Anemia after solid organ transplantation is a relatively common phenomenon that has been mostly overlooked in the last decade (1). Nearly all transplant recipients develop anemia during the early posttransplant period and experience resolution of the anemia by three to six months posttransplant (1). In the only longitudinal study of posttransplant anemia in adult renal transplant recipients (1), the incidence of anemia was reported to be 12% at one year after transplantation, but increased to 26% at five years posttransplantation. The etiopathogenesis of posttransplant anemia is multifactorial. The most common cause is iron deficiency (35%), followed by defective erythropoietin (EPO) production (17%), iron deficiency superimposed to defective EPO production (14%), infection (12%), immunosuppressive drug toxicity (9%), and allograft dysfunction (5%) (2–3). Drug-induced bone marrow suppression is commonly noted after solid organ transplantation particularly in patients treated with antiproliferative agents (3).
Sirolimus, an immunosuppressive drug recently introduced in the antirejection therapy of renal transplant recipients, is a macrolide that inhibits cytokine-stimulated T-cell proliferation (4). Sirolimus initially binds to FK-binding protein-12 (FKBP-12). This complex in turn binds to a specific cell cycle regulatory protein, the mammalian target of rapamycin (mTOR), inhibiting its activation (4). This serine-threonine kinase controls several growth-related readouts including translation initiation, organization of actin cytoskeleton and membrane protein trafficking (5). Thus, mTOR inhibition by sirolimus prevents cell cycle progression from G1 to S phase in T-cells significantly reducing antigen-driven T cell clonal expansion (6). The anti-proliferative effect of sirolimus is evident also on bone marrow cells, as shown by the relatively common occurrence of thrombocytopenia and/or leukopenia (7). The anti-proliferative effect was suggested to underlie sirolimus-induced anemia, observed in 25 to 37% of patients receiving this drug (6). Recently, Thaunat et al. suggested that sirolimus-induced anemia might be due to the development of an inflammatory milieu (8). Indeed, they retrospectively analyzed forty-six renal transplant recipients, who had been converted from calcineurin inhibitors to sirolimus and they observed a microcytic anemia with low serum iron, despite high ferritinemia, consistent with anemia of chronic inflammatory states, associated with increased C reactive protein levels (8).
A hallmark of inflammation-related anemia is the development of disturbances of iron homeostasis, with increased uptake and retention of iron within cells of the reticuloendothelial system (9). This leads to a diversion of iron from the circulation into storage sites, subsequent limitation of the availability of iron for erythroid progenitor cells and iron-restricted erythropoiesis (9). Human hepcidin, a 25-amino acid peptide produced by hepatocytes, is a new mediator of innate immunity and the long-sought iron-regulatory hormone (10). Evidence from transgenic mouse models indicates that hepcidin is the predominant negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from macrophages (10). The synthesis of hepcidin is greatly stimulated by inflammation or by iron overload. Recent evidence shows that in anemia of inflammation, hepcidin production is increased up to 100-fold and this may account for the defining feature of this condition, sequestration of iron in macrophages (10). Thus the aim of the present study was to investigate whether late sirolimus introduction in the therapy of renal transplant recipients may influence iron homeostasis and hepcidin serum levels.
PATIENTS AND METHODS
This is a prospective, open-label, single-center study. Forty-two renal transplant recipients (35 male, 11 female) with biopsy-proven chronic allograft nephropathy (CAN), having given their informed consent, were included in the study. The median time between transplantation and histological CAN diagnosis was 18 months (range 12–75 months). All patients received corticosteroids (methylprednisolone 500 mg intraoperatively and then prednisone 250 mg daily, tapered to 25 mg by day 8), a chimeric anti-CD25 monoclonal antibody (basiliximab, 2 doses of 20 mg intervenously [iv] at day 0 and 4). As maintenance immunosuppression patients received cyclosporine (aiming at C2 blood levels of 600–800 ng/ml), prednisone 5 mg/day and mycophenolate mofetil (MMF; 1 g BID).
At diagnosis of CAN, patients were randomly assigned (in a 1:2 ratio) either to receive a 40% cyclosporine dose reduction (14 patients, group A) or to immediately withdraw cyclosporine and to receive 0.15 mg/kg of sirolimus (28 patients, group B) as a single loading dose followed by 0.04–0.06 mg/kg/day, aiming at blood through levels of 6–10 ng/ml. The main clinical features of the study population are summarized in Table 1. The study was carried out according to the Declaration of Helsinki and was approved by our institutional review board.
