Post-transplant anemia and associated risk factors: the impact of steroid-free therapy

CONTEXT AND OBJECTIVE: The prevalence of post-renal transplant anemia (PTA) reported in the literature is variable and several factors contribute towards its pathophysiology. This study aimed to investigate the prevalence of PTA, its associated risk factors and the impact of therapy without steroids. DESIGN AND SETTING: Retrospective cohort study in a renal transplantation unit at a tertiary hospital. METHODS: Anemia was defined as hemoglobin (Hb) < 12 g/dl in female adult recipients and < 13 g/dl in males. Donor and recipient age and gender, type of donor, creatinine, delayed graft function, acute rejection, use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) and therapy without steroids were investigated as risk factors for PTA through multivariate logistical regression analysis. RESULTS: Evaluations were performed on 258 recipients (mean age: 38.8 years; 60.5% males; 35.7% did not receive steroids). Anemia was diagnosed in 38% of the patients (at the sixth month, M6), 28% (M12), 32% (M24) and 45% (at last follow up). Donor age > 50 years was associated with greater risks of PTA at M6 (odds ratio (OR) = 4.68) and M24 (OR = 6.57), as well as with therapy without steroids at M6 (OR = 2.96). Delayed graft function was independently associated with PTA at M6 (OR = 3.66) and M12 (OR = 2.85). CONCLUSION: The lowest prevalence of PTA was observed between M9 and M24 after renal transplantation. Delayed graft function, donor age and therapy without steroids were the most important factors associated with PTA.


INTRODUCTION
The prevalence of post-renal transplant anemia reported in the literature is variable (13-70%) 1,2 and its management remains poorly explored. The variable prevalence of post-renal transplant anemia depends on the definition of anemia that is applied, the characteristics of the population studied and the length of time after transplantation.
Post-renal transplant anemia may affect quality of life and cardiovascular morbidity/mortality. It contributes towards left ventricular hypertrophy and heart failure, thus increasing the risk of cardiovascular events, which are considered to be the main causes of death after transplantation. 3,4 Several factors such as graft function, use of azathioprine, mycophenolate mofetil or angiotensin inhibitors, the donor's age, viral infections (cytomegalovirus and parvovirus B19), inflammation, hemolytic anemia, hemolytic-uremic syndrome and acute rejection 1,5,6 contribute towards the pathophysiology of post-renal transplant anemia.

OBJECTIVE
This study was designed to evaluate the prevalence of anemia at different times after transplantation; to investigate the associated risk factors; and to evaluate the association between anemia and therapy with or without steroids.

METHODS
We conducted a retrospective cohort study on patients who underwent kidney transplantation in a single hospital between January 2001 and May 2006. The study included all patients who received a transplant during the period of the study and who were older than 18 years, with at least two years of post-transplant follow-up. This study was approved by the Research Ethics Committee of Hospital Geral de Fortaleza, and data were obtained using a post-transplant follow-up form. Pregnant patients were excluded. Data were obtained from the patient's medical files. Over the period of the study, 378 transplantations were performed, and 120 recipients were not included because they did not meet the inclusion criteria.
Donor and recipient age and gender, type of donor, creatinine, delayed graft function (DGF), acute rejection and therapy without steroids were investigated as risk factors for post-renal transplant anemia at 6, 12 and 24 months after transplantation.
Data were expressed as the mean ± standard deviation (SD).

The correlation between Hb and creatinine was assessed through
Pearson's correlation coefficient. P values < 0.05 were considered statistically significant. We first determined the parameters that were associated with post-renal transplant anemia at 6, 12 and 24 months using univariate analysis between the groups by means of a twotailed independent-sample Student's t test or Fisher's test with significance taken at P = 0.10. The variables that reached a P value < 0.10 in the univariate analysis were included in a multivariate, stepwise logistic regression analysis to investigate the risk factors associated with post-renal transplant anemia at 6, 12 and 24 months after transplantation in the study population. All odds ratios (ORs) were reported with a 95% confidence interval. The Statistical Package for Social Sciences (SPSS) 14.0 software was used for statistical analysis.

