Metabolic
Renin–Angiotensin System Polymorphisms and Hemoglobin Level in Renal Allografts: A Comparative Study Between Losartan and Enalapril M. Noroozianavval, H. Argani, M. Aghaeishahsavari, P. Veisi, A. Ghorbanihaghjo, N. Rashtchizadeh, M. Jabbarpourbonyadi, and H. Hamzeiy ABSTRACT Introduction. In this study, hemoglobin (Hb) concentrations secondary to enalapril (E) or losartan (L) therapy were evaluated with respect to renin–angiotensin system (RAS) polymorphisms in renal transplant recipients. Materials and Methods. After determination of RAS polymorphisms [angiotensinconverting enzyme (DD, non-DD), angiotensinogen (TT, non-TT), and angiotensin receptor type1 (CC, non-CC)] by polymerase chain reaction, 70 renal transplant recipients were recruited to four groups randomly: first and second groups were treated with E (10 mg/d, 15 patients) and L (50 mg/d, 20 patients) alone, respectively. The third group received E⫹L (10 mg/d ⫹ 50 mg/d, 13 patients) and the fourth group (22 patients) received no medication. The treatment protocol was followed for 16 weeks. Complete blood counts were checked before treatment and every 2 months. P ⬍ .05 was considered to indicate statistical significance. Results. Treatment for 4 months decreased the Hb level in the E⫹L (14.15 ⫾ 0.94 to 12.06 ⫾ 0.66 g/dL, P ⫽ .000), E (14.00 ⫾ 0.86 to 13.11 ⫾ 0.82 g/dL, P ⫽ .02), and L (14.12 ⫾ 0.90 to 12.10 ⫾ 2.35 g/dL, P ⫽ .01) groups, but not in the control group (13.55 ⫾ 0.70 to 13.36 ⫾ 0.69 g/dL, P ⬎ .05). None of these regimens showed greater Hb reducion than the others (P ⬎ .05). None of the RAS polymorphisms predicted the intensity of the reduced Hb according to the type of treatment (P ⬎ .05). Any other sets of RAS polymorphisms (alone or together) did not impact on Hb levels pre- or post-intervention (P ⬎ .05). Conclusion. Our findings suggest that low dosages of E and/or L decrease Hb levels regardless of the RAS polymorphisms.
From the Drug Applied Research Center, Tabriz Medical University, Tabriz, Iran. Address reprint requests to M. Noroozianavval, Drug Applied Research Center (DARC), Tabriz Medical University, Tabriz, Iran.
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0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.02.016
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1018
Transplantation Proceedings, 39, 1018 –1022 (2007)
LOSARTAN VS ENALAPRIL
E
RYTHROCYTOSIS HAS been reported to be a common complication of renal transplantation since 1965.1 Based on the observation that angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) cause anemia in renal transplant recipients, several groups have used these treatments to manage erythrocytosis.1–3 The mechanism underlying transplantassociated erythrocytosis and reduction of hemoglobin (Hb) concentrations with these drugs have remained controversial.4 The unpredictable efficacy of ACEI and ARB to lower Hb may be secondary to renin-angiotensin system (RAS) polymorphisms.5,6 The aim of the study was to compare the safety and efficacy of ACEI (Enalapril [E]) and/or ARB (losartan [L]) to reduce Hb and to evaluate the effect of RAS polymorphisms on Hb concentrations and responses to therapy.
1019 analyzed by electrophoresis in 8% polyacrylamide gel and visualized by ethidium bromide staining. Determination of angiotensin II type 1 receptor (ATR1) A1166C genotype was performed by amplifying a DNA fragment, encompassing the polymorphism, using the following primers (Invitrogen): 5=-AATGCTTGTAGCCAAAGTCACCT-3= and 5=-GGCTTTGCTTTGTCTTGTTG-3=. PCR amplification was obtained using 30-L reaction containing 250 ng genomic DNA, 50 mmol KCl, 1 mmol MgCl2, 10 mmol Tris-HCl pH 9, 0.1% Triton X-100, 200 mol dNTPs, 1 pmol primers, and 0.5 U of Taq polymerase (Cinnagen). Thermocycling consisted of 2 minutes of denaturation at 94°C, followed by 40 cycles of 1 minute at 94°C, annealing 1 minute at 60°C, extension of 2 minutes at 72°C, and final extension of 10 minutes at 72°C. After enzymatic amplification of genomic DNA, each product was digested with Ddel (Roche Inc., Mannheim, Germany). The genotypes were detected on 3% agarose gels stained with ethidium bromide.8,9
Procedures MATERIALS AND METHODS Patients After obtaining written informed consent we enrolled 70 patients— 40 men and 30 women— of overall mean age 36.47 ⫾ 6.26 years. They had received allografts at least 6 months earlier and displayed stable graft function, defined as a serum creatinine ⬍2.2 mg/dL. They received two types of immunosuppressive therapy, cyclosporine ⫹ prednisolone ⫹ azathioprine or cyclosporine ⫹ prednisolone ⫹ mycophenolate mofetil. None of the patients were diabetics, smokers received diuretic therapy in the last 3 months, or displayed allograft renal artery stenosis or had received a retransplantation.
