levels of renal Epo, serum Epo, Erfe in BM and spleen at day 14 upon 5-FU injection (Figure S8C,D, Supporting Information; Figure 5G-L).Importantly, despite higher levels of renal Epo and serum Epo at day 14 upon FG-4592 or hypoxia treatment and higher Erfe at day 14 upon hypoxia treatment, compared to the untreated 5-FU-injected group, hepatic Hamp and serum hepcidin levels were unchanged or even higher upon hypoxia or FG-4592 treatment at either day 14 or day 21 (Figure 5G-L).However, the levels of serum iron and hepatic iron were increased at day 14 upon hypoxia or FG-4592 treatment and at day 21 upon hypoxia treatment (Figures 5M; S8E, Supporting Information), compared to the simple 5-FU injected group at day 14 or 21, respectively.Besides, duodenal iron levels increased at day 14 post 5-FU treatment but recovered at day 21 post 5-FU treatment (Figure 5N).

FG-4592 Improves IRIDA Mainly through HIF2𝜶 Upregulated Duodenal FPN Expression
To further explore the mechanisms underlying FG-4592mediated regulation of Fpn expression, we treated Caco-2 cells, a widely used human intestinal epithelial cell line, with FG-4592.FPN mRNA levels were significantly greater at the 1 h-and 3 htime points post FG-4592 treatment compared to the 0 h-time point (Figure 6A), suggesting FG-4592 regulates FPN expression Hypoxia and FG-4592 improve anemia phenotypes in acute and chronic TO-induced anemia of inflammation in mice.A) A schematic diagram illustrats the experimental design of turpentine oil (TO, 5 ml kg −1 )-induced acute inflammation in 8-week-old wild-type mice.After 2 h of TO induction, the mice were fed with FG-4592 (30 mg kg −1 ) or placed in a hypoxia chamber for 14 h and all mice were sacrificed at 16 h after the first injection of vehicle or turpentine.B-E) Serum IL-6 B), hepatic Hamp C), serum hepcidin D) and serum iron E) levels were detected in vehicle-treated, TO-treated, hypoxiaexposed and TO-treated, and FG-4592-fed and TO-treated mice.F) A schematic diagram illustrats the experimental design of TO (5 ml kg −1 , once a week, 3 weeks, 4 injections in total)-induced chronic AI in 8-week-old wild-type mice.After 2 weeks post the last injection of TO, the mice were placed in a hypoxia chamber or gavaged with FG-4592 (30 mg kg −1 , once a day) for 3 weeks.G-P) WBC G), RBC H), HGB I) and HCT J), serum Epo K), hepatic Hamp mRNA L), serum hepcidin M), splenic iron N), serum iron O) and hepatic iron levels P) were detected in vehicle-treated, TO-treated, hypoxia-exposed and TO-treated, and FG-4592-fed and TO-treated mice.Q,R) Representative duodenum images of Hif2, Hif1, and Fpn immunohistochemistry Q), and duodenal Fpn mRNA levels R) were shown in the four groups of mice in the chronic AI model.n = 4-8 mice per group.Scale bars, 100 μm.One-way ANOVA with Tukey's post hoc test (for multi-group comparisons).
at the transcriptional level.Besides, the protein levels of HIF2, HIF1, and FPN were significantly greater in a dose-dependent manner at the 6 h after FG-4592 treatment compared to untreated control cells (Figure 6B,C).To measure intracellular iron levels, we used the iron-sensitive fluorophore Calcein-AM, which is quenched upon binding iron. [38]Intracellular iron levels were significantly lower at 24 h post FG-4592 treatment compared to respective controls, either with or without ferric citrate (FAC) treatment (Figure 6D,E), consistent with upregulated FPN levels.
