Reninangiotensin blockade reduces serum free testosterone in middle-aged men on haemodialysis and correlates with erythropoietin resistance
Michael DeLong,
Joy L. Logan,
Kim-Chong Yong and
Yeong-Hau H. Lien
Renal Section, Department of Medicine, University of Arizona, Tucson, AZ, USA
Correspondence and offprint requests to: Joy L. Logan, Department of Medicine, Renal Section, PO Box 245022, Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724, USA. E-mail: jllogan{at}email.arizona.edu
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Abstract
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Background. There are conflicting reports about the importance of the reninangiotensin system (RAS) on erythropoietin (Epo) sensitivity in haemodialysis patients, but the role of gender has not been studied specifically. The hypothesis underlying this study is that Epo resistance associated with RAS blockade (RASB) is specific to men and related to drug-induced lowering of circulating testosterone.
Methods. Men and women undergoing chronic haemodialysis were divided into groups according to whether or not they were receiving RASB. Serum was collected pre-dialysis for determination of free testosterone levels by enzyme immunoassay. Routine laboratory data and Epo doses were collated and analysed for the 3 month period prior to the measurement of the hormone level.
Results. Control women required more Epo than control men (P = 0.002), but the Epo doses between men and women with RASB were similar. Men with RASB required more Epo than control men (P = 0.002), but RASB had no effect on Epo requirements in women. There was a significant relationship between age and testosterone levels in control men (P = 0.01) that was not present in men taking RASB. RASB was associated with lower levels of serum testosterone in men <60 years old (P = 0.02), but had no effect on serum testosterone levels in older men or women. Multiple regression analysis demonstrated that serum testosterone negatively correlated with Epo dose (P = 0.045) when all groups of patients were considered together.
Conclusions. These data suggest that androgens may participate in Epo resistance associated with RASB in patients on haemodialysis, and that the effect is related to both gender and age.
Keywords: anaemia; erythropoietin; haemodialysis; reninangiotensin system; testosterone
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Introduction
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Anaemia is a major cause of morbidity in patients with end-stage renal disease, and the identification of factors that cause resistance to erythropoietin (Epo) is important. Pharmacological blockade of the reninangiotensin system (RAS) may be one cause of Epo resistance, since angiotensin II regulates circulating Epo levels in normal individuals [1] and in patients on haemodialysis [2]. However, clinical studies of the impact of pharmacological RAS blockade (RASB) on Epo resistance in haemodialysis patients have yielded conflicting results [36].
The hypothesis underlying this study was that the inconsistent effects of RASB on the haematological response to Epo are related to the confounding influence of androgens. Men on haemodialysis require less Epo than women to achieve target haemoglobin concentrations [7], an effect that may be the result of androgen-stimulated erythropoiesis. Angiotensin-converting enzyme (ACE) inhibitors have been shown to reduce serum free testosterone levels in men but not women with normal renal function [8]. Therefore, we hypothesized that treatment with ACE inhibitors or angiotensin-receptor blockers (ARBs) might reduce testosterone levels in men on haemodialysis and cause Epo resistance. Since women already have low circulating testosterone levels, treatment with RASB would not be expected to have a significant impact on androgen levels and, therefore, not influence Epo sensitivity. The objective of this study was to test this hypothesis by examining the gender specificity of Epo resistance related to RASB and explore associations with drug-induced testosterone deficiency.
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Subjects and methods
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We evaluated the effect of RASB on serum-free testosterone levels and Epo requirements in patients on haemodialysis. The project was approved by the Institutional Review Board for studies involving human subjects. Informed consent was obtained and serum drawn pre-dialysis from men and women for determination of free testosterone levels. We analysed clinical data from the 3 month period prior to determination of the testosterone level.
Patients were included if they were between the ages of 20 and 80 years, had been on chronic haemodialysis for
6 months and had been managed by the same Epo and iron protocols. Since the incidence of age-associated deficiency of serum testosterone increases sharply in normal men above the age of 60 years [9], subgroups of men below and above this age also were evaluated. Pharmacological blockade of the RAS was considered to be present if the patient had been receiving either ACE inhibitors or ARBs for
2 weeks prior to and during the survey period, i.e. 3 months and 2 weeks prior to collection of blood for serum testosterone. Exclusion criteria included a history of pituitary disease, orchiectomy, sleep apnoea, prostate cancer, active alcohol abuse, morbid obesity (body mass index >40), HIV, bone marrow disease, recent bleeding or transfusion. Patients taking drugs that would suppress the bone marrow or testosterone production (corticosteroids, cimetadine, fluconazole, narcotics and spironolactone) also were excluded.
The following information was extracted from each patient's record: age, gender, mean haemoglobin, iron saturation, ferritin, albumin, intact parathyroid hormone (PTH), single pool Kt/V, post-dialysis weight, pre-dialysis blood pressure and use of other classes of antihypertensive medication.
