Cyclosporine A (CsA) and tacrolimus, both acting through calcineurin inhibition, have become the mainstays for the prevention of acute organ rejection. Their use, however, is associated with increased blood pressure, reduced glomerular filtration, and chronic nephrotoxicity, which may lead to chronic allograft nephropathy and graft loss (1). Given the negative side effects associated with calcineurin inhibitors (CNIs), finding alternatives to their use has remained a goal in transplantation research.
Sirolimus (SRL) is a macrolide with a novel mechanism of imunosuppression. It blocks the IL2 postreceptor signals mediating T-cell proliferation by binding to the mammalian target of rapamycin (mTOR), thus arresting cell cycle progression from the G1 to the S phase (2). In animal models, SRL has been shown to be a potent immunosuppressant with antitumor properties (2). It has not been associated with nephrotoxicity in phase I or II studies (3). It could be of interest in the prevention of chronic allograft nephropathy because experimental studies in the nonhuman primates have shown that SRL inhibits the arterial intimal thickening that follows alloimmune injury (4).
Most of the secondary effects of SRL on bone marrow cells production are thought to be on the nonerythroid progenitors such as megakaryocytes and granulocytes (5,6). In contrast, the responsibility of SRL in the anemia frequently observed in renal transplanted patients has never been demonstrated, because it is commonly used in combination with myelotoxic drugs such as mycophenolate mofetil (MMF) or azathioprine (Aza).
We previously reported an unusually high rate of anemia in patients undergoing late introduction of sirolimus for chronic allograft nephropathy (7).
In the present work, we undertook a retrospective longitudinal monocentric cohort study, to determine if the SRL was responsible for the occurrence of anemia in these patients. We show that anemia is common in patients who were switched from a CNI to SRL late after renal transplantation. Unexpectedly, this sirolimus-associated anemia exhibits features of the “anemia of chronic inflammatory states” (ACIS). Occurrence of inflammatory state in patients on SRL therapy might have resulted from the impairment of interleukin 10 (IL10)-dependent inflammatory autoregulation.
PATIENTS AND METHODS
Study Population
We retrospectively reviewed the medical records of the 48 patients who had been switched from CNI to SRL at the Necker Hospital in Paris (France) between 1997 and 2001.
Indications for the switch were biopsy-proven chronicallograft nephropathy with CNI nephrotoxicity in all patients. Because sirolimus was given as based therapy, we set the target blood trough level between 12 to 20 ng/ml.
Two patients were lost to follow-up. Forty-six patients (24 women and 22 men), with mean age of 45±12 years, switched from CNI to SRL after a mean graft duration of 55±49 months and were considered for the first part of the study. To determine whether SRL introduction was followed by the occurrence of anemia, hemoglobin levels measured before the switch and on SRL therapy were compared.
Considering that before the switch 14 out of the 46 patients had a hemoglobin level less than 12 g/dl, we decided to focus our study on the variations in the level of hemoglobin rather than on its absolute value. The last hemoglobin measure before the switch was taken as reference. Assuming that the characteristics of SRL-induced anemia would be better determined if we focused on the most typical patients, we arbitrarily fixed the variation cutoff value at 2 g/dl. Thus, anemia’s biological features for the 24 patients, whose hemoglobin dropped by at least 2 g/dl after SRL introduction, were collected when their hemoglobin was at its lowest level. The attribution of causality to sirolimus for the anemia was relying on the standard World Health Organization causality criteria (8).
In the last part of the study, we aimed at unraveling the pathophysiology of SRL-induced anemia. We screened the above 24 patients for confounding factors (ongoing infection or inflammatory events; treatment with angiotensin-converting enzyme inhibitors, angiotensin II subtype 1 receptor antagonists, mycophenolate mofetil, azathioprine, or erythropoietin). We identified eight patients whose anemia had no other detectable explanation than SRL introduction, and which disappeared after drug withdrawal. For six of these eight patients, serum samples collected before the switch, on SRL therapy and after SRL withdrawal, were available for analysis. Samples collected before SRL introduction and after SRL withdrawal were used as controls.