In the promoter of the Fpn gene, there are two putative hypoxia response elements (HREs). [29]To further test whether FG-4592 regulates Fpn transcription through directly modulating these two HREs, we analyzed the wild-type Fpn promoter and its HRE site-specific mutants by the dual-luciferase reporter assay (Figure 6F).Co-transfection with a mammalian Hif2 expression construct could strongly increase the luciferase activity of Fpn in HEK293T cells compared to those of empty vector controls (Figure 6G).Whereas the putative HRE mutants showed a significant inhibitory-effect on Fpn expression upon Hif2 induction, which functionally validated these HREs sites were specific HIF-binding sites (Figure 6G).Interestingly, FG-4592 upregulated Fpn expression upon Hif2-mediated induction (Figure 6G).To a lesser extent, similar effects were also observed when co-transfected with a Hif1 expression construct (Figure 6H).However, FG-4592 didn't further change Fpn expression upon Hif1-mediated induction (Figure 6H).Importantly, FG-4592 could directly upregulate the luciferase activity of Fpn through HREs (Figure 6I).Consistently, the chromatin immunoprecipitation (ChIP) assay showed increased binding activity of HIF2 at the FPN promoter at 3 h post FG-4592 treatment, but not of HIF1 in both HEK293T and Caco2 cells (Figure 6J-K).

Intestinal Hif2𝜶 Is Required for the Recovery from 5-FU-Induced Anemia in Mice
To confirm the effect of FG-4592 on Hif2-mediated Fpn expression, we generated intestinal-specific Hif2 knockout mice (Hif2-IKO) by crossing Hif2 flox/flox mice with Villin-Cre mice (Figure S10A, Supporting Information), and examined the function of duodenal Hif2 by cultured intestinal organoids in vitro and 5-FU administration in vivo as indicated (Figure 7A).Unlike control organoids upon FG-4592 treatment, upregulation of Fpn expression (Figure 7B), and Hif2 as well as Fpn protein levels (Figure 7C), were significantly blunted in Hif2-IKO-derived intestinal organoids.And Hif2 has the expression pattern similar to Fpn, both could be enhanced mainly in the basolateral of control organoids treated with FG-4592 (Figure 7D; Figure S10B, Supporting Information).Besides, Hif2-IKO mice showed significantly delayed recovery from 5-FU-induced anemia at day 21 compared to littermate controls (Figure 7E-G).Consistently, Hif2-IKO mice had significantly lower levels of R5 at day 21 in the BM (Figure 7H), and Hif2 is required for the effectiveness of FG-4592 in improving R5 levels in 5-FU-induced anemia (Figure 7I).Moreover, other than similar inflammation levels (Figure S10C, Supporting Information), 5-FU-treated Hif2-IKO mice had significantly higher levels of renal Epo, serum Epo, Erfe in BM and spleen, and lower levels of hepatic Hamp, serum hepcidin, serum iron, and hepatic iron, compared to those of control mice (Figure 7J-Q).Importantly, the rescue effects of FG-4592 on anemia-related and iron-related parameters, especially duodenal Hif2, Fpn mRNA, and protein levels in 5-FU treated control mice could not be replicated in Hif2-IKO mice (Figure 7R-T; Figure S10D, Supporting Information).In comparison, loss of intestinal Hif1 didn't affect the degree of 5-FU-induced anemia (Figure S11, Supporting Information).Taken together, these data demonstrate an indispensable role for an intestinal Hif2-Fpn axis in the protective activity of FG-4592 against perturbed iron absorption-related anemias.

Discussion
In this study, we report hypoxia treatment or its pharmacologically mimetic drug that improves hepcidin-activated anemias, including IRIDA, CKD-associated anemia, AI, and CRA.It is wellknown that HIFs are the master regulators of cellular adaptation to hypoxia.A previous study reported that Hif1 could directly repress Hamp transcription. [40][43] However, this Figure 5. FG-4592 or hypoxia treatment improves 5-fluorouracil-induced chemotherapy-related anemia.A) A schematic diagrasm illustratsings the experiment design of 8-week-old wild-type mice treated with 5-FU (150 mg kg −1 ) to model CRA.As indicated, the treated mice were sacrificed on either day 14 or day 21 after the first injection of 5-FU.Mice were randomly placed in either a hypoxia chamber or gavaged with FG-4592 (30 mg kg −1 , once a day) for one week from day 7 to day 14 after the first injection of 5-FU; or randomly assigned to either a hypoxia chamber or gavaged with FG-4592 (30 mg kg −1 , once a day) for two weeks from day 7 to day 21 after the first injection of 5-FU.B-E) RBC B), HGB C), HCT D) and RET E) in vehicle-treated, 5-FU-treated, hypoxia-exposed and 5-FU-treated, and FG-4592-fed and 5-FU-treated mice.F) Flow cytometry analysis of erythroid cell populations (R1-R5) in bone marrow at day 0, 14, and 21 post 5-FU injection.R2-R5: day 14 of 5-FU group versus control group; R2, R4-R5: day 14 of 5-FU group versus day 21 of 5-FU group; R1-R5: comparison between hypoxia/FG-4592 treatment at day 14 and 21 group, and untreated 5-FU group at day 14 and 21, respectively.G-O) Renal Epo mRNA G), serum Epo H), Erfe mRNA normalized to GypA in the bone marrow I) and spleen J), hepatic Hamp mRNA K), serum hepcidin L), serum iron M), duodenal iron N) and serum IL-6 O) levels were measured in the indicated groups.P) Representative images of immunohistochemistry for duodenal Hif2, Hif1, and Fpn from the indicated groups at day 14 and day 21.Scale bars, 100 μm.Q) Duodenal Fpn mRNA levels were tested in the indicated groups.n = 4-15 mice per group.One-way ANOVA with Tukey's post hoc test (for multi-group comparisons).