Determination of RAS Polymorphisms For determination of RAS polymorphisms, genomic DNA was isolated by phenol-chloroform extraction from 10 mL of whole blood drawn into tubes containing potassium EDTA.7 Polymerase chain reaction (PCR) was used to amplify a DNA fragment of the ACE gene in a 20-L reaction, containing 250 g genomic DNA, 50 mmol KCl, 1 mmol MgCl2, 10 mmol Tris-HCl pH 9, 0.1% Triton X-100, 200 mol dNTPs, 1 pmol primers, and 0.5 U of Taq polymerase (Cinnagen Inc., Tehran, Iran). Primers (Invitrogen Inc., Carlsbad Calif) flanking the insert sequence were 5=-CTGGAGACCACTCCCATCCTTTCT-3= and 5=-GATGTGGCCATCACATTCGTCAGAT-3=. PCR amplification was performed on a programmable thermal controller (Eppendorf, Krackeler Scientific, Inc.) for 30 cycles with denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute. Genotypes of ACE were detected on an ethidium bromide stained 2% agarose gels. Angiotensinogen (AGT) M235T polymorphism was investigated by PCR of genomic DNA followed by restriction endonuclease digestion. For PCR, genomic DNA (250 ng) was used in a final volume of 50 L containing 2.5 mmol/L MgCl2 50 mmol/L KCl, 10 mmol/L Tris hydrochloride (pH 9.0), 1 pmol of each primer (Invitrogen) 5=-GATGCGCACAAGGTCCTG-3= and 5=-CAGGGTGCTGTCCACACTGGCTCGC-3=, 200 mol each of the four dNTPs, and 0.4 U Taq polymerase (Cinnagen). There was an initial denaturation at 94°C, followed by 25 cycles of 1 minute at 94°C, 1 minute at 61°C, and 1 minute at 72°C. After enzymatic amplification of genomic DNA, each product was digested with SfaNl (Fermentas Inc., Burlington, Canada). The genotypes were
Triglyceride, cholesterol, urea, and creatinine were measured by standard enzymatic methods. Cyclosporine (CsA) trough levels (C0) were estimated by radioimmunoassay (Kontron, Laboregerätebörse Inc., Switzerland).
Study Protocol After determination of RAS gene polymorphisms and switching of antihypertensive drugs (calcium channel blockers) in hypertensive cases to ACEI and/or ARBs, the patients were randomly recruited in to four groups: the first and second groups were treated with enalapril (10 mg/d; n ⫽ 15) or losartan (50 mg/d, n ⫽ 20) alone, respectively. The third positive control group received enalapril ⫹ losartan (10 mg/d ⫹ 50 mg/d, n ⫽ 13) and the fourth negative control group (n ⫽ 22) received no medication. The medications were commenced at low dosage (2.5 mg/d for enalapril and 25 mg/d for losartan) and gradually increased with serial examinations of urea, and creatinine, blood pressure control. The patients were treated for 16 weeks. Before starting treatment, we checked the complete blood counts (CBC), electrolytes (Na, K), urea, creatinine, and CsA levels. These laboratory parameters were rechecked after 2 and 4 months of treatment. Every 2 weeks the patients were followed for blood pressure, renal function tests, and drug complications, such as hyperkalemia, dry cough, and other allergic reactions.
