Hypoferremic Response to Chronic Inflammation Is Controlled via the Hemojuvelin/Hepcidin/Ferroportin Axis and Does Not Involve Hepcidin-Independent Regulation of Fpn mRNA

Abstract

The iron regulatory hormone hepcidin contributes to the pathogenesis of anemia of inflammation (AI) by inhibiting the iron exporter ferroportin in target cells, causing hypoferremia. Under acute inflammation, hepcidin induction requires hemojuvelin (Hjv), a bone morphogenetic protein co-receptor, while Fpn mRNA is also suppressed in a hepcidin-independent manner. However, it is unclear whether, during chronic inflammation, Hjv and hepcidin-independent Fpn mRNA regulation are critical for hypoferremia and AI. To address these questions, wild type and Hjv^-/- mice, a model of hemochromatosis, were fed for 8 weeks an adenine-rich diet to develop chronic kidney disease (CKD). Renal inflammation, accessed by increased Il6 mRNA expression, did not differ among genotypes. Hjv disruption did not mitigate the severity of kidney injury but suppressed the inflammatory induction of liver hepcidin. CKD triggered hypoferremia and mild anemia in wild type mice; however, Hjv^-/- littermates maintained high serum iron and normal hemoglobin, consistent with a protective effect of Hjv/hepcidin deficiency. Notably, tissue Fpn mRNA levels were not affected by the inflammatory milieu of CKD. Following injection of wild type or Hjv^-/- mice with heat-killed Brucella abortus, Fpn mRNA was suppressed during the acute phase of inflammation but quickly recovered and persisted in the chronic phase. We conclude that Hjv deficiency reduces hepcidin levels and mitigates anemia in the CKD model, providing further support for pharmacological targeting of Hjv for the treatment of AI. Moreover, our data demonstrate that Fpn mRNA suppression only occurs under acute but not chronic inflammatory conditions and therefore cannot substantially contribute to AI pathogenesis.

Keywords: anemia of inflammation; chronic kidney disease; ferroportin; hemojuvelin; hepcidin; iron metabolism.

FIGURE 1

Development of CKD in wild type and Hjv ^−/− mice. Five‐weeks old male wild type and Hjv ^−/− littermate mice (n = 8–10 per group) were fed for 8 weeks standard rodent chow (as control) or an adenine‐rich diet to induce CKD. At the endpoint, after recording of biometric data, the mice were sacrificed, and blood and tissues were collected for hematological, biochemical and histological analysis. (A) Growth rates of mice during the dietary intervention; (B) Body weight at endpoint; (C) Body length without tail at endpoint; (D) Tibia length at endpoint; (E) Blood urea nitrogen (BUN); (F) Serum creatinine; (G) Serum phosphorus; (H) Histological analysis of kidney sections with H&E, Masson's trichrome, Sirius Red and Perls (magnification: 5×); (I) Renal Col1α1 mRNA; (J) Renal Il6 mRNA; (K) Renal Fpn(+IRE) mRNA. Quantitative data are presented as the mean ± SEM. Statistically significant differences are indicated in p values (ns: non‐significant). Analysis was performed with two‐way ANOVA and Tukey's multiple comparison test. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Hjv ^−/− mice are spared from CKD‐induced hypoferremia and anemia. Blood and serum from mice described in Figure 1 were used for hematological and biochemical analysis. (A) Serum iron; (B) Transferrin saturation; (C) Total iron binding capacity (TIBC); (D) Serum ferritin; (E) Serum hepcidin; (F) Hemoglobin; (G) Mean corpuscular volume (MCV); (H) Red cell distribution width (RDW); (I) Hematocrit. All data are presented as the mean ± SEM. Statistically significant differences are indicated in p values (ns: non‐significant). Analysis was performed with two‐way ANOVA and Tukey's multiple comparison test; differences in serum hepcidin between control and CKD Hjv ^−/− mice were also analyzed by the Student's t test.

FIGURE 3

In mice with CKD, hepatic and splenic ferroportin levels are solely controlled post‐translationally via the hemojuvelin/hepcidin axis. Liver and spleen samples from mice described in Figure 1 were used for biochemical and histological studies. (A) Liver Hamp mRNA; (B) Liver Hjv mRNA; (C) Western blot analysis of liver phospho‐Stat3, Stat3, and β‐Actin; (D) Liver Socs3 mRNA; (E) Liver Fpn(+IRE) mRNA; (F) H&E staining, immunohistochemical detection of ferroportin and Perls staining in liver sections (magnification: 10×); (G) Western blot analysis of liver ferroportin and β‐Actin; (H) Splenic Fpn(+IRE) mRNA; (I) H&E staining, immunohistochemical detection of ferroportin and Perls staining in spleen sections (magnification: 10×); (J) Western blot analysis of splenic ferroportin and β‐Actin. Quantitative data are presented as the mean ± SEM. Statistically significant differences are indicated in p values (ns: non‐significant). Analysis was performed with two‐way ANOVA and Tukey's multiple comparison test. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Differential responses of Fpn and Hjv mRNAs to acute and chronic inflammation. Nine‐weeks old male wild type mice (n = 5–7 per group) and Hjv ^−/− mice (n = 4–14 per group) were injected intraperitoneally with 5 × 10⁸ particles/200 μL of heat‐killed B. abortus and sacrificed after 4, 8 or 24 h, or after 7, 14, 21 or 28 days. Control mice were injected with phosphate‐buffered saline and sacrificed after 4 h. At the endpoints, blood and liver were collected and used for hematological and biochemical studies. (A) Serum iron; (B) Transferrin saturation; (C) Total iron binding capacity (TIBC); (D) Serum ferritin; (E) Serum hepcidin; (F) Hemoglobin; (G) Hematocrit; (H) Mean corpuscular volume (MCV); (I) Liver Il6 mRNA; (J) Liver Saa1 mRNA; (K) Liver Ifng mRNA; (L) Liver Socs3 mRNA; (M) Liver Inhbb mRNA; (N) Liver Hamp mRNA relative to liver iron content (LIC); (O) Liver Fpn(+IRE) mRNA; (P) Liver Hjv mRNA. All data are presented as the mean ± SEM. Statistically significant differences vs. saline‐injected control are indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001) or **** (p < 0.0001). Analysis was performed separately for each genotype with one‐way ANOVA and Dunnett's multiple comparison test.