Transgenic augmentation of erythroferrone in mice ameliorates anemia in adenine-induced chronic kidney disease

Abstract

Anemia is a common and disabling complication of chronic kidney disease (CKD). Current therapies can be burdensome, and full correction of anemia is limited by their cardiovascular side effects. New approaches that may offer additional therapeutic options are needed. We explored the antianemic effects of erythroferrone, an erythroid hormone that induces iron mobilization by suppressing the master iron-regulatory hormone hepcidin. In a preclinical murine model of adenine-induced CKD, transgenic augmentation of erythroferrone mobilized iron, increased hemoglobin concentrations by approximately 2 g/dL, and modestly improved renal function without affecting systemic or renal inflammation, fibrosis, or markers of mineral metabolism. This study supports the concept that therapeutic augmentation of erythroferrone is a promising approach for alleviating CKD-associated anemia.

Keywords: Bone marrow differentiation; Chronic kidney disease; Hematology; Nephrology.

Figure 1. ERFE augmentation ameliorates CKD-associated anemia.

(A) Pilot experimental design: 8-week-old WT littermate (n = 4) and ERFE-overexpressing transgenic (TG; n = 7) mice were fed 100 ppm iron (Fe) diet with 0.2% adenine for 8 weeks and then analyzed. (G) Final experimental design: Mice were weaned at 3 weeks, then WT (n = 7) mice placed on 100 ppm Fe diet and TG (n = 6) placed on 4 ppm Fe diet to prevent systemic iron loading prior to the initiation of adenine diet. At 7 weeks of age, WT and TG mice were placed on 0.2% adenine diet with 100 ppm Fe for 8 weeks and then analyzed. For both experiments, quantitative PCR (qPCR) analysis of (B and H) Erfe expression in bone marrow tissue (C and I), blood hemoglobin concentration, (D and J) RBC count, and (E and K) mean corpuscular hemoglobin (MCH) were determined. (F and L) Mice were weighed weekly. Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.

Figure 2. ERFE augmentation improves kidney function but does not alter injury markers in adenine-induced CKD.

WT and TG mice from Figure 1G (n = 6–7 mice/group) were analyzed after 8 weeks on adenine diet. (A) Body weight, (B) kidney weight, (C) serum creatinine, (D) blood urea nitrogen (BUN). qPCR analysis of (E) Kim1, (F) Ngal, (G) Krt20 and (H) Slc5a2 expression in kidney tissue. (I) Representative H&E-stained kidney sections from WT and TG mice after 8 weeks on adenine diet (black asterisks, dilated tubule; black arrows, glomerular atrophy). Original magnification, ×10 (left) and ×40 (right). Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Figure 3. ERFE augmentation lowers hepcidin levels to enhance iron mobilization in adenine-induced CKD.

WT and TG mice from Figure 1G (n = 6–7 mice/group) were analyzed. (A) Liver iron concentration (LIC). (B) Serum iron. (C and D) Tissue iron concentrations in (C) spleen and (D) kidney. (E) Serum hepcidin levels. (F) Liver hepcidin mRNA expression. (G) Liver hepcidin mRNA/LIC. Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). *P < 0.05, **P < 0.01, ****P < 0.0001. NS, not significant.

Figure 4. ERFE augmentation enhances systemic oxygenation without significantly impacting kidney oxygenation in adenine-induced CKD.

WT and TG mice from Figure 1G (n = 6–7 mice/group) were analyzed. (A) Serum VEGF levels. (B–E) Kidney mRNA concentrations by qPCR for (B) Vegfa, (C) Gapdh, (D) Angptl1, and (E) Epo. Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). *P < 0.05. NS, not significant.

Figure 5. ERFE augmentation does not affect kidney fibrosis in adenine-induced CKD.

In WT and TG mice from Figure 1G (n = 6–7 mice/group), kidney tissues were analyzed by qPCR for (A) Acta2, (B) Col1a1, (C) Col3a1, (D) Tgfb1, and (E) Fn1. (F) Representative Masson’s trichrome–stained kidney sections from WT and TG mice after 8 weeks on adenine diet (black arrows, interstitial fibrosis; red arrows, glomerulosclerosis). Original magnification, ×10 (left) and ×40 (right). Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). NS, not significant.

Figure 6. ERFE augmentation does not substantially change mRNA markers of liver or kidney inflammation in adenine-induced CKD.

WT and TG mice from Figure 1G (n = 6–7 mice/group) were analyzed by qPCR for (A) Saa1 expression in liver tissue and (B–D) Tnfa, Il6, and Il1b expression in kidney tissue. Data are mean ± SEM, analyzed by unpaired t test with Welch’s correction (2-tailed). **P < 0.01. NS, not significant.