Statistical Analysis For statistical analysis, we used SPSS for windows 11.0 (SPSS Inc., Chicago, Il). Categorical data were measured by frequency analysis. Clinical characteristics were compared by parametric or nonparametric tests for independent samples as appropriate. Data are presented as percentages, mean values ⫾ SDs, or medians (ranges). In this study, P ⬍ .05 was considered to indicate statistical significance.
RESULTS
Table 1 shows the demographic data of all patients in the four drug groups. There were no significant differences in age, gender, duration of transplant, hemodialysis period before renal transplantation, causes of chronic renal failure, type of immunosuppressive therapy, or RAS polymorphisms according to the different drug regimens (P ⬎ .05).
1020
NOROOZIANAVVAL, ARGANI, AGHAEISHAHSAVARI ET AL Table 1. Demographic Features Parameters
Enalapril (n ⫽ 35)
Losartan (n ⫽ 20)
Positive Control (n ⫽ 13)
Negative Control (n ⫽ 22)
P Value
Age (yr) Sex (male/female) Time after RTx (mo) Duration of dialysis before RTx (mo) Underlying diagnosis Chronic GN Chronic PN ADPKD DM CVD Alport HTN Unknown causes Immunosuppressive therapy CsA⫹PRD⫹AZA CsA⫹PRD⫹Mycophenolate mofetil RAS polymorphisms ACE genotype DD Non-DD AGT genotype TT Non-TT ATR1 genotype CC Non-CC
32.41 ⫾ 8.82 9/6 50.08 ⫾ 22.06 21.62 ⫾ 5.68
37.68 ⫾ 1.97 12/8 29.10 ⫾ 10.58 19.84 ⫾ 8.42
37.33 ⫾ 6.42 9/4 39.54 ⫾ 16.24 13.57 ⫾ 8.83
37.33 ⫾ 6.27 8/14 44.94 ⫾ 12.54 20.20 ⫾ 9.01
NS NS NS NS NS
3 2 2 1 1 — 2 4
8 2 1 2 1 1 1 4
4 2 1 1 1 — — 4
4 3 2 2 1 1 2 7
7 8
10 10
7 6
12 10
8 7
7 13
6 7
10 12
9 6
14 6
5 8
10 12
4 11
4 16
2 11
6 16
NS
NS
NS
NS
RTx, renal transplantation; GN, glomrulonephritis; PN, pyclonephritis; ADPKD, autosomal dominant polyeystic kidney disease; DMgen, diabetes mellitus; CVD, collagen vascular disease; HTN, hypertension; CsA, cyclosporine; PRD, prednisolone; AZA, azathioprine; NS, not significant.
Before drug intervention, no difference was observed among the patients regarding the treatment regimen and baseline levels of Hb (P ⬎ .05). After initiating treatment, three cases were withdrawn because of drug complications, such as hyperkalemia (n ⫽ 2) and dry cough (n ⫽ 1). After 2 months of treatment, the Hb level was significantly decreased in the enalapril plus losartan group (from 14.15 ⫾ 0.94 g/dL to 12.39 ⫾ 0.92 g/dL; P ⫽ .009; Fig 1). In the enalapril, losartan, and negative control groups, Hb
Fig 1. Changes of mean Hb levels in renal transplant recipients in positive control group (n ⫽ 13), enalapril group (n ⫽ 14), losartan group (n ⫽ 18), and negative control group (n ⫽ 22). Values are means ⫾ SD. *† Is significant in 8th and 16th weeks, *^ Is significant only in 16th week.
levels before intervention were 14.00 ⫾ 0.86 g/dL, 14.12 ⫾ 0.90 g/dL, and 13.55 ⫾ 0.70 g/dL, respectively. After 2 months of treatment, they reached 13.20 ⫾ 1.01 g/dL (P ⬎ .05), 13.27 ⫾ 0.78 g/dL (P ⬎ .05), and 13.39 ⫾ 0.76 g/dL (P ⬎ .05), but after 4 months of therapy with enalapril plus losartan, the Hb level decreased significantly to 13.15 ⫾ 0.82 g/dL (P ⫽ .02) and 12.10 ⫾ 2.35 g/dL (P ⫽ .01), respectively (Fig 1). Also, mixed treatment with enalapril plus losartan significantly decreased Hb concentration to
LOSARTAN VS ENALAPRIL
1021
Table 2. Baseline Hb and Hct of Patients Between RAS Polymorphisms RAS Polymorphisms
ACE genotype DD Non-DD AGT genotype TT Non-TT ATRI genotype CC Non-CC
Hb (g/DL)
Hct (%)
14.29 ⫾ 0.41* 13.44 ⫾ 0.76
40.01 ⫾ 1.15* 37.63 ⫾ 2.12
13.75 ⫾ 0.39 13.73 ⫾ 0.49
38.50 ⫾ 1.09 36.44 ⫾ 2.12
13.40 ⫾ 0.70 13.77 ⫾ 0.32
37.52 ⫾ 1.96 38.55 ⫾ 0.89
*Significant.