regulatory mechanism doesn't apply to these hepcidin-activated anemia scenarios.Instead, we functionally characterize here that targeting the duodenal Hif2-Fpn axis could directly promote iron absorption independent of hepcidin, especially in hepcidinactivated and iron-refractory anemias.
To investigate its potential translational applications, we examined the efficacy of FG-4592 in animal models of hepcidinactivated anemias.FG-4592 functions by stabilizing HIFs through the potent inhibition of PHD activity.Notably, among the available PHD inhibitors, FG-4592 has received clinical approval for the treatment of anemia of CKD in certain countries, such as China and Japan.Previous studies showed FG-4592 improved anemia of CKD by increasing Epo and reducing serum hepcidin in the patients. [32,33]Interestingly, we found intestine serves as the additional important effective organ of FG-4592, which functions to activate duodenal Fpn expression by stabilizing Hif2.This defines an additional important compensatory mechanism in the intestine that underlies the hypoxia/FG-4592induced accelerated recovery from hepcidin-activated anemias in the murine models, consistent with the fact that mice derive a greater proportion of their daily iron needs from dietary intake versus erythroid turnover. [44]In addition, Hif2 is sensitive to cellular iron and oxygen levels, and regulated by the hepcidin-Fpn axis through limiting the activity of iron-dependent PHD enzymes. [23]In cases of hepcidin-activated anemias, hepcidin levels were increased accompanied by decreased duodenal Fpn expression and accumulated duodenal iron.This dysregulation could ultimately result in suppressed Hif2 expression.Consequently, there is a lack of an appropriate intestinal HIF response, which is a major mechanism underlying the development of anemia.Therefore, targeting Hif2 in the duodenum through hypoxia or FG-4592 treatment may offer a potential mechanism to bypass iron regulation in these conditions.
Although Dmt1 has been previously shown to be a Hif2 target gene under low iron conditions, [21] using other stimuli such as PHD inhibition or hypoxia, the duration, concentration, and extent of the treatment could lead to variable results with Dmt1 in vivo, unlike what is seen during iron deficiency.Notably, the transcriptional regulation of Fpn by Hif2 has also been well described, [20,22,29] these important findings have created new therapeutic opportunities for the treatment of hemochromatosis and anemia.For example, intestinal Hif2-Fpn axis is essential for the local absorptive response to systemic iron overload.Deletion of intestinal Hif2 or pharmacological blockade of Hif2 using a clinically relevant inhibitor PT2385 successfully reduced iron accumulation in a mouse model of hepcidin-deficient hemochromatosis. [23,45]Besides, intestine-specific disruption of Hif2 [46,47] or dietary iron restriction [48] or use of the oral Fpn inhibitor vamifeport [49][50][51] has been reported to improve ironoverloaded anemias, such as sickle cell disease and -thalassemia in mice.In this study, we functionally demonstrate stabilizing duodenal Hif2 by hypoxia or FG-4592 treatment results in significantly increased Fpn expression and subsequent improvement of many types of anemias.Therefore, targeting the duodenal Hif2-Fpn axis serves as a promising therapeutic strategy for modulating perturbed iron absorption-related pathogenic conditions.One of the limitations of our study is that inducible intestinal Fpn or Dmt1 knockout mice might be further tested for exploring the precise mechanism of FG-4592.
