Rebalancing Iron Homeostasis and Erythropoiesis through Tfr2 Inhibition for Correction of Anemia in CKD

Background

Transferrin Receptor 2 (TFR2) is a protein expressed in the liver and in the erythroid compartment. Hepatic TFR2 activates the transcription of hepcidin, the master regulator of iron homeostasis, and its inactivation causes iron overload. Erythroid TFR2 interacts with Erythropoietin (EPO) receptor and its deletion enhances erythropoiesis increasing EPO sensitivity of erythroid cells. We recently demonstrated that bone marrow (BM) Tfr2 loss improves anemia in a murine model of β-thalassemia. We hypothesized that the same approach might represent a therapeutic option also for anemias due to insufficient EPO production, as anemia of Chronic Kidney Disease (CKD).

Indeed, anemia is a common complication of CKD, since EPO production is inhibited by progressive renal failure. In addition, chronic inflammation that parallels renal damage decreases EPO responsiveness of erythroid cells and enhances the production of hepcidin. Increased hepcidin levels limit iron absorption from the diet and release from the stores, reducing iron supply to erythropoiesis. All these factors contribute to anemia development.

Replacement therapy with erythropoiesis stimulating agents (ESA), usually combined with iron supplementation, is effective but may lead to cardio-vascular side effects. Thus, novel and more specific strategies are needed.

Aims

Here, we exploit different murine models of selective Tfr2 inactivation to test whether Tfr2 targeting corrects anemia of CKD. In this context, BM Tfr2 inhibition is expected to stimulate erythropoiesis and the simultaneous downregulation of hepatic Tfr2 to correct iron deficiency.

Methods

We induced CKD using an adenine-rich diet in mice with:

(1) BM-specific Tfr2 deletion (Tfr2 ^BMKO), generated through BM transplantation;

(2) reduced Tfr2 hepatic expression, obtained treating wild-type mice with anti-Tfr2 antisense oligonucleotides (Tfr2-ASO);

(3) germline Tfr2 inactivation in the whole organism (Tfr2^KO).

Results

Renal damage was comparable among all the mice analyzed, excluding a differential effect of the diet in the various groups.

Tfr2^BMKO mice showed enhanced erythropoiesis relative to controls, due to the increased EPO responsiveness of erythroid cells lacking Tfr2, as suggested by the over-activation of the EPO-EPOR signaling pathway despite comparable EPO levels. Tfr2^BMKO mice maintained higher red blood cell (RBC) count than controls for the entire protocol. Hemoglobin (Hb) levels, higher in Tfr2^BMKO mice for 6 weeks, reached levels of controls at 8 weeks, concomitant with relative hypoferremia. These results indicate that BM Tfr2 deletion transiently prevents anemia until iron availability is adequate to the enhanced erythropoiesis.

Then we investigated the potential beneficial effect of increasing iron availability through Tfr2-ASOs treatment, which efficiently decreased hepatic (95-97%) but not erythroid Tfr2. As expected, circulating iron levels were increased in Tfr2-ASO mice, maintaining RBC count and Hb levels in the normal range for the first 2 weeks of treatment. However, Hb and RBCs reverted to control levels at 6 weeks, before the end of the protocol. These results show that increased iron availability alone, due to hepatic Tfr2 downregulation, delays anemia development but is not sufficient to boost erythropoiesis on a long term.

In agreement, Tfr2^KO mice, with Tfr2 inactivation both in the liver and in the erythroid compartment, maintained higher RBC count and Hb levels compared to controls until the end of the protocol.

Conclusions

The concomitant targeting of hepatic and erythroid Tfr2, here obtained through Tfr2 germline genetic inactivation, is necessary and sufficient to ameliorate anemia of CKD. On the contrary selective BM Tfr2 deletion that enhances EPO responsiveness of erythroid cells, or hepatic Tfr2 downregulation that increases iron availability, do not correct anemia in a long term. Therefore, a specific approach able to inhibit both hepatic and erythroid Tfr2 could adjust iron availability according to the enhanced erythropoiesis, correcting both drivers of anemia development in CKD. The development of a pharmacologic tool to downregulate Tfr2 might become an alternative to the standard treatment with ESAs plus iron supplementation with limited off-target effects due to the restricted TFR2 expression.