Measurement of Soluble Transferrin Receptor, Inflammatory, and Anti-inflammatory Markers
Blood samples for serum analysis are routinely collected every 3 months for all the patients receiving a renal graft during their follow-up in Necker Hospital.
Blood tubes were allowed to clot for 1 hour at room temperature, centrifuged at 1500 g for 10 min at 20°C, and serum was immediately aliquoted and stored at –80°C until analysis. Quantitative measurement of inflammatory mark—ers IL6, TNFα, anti-inflammatory marker IL10, and soluble transferrin receptor (sTfR) in patients’ serum samples were performed using commercial immunoenzymatic assay kits (for IL6, TNFα and IL10, BioSource, Belgium and sTfR, Deade Behring, Germany). ELISAs were performed at the same time for all the time points. IL6, TNFα, and IL10 were measured before the introduction of SRL, at the nadir of anemia, and 3 months after its withdrawal in 6/8 patients whose anemia was solely due to sirolimus therapy. sTfR was measured before the introduction of sirolimus and at the nadir of anemia for 24 patients, whose Hb level decreased by at least 2 g/dl after the switch from CNI to SRL.
Statistical Analyses
For each patient, the biological parameter measurements obtained before and after the switch, and after SRL withdrawal were compared using the paired t test.
Variations in the serum levels of the inflammatory markers IL6 and TNFα at introduction and withdrawal of SRL were represented as a function of the variation of hemoglobin. The values for hemoglobin and the two inflammatory markers before SRL introduction were taken as reference. The linear regression model was applied to the search for a correlation between these parameters.
For all statistical tests, an alpha probability value of 0.05 or less was considered significant. Values in the manuscript following the ± symbol are standard deviation for the preceding variable. All statistical analyses were performed using SAS software for Macintosh, release 5.0.1 (The SAS Corporation, Inc., Cary, NC).
RESULTS
Decreases in Hemoglobin after Sirolimus Introduction
After the introduction of SRL, the hemoglobin level decreased in 40 of the 46 (86.9%) patients and remained stable in the other six.
The mean decrease in the hemoglobin for the 46 patients after the switch to SRL was 2.5±2.0 g/dl (P<0.001, Fig. 1).
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FIGURE 1.:
Mean hemoglobin level of the 46 renal transplant patients. The mean hemoglobin level of the whole cohort decreased significantly during sirolimus therapy compared to the level measured before its introduction.
Anemia Complicating Late Introduction of Sirolimus Exhibits the Same Characteristics As Anemia of Chronic Inflammatory States
For the 24 patients whose Hb fell by ≥ 2 g/dl, we examined the characteristics of anemia on SRL therapy when hemoglobin levels were the lowest. Biological features of anemia are given in Table 1 (Group A).
TABLE 1: Characteristics of anemia on SRL therapy
Following SRL introduction, anemia reached its nadir after a mean period of 13.7±4.5 weeks and exhibited all the features associated with anemia of chronic inflammatory states, including regenerative microcytic anemia with low serum iron and high serum ferritin levels. Consistent with the diagnosis of anemia of inflammatory states, the difference between sTfR levels before (1.91±2.34 mg/l) and after SRL introduction (2.50±2.01 mg/l) was not significant (P=0.21; Fig. 2A)
FIGURE 2.:
Twenty-four renal transplant patients whose hemoglobin dropped by ≥ 2 g/dl after sirolimus introduction. (A) Serum levels of soluble transferrin receptor (sTfR). (B) Fibrinogen. (C) C-reactive protein (CRP). All were measured before sirolimus (SRL) introduction, at the nadir of anemia, and 3 months after SRL withdrawal. Serum transferrin receptor levels before SRL introduction and at the nadir of anemia were not significantly different.
Anemia Complicating Late Introduction of Sirolimus Is Associated with Systemic Inflammatory Syndrome
Anemia associated with late introduction of SRL displayed the same biological profile as anemia of chronic inflammatory states. To determine whether this was the consequence of a direct effect of sirolimus on erythroblast or if it was due to the development of a systemic inflammation, we compared the serum levels of proteins associated with the acute (CRP) and the late (fibrinogen) phase of inflammation.