From a clinical perspective, hepcidin-activated anemia remains a highly prevalent morbidity in patients with chronic inflammation, posing a major challenge to the development of treatment strategies.In principle, RBC transfusions, iron supplementation and using erythropoiesis-stimulating agents (ESAs) [3] are three treatment options for AI.However, RBC transfusions are restricted to patients with severe anemia (Hb < 8 g L −1 ) and are considered as a temporary strategy due to increased mortality in specific conditions. [52,53]Accordingly, iron supplementation is also ineffective in this case, as iron treatment could further increase not only hepcidin levels but also the pathogenicity of some microbes, which in turn increased the burden of infectious diseases, [3] ESAs, like epoetin or darbepoetin alfa, are associated with cardiovascular side effects and increased risks for thrombosis. [54]Notably, loss of Tmprss6 presents with a drastically blunted hepcidin responsiveness upon Epo treatment. [55]Therefore, there is an urgent need to develop novel safe and effective therapies for hepcidin-activated anemias.Our findings functionally demonstrate the efficacy of FG-4592 as a novel approach for the treatment of IRIDA, AI, and CRA.However, one limitation is that FG-4592 treatment could trigger both iron absorption pathways and other metabolism pathways, [56] we could not completely rule out other potential off-target effects of FG-4592.

Conclusion
In conclusion, the hepcidin independent effect of Hif2-Fpn axis on anemia recovery was demonstrated in multitype of anemia models, including IRIDA, inflammatory anemia, and chemotherapy-induced anemia.Importantly, this study provides compelling evidence of a clinically relevant pharmacological approach to target the duodenal Hif2-Fpn axis as a novel strategy to improve various forms of refractory hepcidin-activated anemias.
Figure 6.FG-4592 upregulates FPN expression via HIF2 binding to HREs.A) Real-time qPCR analysis of FPN levels in Caco2 cells treated with FG-4592 at 0, 1, and 3 h.B,C) Representative western blot analysis of HIF2, HIF1, and FPN levels in Caco2 cells treated with FG-4592 (0, 20, 50, 100 μM) for 1, 3, and 6 h B), and quantitation for the results of 6-h time points, results were shown relative to ACTIN and comparison between 20/50/100 μm with 0 μm C).D,E) Flow cytometry analysis D) and summary of Calcein-AM mean fluorescence intensity (MFI) E) in Caco2 cells treated with FG-4592 for 24 h, with or without ammonium ferric citrate (FAC:100 μm) treatment.F) A schematic diagram of the mouse Fpn promoter illustrating two wild-type or mutant HREs in the regulatory region, which are numbered in relation to the translation initiation site.G,H) HEK293T cells were transiently transfected with wild-type mouse Fpn or the mutant luciferase-reporter construct, and co-transfected with empty vector or Hif2 G) or Hif1 H) expression plasmids or treated with FG-4592.I) Relative light units (RLU) of reporter activity in HEK293T cells that were transiently transfected with the wild-type or the mutant luciferase construct, and treated with FG-4592.J,K) Chromatin immunoprecipitation (ChIP) assay results from HEK293T and Caco2 cells expressing HIF1 and HIF2 relative to normal anti-rabbit IgG treated with FG-4592.L-Q) In control, Tmprss6 −/− , Hif2 LSL/+ and Tmprss6 −/− ; Hif2 LSL/+ mice, duodenal Fpn mRNA levels L), RBC M), HGB N), HCT O), MCV P), and RET Q) were measured, respectively.(n = 7-8 mice per group).A, E, G-Q, one-way ANOVA with Tukey's post hoc test.R) A schematic diagram illustrats that FG-4592 functions as a stabilizer of duodenal HIF2, which binds to two HREs of the FPN promoter to stimulate FPN expression and thus promotes duodenal iron export.Intestine-specific Hif2 knockout reduces recovery from 5-fluorouracil-induced chemotherapy-related anemia.A) A schematic diagram illustrats the use of control and Hif2-IKO to establish in vitro intestinal organoids, that were treated with 100 μM FG-4592 within 24 h for RNA measurement and 1-3 h for protein testing.Additionally, control and Hif2-IKO mice were injected with 5-FU, and fed with vehicle or FG-4592 for 14 days.B-D) Fpn mRNA B), Hif2 and Fpn protein C), and immunofluorescent staining for Hif2 and Fpn D) from the organoids described in A) were detected.E-G) RBC E), HGB F), and HCT G) levels in littermate controls and Hif2-IKO mice at day 21 after 5-FU injection, with or without daily FG-4592 treatment 7 days after 5-FU injection.H,I) Flow cytometry analysis of R1-R5 in bone marrow of control and Hif2-IKO mice at day 21 of 5-FU injection with or without FG-4592 treatment from day 7 to day 21 after 5-FU injection.H): * represents p < 0.05 in R5 between 5-FU treated control and Hif2-IKO mice.J-Q) Renal Epo mRNA J), serum Epo K), Erfe mRNA normalized to GypA in the bone marrow L) and spleen M), hepatic Hamp mRNA N), serum hepcidin O), serum iron P) and liver iron levels Q) were measured in 5-FU administrated control and Hif2-IKO mice with or without FG-4592 treatment.R) Representative images of immunohistochemistry for Hif2 and Fpn in duodenal sections from the indicated four groups.Scale bars, 100 μm.S,T) Duodenal Dmt1 S) and Fpn mRNA T) were detected in the indicated four groups.n = 4-10 mice per group.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons).