12.06 ⫾ 0.66 g/dL (P ⫽ .000) (Fig 1). In the control group, there was no significant change in Hb concentration after 4 months (P ⬎ .05, Fig 1). None of the above regimens was a greater Hb reducer of than the others (P ⬎ .05). Absolute (⫺2.09, ⫺0.84, and ⫺2.02 g/dL; P ⫽ .07) and percent decreases (14.92%, 6%, and 14.42%; P ⫽ .07) in Hb levels were no different between enalapril, losartan, and enalapril plus losartan (P ⬎ .05). Only, the DD genotype of ACE was associated with higher Hb concentration (14.29 ⫾ 0.41 g/dL vs. 13.44 ⫾ 0.76 g/dL; P ⫽ .04; Table 2). None of the RAS polymorphisms displayed an important role in reduction of Hb content according to the type of treatment (P ⬎ .05). Any other sets of RAS polymorphisms (alone or together) did not impact Hb levels in pre- and postintervention (P ⬎ .05). There were no significant changes in serum creatinine, urea, sodium, potassium, CsA levels, and mean arterial blood pressure during follow-up (P ⬎ .05; Table 3). DISCUSSION
Our finding is consistent with previous studies about the effect of ACEI10 –13 and ARB,4,14 –16 to reduce Hb levels in renal transplant recipients. Tylicki et al reported a significant decrease in Hb level after losartan therapy.17 Another
study revealed that ACE inhibition therapy decreased Hb concentrations.18 Moreover, the impact of RAS polymorphisms on response to drug therapy (enalapril and losartan) is unclear.6 The mechanisms underlying transplant-associated erythrocytosis and reduced Hb concentrations with these drugs remain unproven.4,19,20 Losartan and enalapril have been previously reported to reduce Hb levels in healthy individuals as well as in patients with moderate renal insufficiency and those on dialysis.21 Therefore, we compared the combination effects of enalapril and losartan, versus enalapril or losartan alone on Hb content. This comparison showed that 5 mg enalapril and/or 25 mg losartan (twice daily for 4 months) significantly reduced Hb levels. Also combination therapy with enalapril plus losartan for 2 months decreased Hb level significantly, but after 2 months treatment, neither one alone significantly decreased Hb levels. Mechanisms whereby ACEI and ARB decrease Hb are unknown.3 It is well known that the RAS influences erythropoietin (EPO) synthesis. In rodents, hypoxia increased plasma levels of both EPO and renin. Administration of homologous renin increased plasma EPO levels and this response was abolished by pretreatment with