The serum was separated from blood samples and frozen within 3 h. Free testosterone levels were measured using the Diagnostic Systems Laboratories 1049100 ACTIVE Free Testosterone Immunoassay Kit (Webster, TX, USA). This competitive binding biotin immunoassay uses rabbit anti-testosterone antiserum that has a low affinity for sex hormone-binding globulin and albumin.
Statistical analysis
Continuous data were evaluated with analysis of variance followed by the Student's t-test to compare differences between groups of men and women, with or without RAS blockade. Chi-square analysis was used to compare the frequencies at which other classes of antihypertensives were used. The relationships between age and free testosterone were examined by linear regression. Multiple regression analysis (Enter method) was used to evaluate the relationship between serum free testosterone levels and Epo dose (MedCalc Software, Mariakerke, Belgium). A P-value of <0.05 was considered significant.
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Results
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There were 24 men and 14 women included in the control groups that were not taking either ACE inhibitors or ARBs, and 25 men and 13 women in the groups with RASB. The medications taken included lisinopril (580 mg per day), fosinopril (20 mg per day), captopril (2575 mg per day), irbesartan (150300 mg), valsartan (80160 mg per day), losartan (100 mg per day) and olmesartan (40 per day).
The effects of gender and RAS blockade on Epo requirements are shown in Figure 1. The total Epo dose over the 3 month period was divided by the means of the post-dialysis weight and haemoglobin concentration. Control men required significantly less Epo than men with RASB and both groups of women. The Epo dose in women was not influenced by RASB. Comparison of the Epo doses in terms of units/kg or units/g haemoglobin yielded the same result (data not shown).

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Fig. 1. Bars represent the mean (±SEM) Epo dose for groups of control men (n = 24), RASB men (n = 25), control women (n = 14) and RASB women (n = 13). RASB refers to patients taking ACE inhibitors or ARBs vs those who did not (control).
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Table 1 demonstrates clinical characteristics of the groups illustrated in Figure 1. There were no differences in age, weight, haemoglobin, ferritin, intact PTH, Kt/V or blood pressure determinations. There were also no differences in the frequencies of patients taking beta-blockers, alpha-blockers or calcium-channel blockers. The serum free testosterone levels were higher in men than in women and the mean values were similar among controls vs those with RASB within each gender.
The relationship between age and serum testosterone is shown for control men on haemodialysis in Figure 2. This highly significant relationship was lost in men taking RASB (Figure 3). Controls younger than 60 years had significantly higher levels of testosterone than older controls (Figure 4), while there was no difference in testosterone levels between young and old men taking RASB. The effect of age and RASB on Epo doses in men on haemodialysis is shown in Figure 5. Young controls required less Epo than young men with RASB and old men with RASB. There was no clear effect of age on Epo requirements within the controls or men taking RASB. Older men with RASB tended to require more Epo than old controls (Figure 5).

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Fig. 2. Relationship between age and serum free testosterone in men on haemodialysis who are not taking RASB (n = 24).
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Fig. 3. Lack of relationship between age and serum free testosterone level in men on haemodialysis who are taking RASB (n = 25).
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We further explored the relationship between serum testosterone levels and Epo doses in patients on haemodialysis with multiple regression analysis. We considered the Epo dose (units/kg) as the dependent variable and serum testosterone as well as other factors known to influence the Epo dose as independent variables. We included both men and women in the analysis. There was a significant relationship between serum testosterone and Epo dose when ferritin, haemoglobin and age were also considered (Table 2).
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Discussion
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The results of this study support the hypothesis that androgens participate in Epo resistance induced by pharmacological blockade of the RAS. The importance of this effect of RASB appears to be confined to those with the highest levels of circulating testosterone, i.e. men <60 years old. We confirmed the work of Ifudu et al. [7] that showed that women on haemodialysis have higher Epo requirements than men, and we demonstrated that the effect was associated with lower circulating testosterone levels, as expected. RASB treatment in men was associated with an increase in the Epo requirement that was similar to that of women. However, the mean testosterone levels in men taking RASB was not reduced to that of women and, in fact, was not different to that of control men when all ages were considered together. The impact of RASB on serum testosterone was evidenced by disruption of the well-known significant relationship between age and serum testosterone in men. RASB treatment lowered free testosterone levels in men <60 years old to levels similar to those older than 60. This effect of RASB on testosterone in middle-aged men correlated with an increased Epo requirement compared with control men in the same age group. Given the apparent importance of both gender and age, it is not surprising that previous investigations into the effects of RASB on Epo sensitivity have yielded conflicting results.