Hemoglobin (Hb) concentration, red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum iron, transferrin and ferritin levels as well as transferrin saturation (TSAT) were evaluated three times in the 6 months before and three times between three and nine months after the switch. All the results are the mean of three determinations per patient before and after the switch. In addition, hepcidin serum levels were measured in all patients included in the study six months after randomization by enzyme-linked immunosorbent assay (ELISA; DRG, Marburg, Germany).
Anemia was defined according to the World Health Organization (WHO) criteria as Hb less than 12 g/dl in women and 13 g/dl in men. Finally, oral iron supplementation was given when TSAT was less than 20%, according to the European best practice guidelines (11, 12).
Data are presented as means±standard deviation. The comparisons between groups were performed using the unpaired t test (two-sided). For each patient, the biological parameter measurements obtained before and after randomization were compared using the paired t test. P<0.05 was considered statistically significant. The Statview software package, SAS Inc. Co (5.0 version) was used for all analyses.
None of the patients included in the study presented clinically significant anemia before randomization and none of them were on EPO and iron therapy. No difference in Hb concentration or RBC count was observed between group A and B patients before randomization (Fig. 1, A and B). Group A patients did not present any change in Hb concentration and RBC count following randomization (Fig. 1, A and B). On the contrary, a statistically significant reduction in Hb levels was observed in the patients switched to sirolimus (Fig. 1A), without any change in RBC count (Fig. 1B), suggesting that the anti-proliferative effect of the drug does not play a role in Hb reduction. To further confirm this hypothesis, we evaluated circulating reticulocyte number, without observing any significant change after conversion to sirolimus (data not shown). The reduction observed did not correlate either with sirolimus dose or sirolimus through levels (data not shown).
As a consequence of reduced Hb with no change in RBC count, we observed a significant reduction of MCV (Fig. 2A), MCH (Fig. 2B), but not of MCHC (Fig. 2C) after the introduction of sirolimus. On the other hand, the patients continuing on cyclosporine therapy did not present any change in these parameters (Fig. 2, A–C).
Interestingly, iron serum levels were markedly reduced after randomization in the patients assigned to sirolimus conversion (Fig. 3A) without any change in serum transferrin concentrations (data not shown) and with a consensual reduction of TSAT (Fig. 3B). Ferritin serum levels remained stable after sirolimus introduction (Fig. 3C). In the group of patients continuing on cyclosporine we did not observe any significant change in iron, TSAT and ferritin levels (Fig. 3, A–C).
To evaluate whether sirolimus may influence hepcidin synthesis or release from the liver, we measured hepcidin circulating levels in both groups of patients 6 months after randomization. Interestingly, hepcidin serum concentration was lower in sirolimus-treated than in cyclosporine-treated patients (cyclosporine 139.5±61.4 ng/ml; sirolimus 111.8±20.1 ng/ml; P=0.08).
We then analyzed the need of iron supplementation (TSAT<20%) after randomization. None of the patients in group A presented a TSAT<20% in the 6 months following randomization, whereas after sirolimus introduction 8 patients presented a TSAT<20% and were given iron supplementation (TSAT<20). In this small group of patients, oral iron therapy did not significantly influence either Hb (pre, 12.2 g/dl±1.7; post, 11.7 g/dl±1.6) or serum iron levels (pre, 48.1 mg/dl±13.4; post, 34.2 mg/dl±8.8).
Sirolimus is an immunosuppressive drug recently introduced in the antirejection therapy of renal transplantation. It exerts its immunosuppressive effect through the inhibition of cytokine-stimulated T-cell proliferation. Indeed, mTOR inhibition by sirolimus prevents cell cycle progression from G1 to S phase. The anti-proliferative effects of this drug were demonstrated on nonerythroid bone marrow progenitors including megakaryocytes and leukocytes (4). Recently, an inhibitory and dose-dependent effect of sirolimus was suggested also on erythrocyte production (6). In a phase III multicenter trial evaluating sirolimus/cyclosporine combination therapy in renal transplant recipients, anemia was reported in 16% of patients on 2 mg/day of sirolimus and 27% of patients on 5 mg/day. The incidence of anemia in the latter group was significantly higher than in the placebo group, maintained only on prednisone and cyclosporine (13). When higher doses of sirolimus (8–12 mg/m2/day) were used in another study by Groth et al., the incidence of anemia at 12 months after transplantation was further increased to 37% (14). Kreis et al. (15) reported a 43% incidence of anemia in patients on sirolimus/MMF combined therapy compared with 29% on cyclosporine/MMF therapy.