RESULTS
The population studied comprised 258 renal transplant recipients with a mean age of 38.8 ± 11.4 years, and 60.5% of the sample were male. The primary renal disease was unknown in 38% of the cases; glomerulonephritis was diagnosed in 25% of cases; and hypertension was diagnosed in 19%. A total of 53.5% of the patients received a graft from a deceased donor. The initial immunosuppressive therapy consisted of mycophenolate mofetil in 92.2% of the patients, cyclosporine in 45%, tacrolimus in 53% and rapamycin in 2.3%. Prednisone was not used in maintaining the immunosuppressive therapy in 35.7% of the patients. Induction therapy with basiliximab was administered to 153 patients (59.3%), and thymoglobulin was administered to 11 patients (4.3%). Delayed graft function, defined as the need for dialysis in the first week post-transplant, and clinical or biopsyproven acute rejection were observed in 38% and 14.7% of the recipients, respectively. The demographic characteristics of the population studied and the mean hemoglobin and creatinine values over the course of the follow-up are presented in Table 1.
The average hemoglobin level at the time of transplantation was 11.8 g/dl, and 63.2% of patients were classified as having anemia at the time of the surgery. The mean hemoglobin increased from 11.7 g/dl in the first month after transplantation to 13.6 g/dl at 12 months.
Anemia was diagnosed in 83% of the patients at one month, 60.8% at two months, 55% at three months, 47.6% at four months, 43.8% at five months, 38.3% at six months, 32.9% at nine months, 28.3% at one year, 31.7% at two years and 44.9% at the last follow-up (mean time after transplantation: 5.4 ± 1.9 years) (Figure 1). The anemia was classified as moderate in 11.6%, 9.3% and 10.1% of the patients at 6, 12 and A significant negative correlation was found between serum creatinine and hemoglobin levels at all of the time points after transplantation ( Table 3).
The factors that were significantly associated with post-renal transplant anemia at six months after transplantation were donor age, DGF, creatinine at discharge, type of donor and therapy without steroids ( Table 4) Table 4).

DISCUSSION
After kidney transplantation, many factors that contribute towards anemia in chronic kidney disease (CKD) persist, but causes specific to transplantation may predominate. During the  Post-renal transplant anemia may contribute towards damage to and subsequent loss of the graft and progression of CKD. 9,10 Tissue hypoxia stimulates synthesis of transcription factors that promote fibrosis and progression of chronic graft nephropathy. 11 Some authors have suggested that post-renal transplant anemia may contribute towards graft loss and evolution of chronic nephropathy, with a 38% increased risk of graft loss. 12 Moreover, correction of anemia using erythropoietin, which has anti-apoptotic effects on erythrocyte precursor cells in bone marrow, due to inhibition of the caspase pathways, 13  The prevalence of post-renal transplant anemia described in the literature has ranged from 13 to 70% irrespective of the time elapsed after transplantation. A number of studies reporting on varying numbers of patients have been conducted. 1,12,16,[18][19][20][21][22][23][24] This varying prevalence of PTA probably is observed because different criteria have been suggested for the definition of anemia, including Hb < 13 g/dl for male patients and < 12 g/dl for females; 16 Hb < 13.5 g/dl for males and < 12 g/dl for females (K/DOQI); 25 Hb < 12 g/dl for males and < 11 g/dl for females (European Best Practice Guidelines); 26 Hb < 12.5 for males and < 11.5 g/dl for females; 18 Hb < 11 after three months; 19 and hematocrit (Hct) < 33%. 8,16,21 The prevalence of post-renal transplant anemia in the present study was 38.3% at six months and 28.3% at 12 months, and these results are similar to those reported by Turkowski et al., who reported that 35.5% of patients had anemia at six months, and 25% at 12 months after transplantation. 27 However, Shibagakin et al. analyzed 192 patients and found that 41.1% had anemia at six months, and 44.8% at 12 months after transplantation. 15 The prevalence of post-renal transplant anemia follows a bimodal pattern with an initial peak at the time of transplantation, when erythropoiesis is minimal due to various factors, and another peak after the third year, which is associated with the progression of renal disease. Normal hemoglobin levels are expected as early as two months after transplantation in patients without iron deficiency and without graft dysfunction. 28 In our study, two months after transplantation, we observed high prevalence of anemia (60.8%), which was similar to what was detected at the time of transplantation (63.2%). We found a progressive decrease in the prevalence of anemia up to 12 months    Restoration of endogenous secretion of erythropoietin (EPO) and its impact on the outcome of anemia have been assessed in several studies. After an initial peak in EPO levels during the first days post-transplant, restoration of EPO synthesis depends on the recovery of renal function. 28

Creatinine
In this study, creatinine > 2 mg/dl was not predictive of postrenal transplant anemia at any of the time points studied.
EPO synthesis decreases and resistance to its effects increases when renal function deteriorates. 30 Therefore, an inverse correlation between function and hemoglobin is expected. Many studies have found renal function to be a predictor of post-renal transplant anemia, although this was not the case in the present study.
In a multicenter European study, 1  The degree of anemia is about 10 times higher for a given degree of renal dysfunction when recipients of a kidney transplant are compared with native kidney disease patients. 17 The choice of creatinine as a marker of renal function rather than creatinine clearance may have been a limitation of the present study. Creatinine is an insensitive and late marker of renal dysfunction and glomerular filtration rate must be preferred to estimate renal function, 33 which may have been responsible for the results.

Donor age
In the present study, donor age > 50 years was associated with presence of post-renal transplant anemia (OR = 4.68 at six months and OR = 6.57 at 24 months). In the European study, donor age > 60 years was a risk factor for post-renal transplant anemia (OR = 1.41). 1 Since donor age was an independent risk factor for post-renal transplant anemia, the association could not be explained by the lower degree of renal function associated with kidneys from older donors.

