A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats

Abstract

Background: Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients.

Methods: We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model.

Results: Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes.

Conclusions: Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.

Keywords: anemia; chronic kidney disease; hepcidin; iron; mouse model.

FIGURE 1:

Schematic of LDN-193189 treatment strategy in a modified adenine-induced kidney disease model. Eight-week-old Wistar male rats were given either a control diet (open bars) for 6 weeks (control) or a 0.75% adenine supplemented diet (black bars) for 3 weeks followed by a control diet (open bars) for another 3 weeks. The adenine diet group was further treated with either a vehicle alone (modified adenine) or LDN-193189 at a dose of 8 mg/kg (modified adenine LDN), while the control group was treated with vehicle alone, starting at Week 1. Tail-vein blood draws (indicated by triangles) were performed at Weeks 0, 2, 4 and 5 for all groups. Animals were sacrificed (S) and tissues harvested for analysis at Week 6.

FIGURE 2:

Increased survival and maintenance of persistent kidney disease in the modified adenine model compared with the original adenine model. (A) Survival rates were measured at 1 to 2-week intervals for rats on a control diet for 8 weeks (control, CTL, dashed line), an adenine diet for 3 weeks followed by 5 weeks of a control diet (modified adenine, MA, solid line) or a continuous adenine diet for 6 weeks (original adenine, OA, dotted line). Sample sizes of each group at each time point are shown below the panels. The decrease in numbers at later time points is due to planned sacrifice of animals for tissue analysis and mortality. P < 0.01 for the original adenine group in comparison to the control group, P = not significant for the modified adenine group in comparison to the control group. (B and C) A subset of rats from panel A was analyzed by phlebotomy or at the time of sacrifice for serum CRE (B) and serum BUN (C). Sample sizes of each group at each time point are shown below the panels (control, CTL, open squares; modified adenine, MA, closed squares; original adenine, OA, closed triangles). ^##P < 0.01 for the original adenine group in comparison to the control group at the same time point, **P < 0.01 and *P < 0.05 for the modified adenine group in comparison to the control group at the same time point.

FIGURE 3:

Rats treated with the modified adenine diet develop increased hepatic hepcidin mRNA, splenic iron sequestration, decreased serum iron and transferrin saturation, decreased reticulocyte count, decreased Hb content of reticulocytes and persistent anemia compared with rats on a control diet. Rats treated with a modified adenine diet as described in Figure 1 (closed squares or black bars) or a control diet (open squares or white bars) were analyzed for (A) Hb, (B) reticulocyte count, (C) hemoglobin content of reticulocytes (CHr), (D) serum iron, (E) serum transferrin saturation (Tf Sat), (F) spleen iron content, (G) liver hepcidin mRNA (Hamp) relative to hypoxanthine-guanine phosphoribosyltransferase mRNA (Hprt) quantitated by real-time RT-PCR and (H and I) liver pSTAT3 relative to STAT3 protein analysis by western blot every 1–2 weeks either by phlebotomy or at the time of sacrifice. Blood analysis at Week 8 was performed on the Heska HemaTrue at MGH, which does not have the capacity to measure reticulocyte counts or CHr. For panels A–E, sample sizes for each group at each time point are shown below the panels (CTL, control; MA, modified adenine). For panels F–H, sample sizes for each group are 3–12. For panel I, representative blots of three animals per group were shown. For all panels, **P < 0.01, *P < 0.05 and ^P = 0.05 for the modified adenine group in comparison to the control group at the same time point.

FIGURE 4:

LDN-193189 treatment of rats on a modified adenine diet decreases hepcidin expression and mobilizes stored iron into plasma. Rats receiving a modified adenine diet were treated with either vehicle alone (modified adenine, closed squares or black bars, n = 8) or LDN-193189 at a dose of 8mg/kg once daily (modified adenine LDN, gray diamonds or bars, n = 8) starting at Week 1 after initiation of the adenine diet until sacrifice. Mice were sacrificed at 6 weeks and analyzed for (A) hepcidin (Hamp) relative to Hprt mRNA by quantitative real-time RT-PCR and (B) spleen iron content. Blood collected via tail-vein phlebotomy at Weeks 0, 1, 3 and 5, and at the time of sacrifice at 6 weeks was analyzed for serum iron (C) and serum Tf sat (D). For all panels significant changes are shown as (**P < 0.01) for the modified adenine LDN group in comparison to the modified adenine group at the same time point.

FIGURE 5:

LDN-193189 treatment of rats on a modified adenine diet increases the reticulocyte hemoglobin content (CHr), reticulocyte MCVr, RBC MCV and MCH, but does not increase Hb levels. Blood from rats receiving a modified adenine diet treated with either vehicle alone (modified adenine, closed squares, n = 8) or LDN-193189 (modified adenine LDN, diamonds, n = 8) from Figure 4 was analyzed for (A) CHr, (B) MCVr, (C) reticulocyte count, (D) Hb, (E) MCV and (F) MCH. For all panels, **P < 0.01, *P < 0.05, ^P = 0.05 for the modified adenine LDN group in comparison to the modified adenine group at the same time point.

FIGURE 6:

Serum EPO levels were undetectable at Week 1 and unchanged from control animals at Week 6 of the modified adenine diet. Blood from rats receiving a control diet treated with vehicle alone (white bars, n = 6), or a modified adenine diet treated with either vehicle alone (modified adenine, black bars, n = 8) or LDN-193189 (modified adenine LDN, gray bars, n = 8) at Weeks 1 and 6 from Figure 4 were analyzed for serum EPO levels by ELISA. Significant changes are shown as **P < 0.01 in comparison to the control group at the same time point. EPO levels were not significantly changed in the modified adenine LDN group in comparison to the modified adenine group at Week 6 (P = 0.25).