captopril and restored by angiotensin II infusion.21 In transplant recipients, production of renin from native kidneys is increased and concentrations of renin and EPO in the native kidney venous blood are highly correlated.22 These data suggest that both ACEIs and ARBs might blunt erythropoiesis by dampening angiotensin II-driven EPO production in the native kidneys. ACEIs and ARBs might diminish EPO production either by altering renal blood flow and, therefore, oxygen delivery or by indirect suppression of hormone synthesis.21 The effects of ACEI and ARB to reduce Hb concentrations suggest that RAS polymorphisms may play an important role in the erythrocytosis after renal transplantation. The results of previous studies have been limited to just one part of RAS polymorphisms. In one study, the DD genotype of ACE polymorphism was associated with higher plasma levels of ACE and probably a more activated RAS. Both,
Table 3. Biochemical Profiles of Patients Before and After Treatment Enalapril (n ⫽ 14)
Positive Control (n ⫽ 13)
Negative Control (n ⫽ 22)
Baseline
8th Week
Baseline
8th Week
P Value
37.4 ⫾ 2.6 53.4 ⫾ 9.3 1.46 ⫾ 0.23 135 ⫾ 6 4.80 ⫾ 0.4 247 ⫾ 23
40.6 ⫾ 1.4 42.1 ⫾ 16.5 1.35 ⫾ 0.06 140 ⫾ 5 4.00 ⫾ 0.1 250 ⫾ 26
39.9 ⫾ 2.4 41.7 ⫾ 19.2 1.30 ⫾ 0.05 139 ⫾ 8 3.90 ⫾ 0.1 272 ⫾ 34
NS NS NS NS NS NS
95.0 ⫾ 14.8 93.6 ⫾ 10.5 105.0 ⫾ 12.4 100.3 ⫾ 11.8 104.6 ⫾ 9.1 84.0 ⫾ 18.3 94.0 ⫾ 14.1 97.0 ⫾ 13.8 43.1 ⫾ 10.38 54.5 ⫾ 7.84 45.8 ⫾ 6.46 45.5 ⫾ 6.01 46.81 ⫾ 8.19 45.0 ⫾ 5.68 50.8 ⫾ 7.64 56.0 ⫾ 7.25 121 ⫾ 24.3 119 ⫾ 24.6 105 ⫾ 14.8 122 ⫾ 18.0 135 ⫾ 14.7 117 ⫾ 16.8 109 ⫾ 18.6 126 ⫾ 15.6 130 ⫾ 30.3 175 ⫾ 33.1 220 ⫾ 31.1 261 ⫾ 54.0 235 ⫾ 34.3 209 ⫾ 42.6 141 ⫾ 36.2 151 ⫾ 32.3 199 ⫾ 26.8 208 ⫾ 31.3 191 ⫾ 22.4 215 ⫾ 26.8 227 ⫾ 35.2 203 ⫾ 26.2 184 ⫾ 20.4 195 ⫾ 29.8
NS NS NS NS NS
Baseline
Hct (%) Urea (mg/dL) Cr (mg/dL) Na (mEq/L) K (mEq/L) CsA level (ng/mL) MAP (mmHg) HDL-c (mg/dL) LDL-c (mg/dL) TG (mg/dL) Chol (mg/dL)
40.7 ⫾ 2.1 41.3 ⫾ 7.3 1.15 ⫾ 0.07 137 ⫾ 5 4.61 ⫾ 0.3 194 ⫾ 32
8th Week
38.5 ⫾ 2.8 48.4 ⫾ 8.4 1.47 ⫾ 0.15 138 ⫾ 7 4.58 ⫾ 0.2 176 ⫾ 33
Losartan (n ⫽ 18) Baseline
40.5 ⫾ 2.3 37.7 ⫾ 6.5 1.40 ⫾ 0.08 138 ⫾ 9 4.50 ⫾ 0.2 256 ⫾ 25
8th Week
38.7 ⫾ 2.7 43.0 ⫾ 6.1 1.48 ⫾ 0.07 135 ⫾ 5 4.51 ⫾ 0.1 222 ⫾ 24
40.3 ⫾ 1.5 40.2 ⫾ 18.0 1.27 ⫾ 0.12 139 ⫾ 3 4.20 ⫾ 0.3 235 ⫾ 22
Abbreviations: CsA, cyclosporine; MAP, mean arterial pressure; Hct, hematocrit; Cr, creatinine; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; TG, triglycerides; Chol, cholesterol.