The mechanisms of sirolimus-induced anemia in renal allograft recipients remain still largely unclear. In the present study we demonstrated that sirolimus does not modify RBC counts while reducing Hb levels, suggesting that the antiproliferative effects of this immunosuppressive drug do not play a key role in sirolimus-induced anemia. On the other hand, we observed a significant influence of sirolimus on iron homeostasis. The reduction in iron levels together with the stable ferritin serum concentrations may suggest a condition of functional iron deficiency, partially resembling that observed in the inflammation-related anemia. Thus, the results of our prospective study may in part support the recent observation of Thaunat et al. (8). They suggested that sirolimus-induced anemia might be due to the development of an inflammatory milieu (8).
The pathogenesis of the inflammation-related anemia was recently been elucidated. The hallmark of this condition is the development of disturbances of iron homeostasis, with increased uptake and retention of iron within cells of the reticuloendothelial system (9). This leads to a diversion of iron from the circulation into storage sites, subsequent limitation of the iron availability for erythroid progenitor cells, and iron-restricted erythropoiesis (9). Several studies demonstrated that hepcidin, an acute-phase reacting protein, is a key regulator of iron balance in the intestinal mucosa and in the reticuloendothelial system (16). Evidence from transgenic mouse models indicates that hepcidin is the negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from macrophages (10). Investigation of hepcidin production demonstrated a strong correlation with serum ferritin concentration (17). Decreased hepcidin leads to tissue iron overload, while hepcidin overexpression leads to hypersideremia and anemia (10). In vitro there is clear evidence that hepcidin binds to ferroportin, expressed on macrophage and intestinal cells. Ferroportin is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes and placental cells (10). Following hepcidin binding, ferroportin is internalized and degraded leading to a decreased export of cellular iron (17). The posttranslational regulation of ferroportin by hepcidin may thus complete a homeostatic loop: iron regulates secretion of hepcidin, which in turn controls the concentration of ferroportin on the cell surface (17). Hepcidin synthesis and, subsequently, its serum level are dramatically increased by the inflammatory milieu. Thus, this small peptide was suggested to play a key role in the chronic inflammation-related anemia (18, 19). Surprisingly, in our patient population sirolimus induced a decrease in hepcidin serum concentrations, most likely related to the reduction in hemoglobin and serum iron levels. This observation strongly suggests that the changes induced by late sirolimus administration in renal transplant recipients is not due to an increase in proinflammatory and/or a reduction in anti-inflammatory cytokine levels as previously suggested (8).
This may lead to the alternative hypothesis that sirolimus may act downstream of hepcidin, influencing directly ferroportin expression or function, thus directly modulating the cellular iron metabolism. Akt, a serine-threonine kinase upstream of mTOR, is a key component of the growth factor signal transduction cascade regulating cell growth and survival (20–26). Akt is a well established check point in the regulation of cell metabolism and nutrient uptake and these functions are strictly dependent on mTOR activity. Indeed, mTOR has been shown to regulate the trafficking of nutrient transporters in mammalian cells expressing a constitutively active mutant of akt (27). In yeast, mTOR coordinates the cellular response to extracellular nutrient levels and allows yeast cells to respond adaptively to changes in the extracellular environment by modulating nutrient transporter expression (19, 20). Sirolimus treatment in yeast produces a response equivalent to starvation (28, 29). Although, no direct effect of the akt-mTOR pathway has been demonstrated on ferroportin synthesis, it is conceivable that this key signaling axis in the uptake of extracellular nutrients may also modulate the synthesis and/or the trafficking of the main iron transporter, potentially explaining the clinical effect of reduced iron serum levels induced by sirolimus. Two observations in our study may support this hypothesis. The reduced serum iron levels associated with stable ferritin concentrations clearly suggests a defective iron release from hepatic and reticuloendothelial storage. In addition, the lack of response to oral iron supplementation in sirolimus-treated patients indicates a modification in enteric iron adsorption. At both levels, iron storage and intestinal absorption, iron uptake and release are ferroportin-dependent (30).
In conclusion, the present study demonstrates a novel effect of sirolimus on iron homeostasis suggesting an alternative pathogenic mechanism for one of the more frequent hematological complications observed in patients treated with this immunosuppressive drug.
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