We showed that after SRL introduction, the levels of both CRP (17±8.5; P=0.048) and fibrinogen (6.0±1.6; P<0.001) increased significantly as compared with their baseline levels measured just before the switch (respectively 5.8±1.9 and 3.8±0.6), but returned to their initial levels after SRL withdrawal (respectively 6.0±0.9, P=0.685 and 3.2±0.3, P=0.705; Fig. 2B and C)
These data suggested that late introduction of SRL was able to induce systemic inflammatory syndrome in some patients. This hypothesis was further sustained by the finding that five patients among the 24 exhibited, in addition to anemia, sirolimus-associated interstitial pneumonitis (9). In all cases, the decrease of hemoglobin started several weeks before the first pulmonary symptoms. Pulmonary infections were ruled out by specific stainings and cultures of BAL, bronchial aspirates, and blood cultures. Discontinuation of sirolimus led to complete resolution within 3 months.
Assessment of Sirolimus Responsibility in the Occurrence of Postswitch Anemia
Because this study was retrospective, the assessment of the responsibility of sirolimus in the occurrence of postswitch anemia has to deal with numerous confounding factors.
We carefully reviewed the medical record of each of the above 24 patients for the factors which may have played a role in the occurrence of anemia (Table 2).
TABLE 2: Individual characteristics for the 24 patients, whose Hb fell by ≥2 g/dl after a late post-transplant switch from calcineurin inhibitors to sirolimus
Renal function deterioration did not explain anemia, because creatininemia was not significantly different before the switch and at the nadir of anemia (mean=171±69 μmol/L VS. 172±87 μmol/L, P=0.92). However, most of the patients (16/24) exhibited at least one confounding factor: ongoing infection (1/24); treatment with angiotensin II subtype 1 receptor antagonists (AIIRA; 7/24), mycophenolate mofetil (MMF; 10/24), azathioprine (AZA; 3/24), or erythropoietin (EPO; 5/24).
To determine whether the anemia could be attributed to sirolimus introduction, we relied on WHO causality criteria (8). We identified eight patients (Table 2, Patients 1–8) whose anemia occurred in a plausible time relation to SRL administration, cannot be explained by concurrent disease or other drugs, and disappeared after SRL withdrawal.
For these eight patients, the responsibility of SRL in the occurrence of anemia was certain. The characteristics of their anemia did not differ significantly from those observed in the other 18 (Table 1, Group B).
Additionally, we found that SRL causality for anemia was probable for 11 other patients (Table 2, Patients 9–19) and possible for the remaining five patients (Table 2, Patients 20–24).
For 4/24 patients (Table 2, Patients 3, 4, 14, and 15) a rechallenge procedure had been performed and was positive in all cases.
Occurrence of Anemia and Pro-inflammatory Marker Elevation on Sirolimus Therapy Are Synchronized
In the last part of the work, we tried to unravel the mechanism by which late introduction of sirolimus led to the development of a systemic inflammatory syndrome resulting in anemia. We focused our study on six of the above eight patients whose anemia was attributable to sirolimus because serum samples collected before the switch, at the nadir of anemia and three months after SRL withdrawal, were available for analysis. The levels of inflammatory markers IL6 (42.82±16.54 vs, 5.77±3.19 ng/ml; P=0.048) and TNFα (160.33±62.32 vs. 52.61±18.31 ng/ml; P=0.029) were increased at the nadir of anemia, but returned to their initial levels after SRL withdrawal. (Fig. 3A and B)Furthermore, the variations in IL6 and TNFα levels were compared with those of hemoglobin level using the linear regression model and were significantly correlated, despite the low number of patients available for analysis. (Fig. 4)
FIGURE 3.:
Six renal transplant patients with sirolimus-induced anemia. (A) Tumor necrosis factor α (TNFα). (B) Interleukin (IL6). (C) Interleukin 10 (IL10). (D) Hemoglobin levels. All were measured before sirolimus (SRL) introduction, at the nadir of anemia, and 3 months after SRL withdrawal. TNFα and IL6 levels were significantly increased at the nadir of anemia, but returned to their initial levels after SRL withdrawal. In contrast, the IL10 serum level remained unchanged.