1022
the response rate to renin–angiotensin–aldostrone blockade as well as the absolute and percent decrease in Hb levels were not different between DD and non-DD patients.19 In another study, patients with the DD genotype displayed lower Hb concentrations compared with the non-DD genotype.6 Our study examined three different parts of the RAS–ACE, AGT, and ATR1 polymorphisms. The current study demonstrated that only the DD genotype of ACE should higher Hb levels but none of the RAS polymorphisms displayed an important role to reducts Hb concentration according to the type of treatment. In conclusion, our study suggested that low dosages of ACEI and/or ARB decreased Hb levels within 4 months. Additionally, cotreatment with losartan and enalapril reduced Hb concentration within 2 months. Although our research demonstrated a relationship between RAS polymorphisms and Hb level, further studies in a larger sample are warranted to investigate the exact mechanisms by which ARB and ACEI reduce Hb levels. REFERENCES 1. Torregrosa JV, Campistol JM, Montesinos M, et al: Efficacy of captopril on posttransplant erythrocytosis. Long-term follow-up. Transplantation 58:311, 1994 2. Inigo P, Torregrosa JV, Campistol JM, et al: Treatment with losartan in kidney transplant recipients with posttransplant erythrocytosis. Transplant Proc 31:2321, 1999 3. Danovitch GM, Jamgotchian NJ, Eggena PH, et al: Angiotensin-converting enzyme inhibition in the treatment of renal transplant erythrocytosis. Clinical experience and observation of mechanism. Transplantation 60:132, 1995 4. Wang AY, Yu AW, Lam CW, et al: Effects of losartan or enalapril on hemoglobin, circulating erythropoietin, and insulinlike growth factor-1 in patients with and without posttransplant erythrocytosis. Am J Kidney Dis 39:600, 2002 5. Torregrosa JV, Poch E, Oriola J, et al: Postrenal transplant erythrocytosis and insertion/deletion polymorphism of angiotensin converting enzyme gene. Transplant Proc 31:2319, 1999 6. Yildiz A, Cine N, Akkaya V, et al: Comparison of the effects of enalapril and losartan on posttransplantation erythrocytosis in renal transplant recipients. Transplantation 72:542, 2001 7. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988
NOROOZIANAVVAL, ARGANI, AGHAEISHAHSAVARI ET AL 8. Frishberg Y, Becker-Cohen R, Halle D, et al: Genetic polymorphisms of the renin-angiotensin system and the outcome of focal segmental glomerulosclerosis in children. Kidney Int 54:1843, 1998 9. Ohno T, Kawazu S, Tomono S: Association analyses of the polymorphisms of angiotensin-converting enzyme and angiotensinogen genes with diabetic nephropathy in Japanese noninsulin-dependent diabetics. Metabolism 45:218, 1996 10. Lal SM, Trivedi HS, Ross G: Long term effects of ACE inhibitors on the erythrocytosis in renal transplant recipients. Clin Nephrol 18:13, 1995 11. Kessler M, Hestin D, Mayeux D, et al: Factors predisposing to post-renal transplant erythrocytosis. A prospective matched-pair control study. Clin Nephrol 45:83, 1996 12. Gaston RS, Julian BA, Barker CV, et al: Enalapril: safe and effective therapy for posttransplant erythrocytosis. Transplant Proc 25:1029, 1993 13. Beckingham IJ, Woodrow G, Hinwood M, et al: A randomized placebo-controlled study of enalapril in the treatment of erythrocytosis after renal transplantation. Nephrol Dial Transplant 10:2316, 1995 14. Klaassen R, Gelder T, Rischen-Vos J, et al: Losartan, an angiotensin II receptor antagonist, reduces hematocrits in kidney transplant recipients with posttransplant erythrocytosis. Transplantation 64:700, 1997 15. Julian BA, Brantley JR, Barker CV, et al: Losartan, an angiotensin II type I receptor antagonist lowers hematocrits in posttransplant erythrocytosis. J Am Soc Nephrol 9:1104, 1998 16. Midtvedt K, Stokke ES, Hartmann A: Successful long-term treatment of post- transplant erythrocytosis with losartan. Nephrol Dial Transplant 11:2495, 1996 17. Tylicki L, Biedunkiewicz B, Chamienia A, Wojnarowski K, Zdrojewski Z, Rutkowski B. Randomized placebo-controlled study on the effects of losartan and carvedilol on albuminuria in renal transplant recipients. Transplantation 81:52, 2006 18. Plata R, Cornejo A, Arratia C, et al: Angiotensin-converting-enzyme inhibition therapy in altitude polycythaemia: a prospective randomised trial. Lancet 359:663, 2002 19. Gaston RS, Julian BA, Diethelm A, et al: Effects of enalapril on erythrocytosis after renal transplantation. Ann Intern Med 115:954, 1991 20. Julian BA, Gaston RS, Barker CV, et al: Erythropoiesis after withdrawal of enalapril in posttransplant erythrocytosis. Kidney Int 46:1397, 1994 21. Ducloux D, Fournier V, Bresson-Vautrin C, et al: Long-term follow-up of renal transplant recipients treated with losartan for post-transplant erythrocytosis. Transpl Int 11:312, 1998 22. Aeberhard JM, Scneider PA, Vallotton MB, et al: Multiple sites estimates of erythropoietin and renin in polycythemic kidney transplants patients. Transplantation 50:613, 1990