That androgens augment red blood cell production in haemodialysis patients has been appreciated since the pre-Epo era, but this remains poorly understood. Androgen effects on haematopoiesis might be related to Epo, but they are not strictly dependent on circulating Epo levels [10]. Recent evidence indicates that insulin-like growth factor type I may mediate the effects of androgens on red blood cell production [11]. Androgens produced similar levels of haemoglobin as that achieved with Epo treatment in peritoneal dialysis patients [11] and also have been shown to increase the sensitivity to treatment with Epo in haemodialysis patients [12]. There is one study in which androgen treatment did not enhance the effect of Epo, but the interpretation of the results was limited by having only seven patients in the treatment group, three of whom did not complete the 16 week course of treatment [13]. Our study provides a different evaluation of the relationship between androgens and Epo responsiveness in haemodialysis patients, but confirms a synergistic effect. Multiple regression analysis demonstrated that serum testosterone is a significant variable in predicting Epo dose when men and women of all ages, with or without RASB, were considered together. This analysis showed that the lower the testosterone level the higher the Epo dose when the effects of haemoglobin, ferritin and age as covariables were considered.
A complete understanding of the relationship between Epo and androgens is complicated by the data indicating that exogenous Epo stimulates testosterone production in men with normal renal function [14] and men on haemodialysis [15]. Therefore, while age and RASB might be driving serum testosterone levels down, the final impact on serum testosterone might be mitigated to some degree by the higher exogenous Epo doses required to achieve the desirable concentration of haemoglobin. Therefore, an evaluation of the effects of RASB on serum testosterone and haemoglobin levels in patients with renal failure who are not receiving exogenous Epo would be helpful.
An important limitation of testing the hypothesis in this cross-sectional evaluation was the fact that the patients were on a wide variety of RAS blockers that had been taken over variable time periods in a broad dosage range. The dose of RASB has been suggested to be a key factor in the mediation of Epo resistance in haemodialysis patients [4,16]. The rationale for studying the effects of both ACE inhibitors and ARBs rested on the evidence that angiotensin II infusions stimulate Epo secretion in individuals with normal renal function and this effect is mediated via its type 1 receptor [1]. Furthermore, Epo resistance has been observed with ARBs [5] as well as ACE inhibitors [3]. However, an Epo-independent mechanism for ACE inhibitor-mediated anaemia in patients with renal failure may involve accumulation of N-acetyl-seryl-aspartyl-lysyl-proline [17], which may explain why some studies have found Epo resistance in haemodialysis patients taking ACE inhibitors but not ARBs [18]. Therefore, a separate evaluation of the effects of ACE inhibitors and ARBs on testosterone levels as well as Epo requirements is needed also.
This study suggests that androgens might be an important link between RASB and Epo resistance; however, the mechanism of the effect of RASB on serum testosterone was not evaluated. Koshida and co-workers [8] found that lisinopril lowered free testosterone levels in men with normal renal function. This was also observed in women with high circulating levels of testosterone from polycystic ovary syndrome [19], but neither investigation included elucidation of a mechanism. One recent study in rats demonstrated that RASB reduced serum testosterone in association with depressed activity of a hepatic P-450 enzyme, 17-testosterone hydroxylase, which regulates testosterone metabolism [20]. Therefore, studies investigating the effects of RASB production, regulation and metabolism of testosterone are needed.
The most important conclusion that can be drawn from the present study is that the effect of RASB on Epo resistance in haemodialysis patients is specific to the male gender. While other studies of the effects of RASB on Epo requirements have included women, we are unaware of any previous evaluation of the gender specificity of the effect. Another setting in which RASB reduces the haemoglobin is in patients who develop post-kidney transplant erythrocytosis. It is interesting that this problem is much more common in men than in women [20], but the gender specificity of the effects of RASB on erythrocytosis in this condition also has not been studied and would be interesting.
Androgens might be at least part of the explanation of the gender-specific effect of RASB on Epo resistance in haemodialysis patients, since these drugs lower testosterone levels in middle-aged men. The age-dependence of the effect of RASB on serum testosterone is difficult to understand. It is possible that the function of the pituitarytesticular axis in younger men is more dependent on an intact RAS than that of older men in whom the effects of aging are already contributing to hypogonadism. The observation that older men treated with RASB tended to require more Epo than older control men may be explained either by a subtle effect on androgen production that was not detectable in a single pre-dialysis serum testosterone measurement or by the fact that RASB also has effects on erythropoiesis that are independent of androgens. Further work is needed to eliminate some of the confounding variables and clarify the mechanism. Finally, the effect of RASB on testosterone may have important implications for anaemia management as well as other important problems in patients on haemodialysis, such as bone disease and depression.
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Acknowledgments
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This work is supported by a grant from the Dialysis Clinic Inc., Nashville, Tennessee, a non-profit organization.
Conflict of interest statement. None declared.
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Received for publication: 9. 2.04
Accepted in revised form: 11. 8.04