FIGURE 4.:
Variations in tumor necrosis factor α (TNFα) and interleukin 6 (IL6) levels compared to the variations of hemoglobin in six renal transplant patients with sirolimus-induced anemia using the linear regression model. Variations of both TNFα and IL6 levels correlated significantly with those of hemoglobin.
Evidence for Impairment of IL10-Dependent Inflammatory Autoregulation
The anti-inflammatory marker IL10 was also measured in the serum simultaneously with the inflammatory markers in the same six patients. Despite the development of systemic inflammatory syndrome as assessed by the rise in mean fibrinogen and CRP in response to increased levels of IL6 and TNFα, the IL10 serum level remained unchanged (3.5±1.98 VS 1.17±1.17 ng/ml; P=0.309; Figure 3C). This suggests the impairment of IL10-dependent inflammatory autoregulation in patients under SRL therapy, which might be responsible for the development of systemic inflammation leading to anemia.
DISCUSSION
In this study, we showed that late introduction of sirolimus in renal transplant recipients is responsible for the occurrence of anemia in some patients, that sirolimus-associated anemia exhibits the characteristics of anemia of chronic inflammatory states, and we hypothesize that their pathogenesis may be due to the development of a systemic inflammatory syndrome because of an interference of sirolimus with IL10-dependent inflammatory autoregulation.
Because the basal level of hemoglobin in our cohort was low (11.06±2.19 g/dl), we decided to focus on the decrease of hemoglobin rather than on the absolute hemoglobin level, which is usually use to define anemia (10). Relying on the standard WHO causality criteria (8), the responsibility of SRL in the occurrence of anemia was certain for eight patients among the 24, whose hemoglobin level decreased by at least 2 g/dl after SRL introduction. About 14 weeks after SRL introduction, all eight patients indeed experienced a dramatic decrease in their hemoglobin level, which normalizes after drug withdrawal. These eight patients were carefully selected for the absence of other confounding factors, which could have helped to generate anemia. In particular, anemia could not be explained by concurrent disease and none of the patients was given MMF, AZA, ACEI, AIIRA, or EPO. Additionally, we found that SRL causality for anemia was probable for 11 other patients, and possible for the remaining five patients. Furthermore, four of the 19 patients with certain or probable SRL-related anemia had a positive rechallenge test, usually considered as the ultimate proof for the imputation of a side effect to a drug. In contrast with what has been reported for the other hematologic adverse effects of SRL (6), we found no correlation between SRL trough concentration and the decrease of hemoglobin in our 46 patients.
Although this retrospective study does not allow us to draw definitive conclusions concerning the pathogenic mechanisms leading to the development of anemia following late SRL introduction, we believe that it provides interesting clues towards this goal. Indeed, although we anticipated that SRL might induce central aregenerative anemia by inhibiting the cytokine-driven proliferation of erythroid cells (11), and although previous studies documented a bone marrow toxicity of sirolimus responsible for leukopenia and thrombocytopenia (6), we found that sirolimus-induced anemia displays features rather suggestive of an inflammatory origin. Indeed, the anemia induced by sirolimus therapy was microcytic and aregenerative, whereas serum iron levels were low despite the adequate iron stores reflected by the high serum ferritin level.
To further document the absence of direct bone marrow toxicity of sirolimus in the pathogenesis of anemia, we analyzed the variations of soluble transferrin receptor (sTfR) level, which has been recently introduced as a reliable marker for the evaluation of erythropoiesis (12). Indeed, because most of the bulk of sTfR measured in serum originates from erythroblasts, sTFR levels decrease when erythropoietic activity is reduced. In contrast, sTfR levels at the nadir of anemia remained unchanged compared to those measured before SRL introduction (Fig. 2A). Interestingly, the occurrence of anemia of inflammatory states have already been suspected in two previous studies, in which the possible benefit of late SRL introduction for the prevention of chronic rejection after lung (13) and liver (14) transplantation was explored.