Figure 1 .
Figure 1.Global Tmprss6 knockout mice display improved phenotypes of IRIDA upon hypoxia exposure for 4 weeks.A) A schematic diagram illustrates the key scientific question of the study, which is to investigate the potential effect of hypoxia on a mouse model of hepcidin-activated anemia.B) Representative images of 8-week-old control and Tmprss6 −/− mice before and after placement in a hypoxia chamber (O 2 :10%) for 4 weeks.C-Q) RBC C), HGB D) HCT E), MCV F), RET G), renal Epo mRNA H), serum Epo I), Erfe mRNA normalized to the erythroid marker GypA in the bone marrow J) and spleen K), hepatic Hamp mRNA L), serum hepcidin M), splenic iron N), serum iron O), hepatic iron P) and duodenal iron Q) levels were detected in normoxia-and hypoxia-exposed control and Tmprss6 −/− mice.R-T) Representative duodenum images of Hif2, Hif1 and Fpn immunohistochemistry R), duodenal Fpn mRNA S), representative western blot results of duodenal Hif2, Fpn and Dmt1 T) were shown in normoxia-and hypoxia-exposed control and Tmprss6 −/− mice.Scale bars, 100 μm.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons), n = 4-9 mice per group.

Figure 2 .
Figure 2. FG-4592 improves phenotypes of IRIDA in global Tmprss6 knockout mice.A) A schematic diagram illustrating the action mode of FG-4592 (left) and representative images of Tmprss6 −/− and their littermate control mice with or without FG-4592 (30 mg kg −1 gavage, once a day) for 4 weeks (right).B-Q) RBC B), HGB C), HCT D), MCV E) and RET F), spleen/body weight (%) G), renal Epo mRNA H), serum Epo I), Erfe mRNA normalized to the erythroid marker GypA in the bone marrow J) and spleen K), hepatic Hamp mRNA L), serum hepcidin M), splenic iron N), serum iron O), hepatic iron P) and duodenal iron levels Q) were detected in saline vehicle-and FG-4592-treated control and Tmprss6 −/− mice.R-U) Representative duodenum images of immunohistochemistry for Hif2, Hif1 and Fpn R), duodenal Dmt1 S) and Fpn T) mRNA levels, and representative western blot results of Hif2, Fpn and Dmt1 U) were shown in vehicle-and FG-4592-treated control and Tmprss6 −/− mice.Scale bars, 100 μm.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons), n = 4-9 mice per group.

Figure 3 .