Delayed graft function
In the pre and immediate post-transplant period, ischemiareperfusion injury and calcineurin inhibitor nephrotoxicity may lead to tubular cell death and alloimmune fibrosis, with consequent DGF, acute cell rejection, tissue fibrosis, poor graft function and graft loss. 34 DGF has a negative impact on long term graft function, since it increases patient morbidity and mortality. 35 DGF is associated with increased biopsy-proven acute rejection as a consequence of increased immunogenicity of the ischemic graft. 34 Few studies have included DGF in the investigation of post-renal transplant anemia predictors.
Chhabra et al. showed that the prevalence of DGF was higher in anemic patients (Hb < 11 g/dl) than in those with Hb > 11 g/dl (32% versus 23%; P = 0.027). 19 The European study did not include DGF in the analysis of post-renal transplant anemia. In the present study, DGF was a predictor of post-renal transplant anemia at six and 12 months after transplantation, independently of acute rejection and renal function. The prevalence of DGF (38%) was similar to what was found in a recent European multicenter study, which evaluated whether the use of high doses of EPO immediately after transplantation would have an impact on the prevalence of DGF. 36

Immunosuppression without steroids
Recently, studies have been conducted on the safety, efficacy and toxicity of kidney transplantation without the use of steroids, and the number of studies in this area has been increasing. [37][38][39][40] Steroid-free therapy for kidney transplant patients has been recognized to be beneficial because of the complications associated with long-term steroid use, such as weight gain, osteopenia, hypertension, hyperglycemia and increased risk of infection. 41 In clinical practice, it has been observed that leukocyte levels in the absence of steroid use are lower than in patients who use steroids. The prevalence of anemia with or without steroid use has been studied and deserves further evaluation. In the present study, steroid-free therapy was found to be a risk factor for post-renal transplant anemia six months after transplantation, thus suggesting that steroids affect erythropoiesis.
The role of glucocorticoids in erythropoiesis is unclear.
Steroids have been used to treat aplastic anemia and can improve the production of red blood cells in cases of congenitally abnormal erythropoiesis (Diamond-Blackfan syndrome). Golde et al.
reported that dexamethasone boosts the proliferation of red blood cell progenitors stimulated by erythropoietin. 42 The effect of dexamethasone may involve increased sensitivity of progenitor cells to erythropoietin in vitro.
It has been shown that steroids increase the growth of colonies in vitro in normal bone marrow, 43,44 and that treatment of non-anemic patients with prednisone appears to increase erythropoiesis. 45

Angiotensin-converting enzyme inhibitor (ACEI) and angiotensin receptor blockers (ARB)
Other drugs that have been associated with post-renal transplant anemia include inhibitors of the renin-angiotensin-aldosterone system (RAAS). RAAS is an important regulator of erythropoiesis, and its inhibition has been shown both to be associated 49

Other variables studied
In our study, the prevalence of acute rejection was low, which may have contributed towards the lack of association with the presence of anemia. The European multicenter study did not find acute rejection to be a risk factor for post-renal transplant anemia, although patients who experienced more episodes of treated acute rejection had lower mean Hb levels. 1 The increased inflammatory response that accompanies rejection seems to downregulate genes that are involved in erythropoiesis, 51 and it has been shown that occurrence of acute rejection in the first month after transplantation interrupts the erythropoietic response. 52 Acute rejection is associated with lower Hb levels, but it is not an independent predictor of post-renal transplant anemia, since it is generally associated with worse renal function, use of higher doses of myelotoxic immunosuppressants to control new episodes of rejection and use of drugs to inhibit RAAS.
The immunosuppressants azathioprine and mycophenolate mofetil have been used in kidney transplant patients to prevent acute rejection. These drugs inhibit lymphocyte proliferation.
They have myelotoxic side effects and are associated with the risks of developing anemia, leukopenia and thrombocytopenia.
Furthermore, they have been clearly associated with the development of post-renal transplant anemia. 1 It was not possible to evaluate this variable in the present study because 92.2% of the patients were taking mycophenolate mofetil.

CONCLUSIONS
Post-renal transplant anemia had a high prevalence in our study, and the lowest prevalence of anemia was observed between nine months and two years after transplantation.
The factors associated with post-renal transplant anemia were donor age > 50 years, therapy without steroids and DGF, in this population. Use of steroid-free therapy has been increasing over the last few years and the consequences of this therapy need to be better understood.
In the literature, there are few studies investigating steroids as predictors of post-renal transplant anemia and the results are divergent. Additional studies investigating the role of steroids in post-renal transplant anemia need to be conducted. To the best of our knowledge, the present study is the first to identify steroidfree therapy as a predictor of post-renal transplant anemia in kidney transplantation cases.