In an attempt to further document the development of a systemic inflammatory state in the patients experiencing late introduction of SRL, we compared the serum levels of CRP and fibrinogen before the switch, on SRL therapy, and after SRL withdrawal. We showed that SRL introduction is associated with a significant increase of the levels of these proteins, which were back to their baseline levels after withdrawal of the drug (Fig. 2B and C). These findings suggest that late sirolimus introduction is able to interfere with the control of inflammation. Interestingly we found that a significant number of patients with sirolimus-associated anemia exhibited also another sirolimus-related inflammatory manifestation: interstitial pneumonitis (5/24, 21%). For these patients, the presence of the pneumonitis raised the question of its role in the occurrence of anemia. A careful review of the medical records showed that the decrease of hemoglobin started in all cases several weeks before the first pulmonary symptoms. Thus, although pneumonitis could have worsened anemia, it could not explain by itself the decrease of hemoglobin and was not considered as concurrent explanation for the occurrence of anemia after late introduction of sirolimus. Instead, we hypothesized that these two sirolimus-associated inflammatory adverse effects could share the same pathogenesis.
Trying to understand how late introduction of sirolimus could unbalance the system of control of inflammation, we focused our study on six patients whose anemia was attributable to sirolimus. Regarding inflammatory status as a balance, the development of an inflammatory syndrome after late sirolimus introduction could result either from an increase production of pro-inflammatory mediators, or from a decrease production of anti-inflammatory mediators.
Among the various pro-inflammatory cytokines, we chose to focus on TNFα and IL6 because both have been involved in the pathogenesis of anemia of chronic inflammatory states. Indeed, TNFα has been shown to stimulate ferritin synthesis, iron acquisition, and iron storage by the reticuloendothelial system, which limits iron availability to the erythron (15). Others studies suggest that TNFα might also inhibit bone marrow erythropoiesis by exerting a direct negative effect on erythroid progenitor cell growth (16) and accelerating the apoptosis of bone marrow erythroid cells (17). More direct evidence for the role of TNFα in the pathogenesis of ACIS recently became available from clinical trials using in vivo TNFα blockade, a treatment which significantly improved ACIS (18). IL6 has also been reported to have a role in ACIS pathophysiology by stimulating the hepcidin production by the liver (19). In turn, hepcidin, an iron regulatory hormone, inhibits the intestinal iron absorption and the release of recycled iron from macrophages (20, 21), decreasing iron delivery to maturing erythrocytes in the bone marrow. Strikingly, serum levels of both TNFα and IL6 were found to rise in our patients on SRL therapy. Furthermore, the decrease in hemoglobin correlated with the rise in these proinflammatory cytokines, suggesting that these two events were linked. However, because our study is retrospective, we were unable to establish the direct responsibility of this rise in the level of inflammatory mediators in the occurrence of SRL-induced anemia. To explore the other arm of the balance we compared the serum levels of IL10 at the same points for the above six patients. Indeed, under physiological conditions, the rise in pro-inflammatory mediators induces, and is then balanced by the production of anti-inflammatory molecules in a counterregulatory fashion. These molecules limit and suppress the inflammatory process throughout the body (22, 23). In this compensatory process, IL10 has been shown to be crucial because of its capacity to inhibit the production of proinflammatory cytokines by monocytes (24). In agreement with these findings, IL10−/− mice exhibited a higher rate of mortality and more severe tissue injury than wild type mice subjected to intestinal ischemia and reperfusion (25). One of the interesting findings of our study was the contrast between the rise of the levels of inflammatory mediators and the dramatic absence of any increase in IL10. This feature suggests a failure in the autoregulatory mechanism that controls inflammation. This hypothesis is in accordance with previous studies, which have shown that SRL inhibits monocyte IL10 production via the p70 S6-kinase pathway (26, 27).
CONCLUSION
Our data show that late introduction of sirolimus after renal transplantation may induce anemia. The occurrence of this newly identified side effect correlates with biochemical evidence of a chronic inflammatory state. Although this study does not demonstrate a causative link between these two findings, it suggests that sirolimus-induced anemia may, however, be due to defective IL10-dependent inflammatory autoregulation.
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