Figure3.FG-4592 ameliorates anemia of CKD in both control and Tmprss6-LKO mice.A) A schematic diagram illustrats the experimental design of sham-operated or 5/6 nephrectomy (5/6Nx)-induced anemia of CKD in Tmprss6-LKO and their littermate control mice with or without treatment of FG-4592 (30 mg kg −1 , once a day) for 4 weeks.B-J) RBC B), HGB C), HCT D), renal Epo mRNA E), serum Epo F), hepatic Hamp mRNA G), serum hepcidin H), serum iron I) and duodenal iron levels J) were detected in vehicle-and FG-4592-treated control and Tmprss6-LKO mice with sham operation or 5/6Nx.K) Representative duodenum images of Hif2, Hif1, and Fpn immunohistochemistry from vehicle-and FG-4592-treated control and Tmprss6-LKO mice with 5/6Nx.Scale bars, 100 μm.L) Duodenal Fpn mRNA levels were tested in vehicle-and FG-4592-treated control and Tmprss6-LKO mice with sham operation or 5/6Nx.M) Representative western blot results of Hif2, Fpn, and Dmt1 from vehicle-and FG-4592-treated control and Tmprss6-LKO mice with 5/6Nx.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons), n = 4-11 mice per group.N) A schematic diagram illustrats the model that FG-4592 promotes erythropoiesis in the high hepcidin context.FG-4592 pharmacologically increases HIF in the duodenum by increasing the transcription levels of the HIF target gene Fpn, which increases iron delivery to marrow erythroblasts to increase erythropoiesis and thus improves anemia.

Figure 4 .
Figure 4. Hypoxia and FG-4592 improve anemia phenotypes in acute and chronic TO-induced anemia of inflammation in mice.A) A schematic diagram illustrats the experimental design of turpentine oil (TO, 5 ml kg −1 )-induced acute inflammation in 8-week-old wild-type mice.After 2 h of TO induction, the mice were fed with FG-4592 (30 mg kg −1 ) or placed in a hypoxia chamber for 14 h and all mice were sacrificed at 16 h after the first injection of vehicle or turpentine.B-E) Serum IL-6 B), hepatic Hamp C), serum hepcidin D) and serum iron E) levels were detected in vehicle-treated, TO-treated, hypoxiaexposed and TO-treated, and FG-4592-fed and TO-treated mice.F) A schematic diagram illustrats the experimental design of TO (5 ml kg −1 , once a week, 3 weeks, 4 injections in total)-induced chronic AI in 8-week-old wild-type mice.After 2 weeks post the last injection of TO, the mice were placed in a hypoxia chamber or gavaged with FG-4592 (30 mg kg −1 , once a day) for 3 weeks.G-P) WBC G), RBC H), HGB I) and HCT J), serum Epo K), hepatic Hamp mRNA L), serum hepcidin M), splenic iron N), serum iron O) and hepatic iron levels P) were detected in vehicle-treated, TO-treated, hypoxia-exposed and TO-treated, and FG-4592-fed and TO-treated mice.Q,R) Representative duodenum images of Hif2, Hif1, and Fpn immunohistochemistry Q), and duodenal Fpn mRNA levels R) were shown in the four groups of mice in the chronic AI model.n = 4-8 mice per group.Scale bars, 100 μm.One-way ANOVA with Tukey's post hoc test (for multi-group comparisons).

Figure 7 .
Figure 7.Intestine-specific Hif2 knockout reduces recovery from 5-fluorouracil-induced chemotherapy-related anemia.A) A schematic diagram illustrats the use of control and Hif2-IKO to establish in vitro intestinal organoids, that were treated with 100 μM FG-4592 within 24 h for RNA measurement and 1-3 h for protein testing.Additionally, control and Hif2-IKO mice were injected with 5-FU, and fed with vehicle or FG-4592 for 14 days.B-D) Fpn mRNA B), Hif2 and Fpn protein C), and immunofluorescent staining for Hif2 and Fpn D) from the organoids described in A) were detected.E-G) RBC E), HGB F), and HCT G) levels in littermate controls and Hif2-IKO mice at day 21 after 5-FU injection, with or without daily FG-4592 treatment 7 days after 5-FU injection.H,I) Flow cytometry analysis of R1-R5 in bone marrow of control and Hif2-IKO mice at day 21 of 5-FU injection with or without FG-4592 treatment from day 7 to day 21 after 5-FU injection.H): * represents p < 0.05 in R5 between 5-FU treated control and Hif2-IKO mice.J-Q) Renal Epo mRNA J), serum Epo K), Erfe mRNA normalized to GypA in the bone marrow L) and spleen M), hepatic Hamp mRNA N), serum hepcidin O), serum iron P) and liver iron levels Q) were measured in 5-FU administrated control and Hif2-IKO mice with or without FG-4592 treatment.R) Representative images of immunohistochemistry for Hif2 and Fpn in duodenal sections from the indicated four groups.Scale bars, 100 μm.S,T) Duodenal Dmt1 S) and Fpn mRNA T) were detected in the indicated four groups.n = 4-10 mice per group.Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons).