Post-Kidney Transplantation Erythrocytosis and Its Relationship to Renal Artery Stenosis, IGF-1, and Its Binding Protein 3 (IGFBP-3) Essam Khedr, MD; Saeed Abdelwhab, MD; Magdy El-Sharkawy, MD; Medhat Ali, MD; Kadry El-Saed, MD; Dalia Dawoud, MD The authors are with the Department of Internal Medicine and Nephrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. BACKGROUND: Erythrocytosis is relatively common following kidney transplantation. It increases the risk of thromboembolism. Insulin-like growth factor-1 (IG-1) and its binding protein 3 (IGFBP-3) may play a role in its pathogenesis. PATIENTS AND METHODS: A total of 120 consecutive kidney-transplanted patients with at least 3 month graft function were studied. Forty-five kidney transplant patients were then selected and divided into 2 groups: 15 patients with post-transplant erythrocytosis (PTE; group I), 30 without erythrocytosis (group II), and 15 healthy subjects matched with the cases as a control group (group III). Full medical history, clinical examination, hematological parameters, routine biochemical tests, IGF-1, and IGFBP-3 were measured in all subjects. Duplex ultrasonography for diagnosis of renal artery stenosis was done. RESULTS: Post-transplant erythrocytosis developed with an incidence rate of 23.3%.The serum of IGF-1 was significantly higher in the renal transplant recipients with and without erythrocytosis (309.4 ⫾ 72.9, 301.6 ⫾ 67.5 ng/mL, respectively) compared to controls (232.5 ⫾ 95.3 ng/mL, p < 0.01), although there was no statistical difference between the first 2 groups. However, the serum of IGFBP-3 was significantly higher in the renal transplant recipients with erythrocytosis than those without erythrocytosis and normal individuals (p < 0.001). Graft function and incidence of acute and chronic rejection were similar between the 2 groups. CONCLUSION: Post-transplant erythrocytosis represents an anomaly of IGF-1 and its major binding proteins in which they may alter erythropoietin homeostatic mechanisms in the denervated transplant kidney which may be superimposed on a setting of multiple risk factors and leads to a state of erythrocytosis that is seen after kidney transplantation. (IGF-1) and insulin-like growth factor binding proteins (IGFBP-3), known to enhance erythropoiesis.2 Although early case reports suggested that post-transplant erythrocytosis was caused by renal ischemia resulting from renal artery stenosis other studies failed to confirm this association.3, 4 Patients and Methods Patients This work is a case-control study. The study included 120 consecutive kidney transplanted patients with at least 3 months of graft function. Then 45 kidney transplanted patients were selected and divided into 2 groups: 15 patients with post-transplant erythrocytosis (group I), 30 without erythrocytosis (group II), and 15 healthy subjects matched with the cases as a control group (group III). All patients were col- lected from the Nephrology Department, Nasser Institute, Cairo, Egypt. After extensive chart review of all weekly transplanted clinics, laboratory data through a period of 36 months were reviewed to define the population of erythrocytosis. A total of 28 patients out of 120 kidney transplanted patients were identified. These 120 consecutive patients who retained a functional graft for at least 3 months were determined to be an at-risk population (3 months was the minimum post-transplant time prior to the onset of erythrocytosis). Post-transplant erythrocytosis was defined as a hematocrit of greater than 51% on 3 consecutive clinic visits after transplantation. From the pool of 28 patients with PTE, the first group of 15 renal transplant recipients with PTE were selected. To identify potential erythrocytosis risk factors and to determine how the role of renal transplantation might influence our results, the second group consisted of œ P ost-transplant erythrocytosis (PTE) is defined as a persistently elevated hematocrit to a level greater than 51% after renal transplantation. It occurs in 10% to 15% of graft recipients. Post-transplant erythrocytosis is associated with an increased risk of complication due to thromboembolic events that occur in 10% to 30% of cases; 1% to 2% eventually die of associated complications.1 Although incompletely understood, the pathogenesis of PTE appears to be multifactorial. Patient-specific factors are undoubtedly involved. Classically, erythropoietin levels in most PTE patients still remain within the “normal range” indicating that erythrocytosis finally ensues by the contributory action of additional growth factors on erythroid progenitors. The non-erythropoietin factors may either enhance the sensitivity to erythropoietin or directly promote erythropoiesis. Implicated proteins include insulin-like growth factor-1 May 2009 Dialysis & Transplantation 1 Post-Transplant Erythrocytosis Methods All patients and controls were subjected to a complete medical history including name, age, sex, occupation, residence, hypertension, smoking history, underlying renal disease, duration of dialysis, duration of use of erythropoietin before transplant, and number of months from transplant to the development of PTE, and the other concomitant risk factors that may be associated with erythrocytosis and drug history including immunosuppressive medication used in the post-transplant setting (including prednisone, azathioprine, mycophenolate, and cyclosporine A in various combinations). All patients and controls were given a thorough clinical examination. The following biochemical evaluations were completed for all patients and controls: blood analysis for glucose, liver function including serum albumin levels were done and serum creatinine and blood urea were obtained, centrifuged, and immediately separated to measure kidney function. Hematological parameters including hemoglobin, hematocrit value, mean corpuscular volume, serum iron, and total iron binding capacity were measured by spectrophotometric methods (AEROSET, Abbott Japan, Tokyo, Japan). An estimation of the serum level of insulin-like growth factor-1 (IGF1), and insulin-like growth factor binding 2 Dialysis & Transplantation May 2009 protein 3 (IGFBP-3) was completed. The blood was drawn after an overnight fast for the determination of serum levels of IGF-1 and IGFBP-3. A total of 5 cc venous blood samples were obtained from each patient and control, separated into a clean tube and serum was divided into aliquots; each of them were put into an eppendorf tube and stored at ⫺20°C till the time of the assay. Estimation of Serum IGF-1 Serum IGF-1 was measured by DSL5600 active IGF-1 coated tube IRMA kit (Diagnostic Systems Laboratories, Webster, Tex.) Principle The procedure employs a 2 site immunoradiometric assay (IRMA) principle.5 The DSL-5600 IGF-1 IRMA includes a simple extraction step in which IGF-1 is separated from its binding protein in serum. This step is considered to be essential for the accurate determination of IGF-1.6 The IRMA is a non-competitive assay in which the analyte to be measured is sandwiched between 2 antibodies. The first antibody is immobilized to the inside wall of the tubes. The other antibody is radio-labeled for detection. The analyte present in the patient samples, standards, and controls is bound by both of the antibodies to form a “sandwich” complex. Unbound materials are removed by decanting and washing the tubes. Materials Supplied and Initial Preparation 1. 2. 3. 4. 5. 6. 7. Six vials of IGF-1 standards A through F. Anti-IGF-1 coated tubes. Anti-IGF-1 (1–125) reagent. Two vials extraction solution. Neutralizing solution. One vial of IGF-1 control level I. One vial of IGF-1 control level II. Extraction Procedure Extraction of IGF-1 from its binding protein may be performed in advance (extracted supernatant may be stored at 2°C–8°C for up to 24 h or at ⫺20°C or lower for longer periods). This procedure yields approximately 90% to 100% recovery of added IGF-1 in samples.7 Neither standards nor controls were extracted. Steps 1. Two polypropylene 12 ⫻ 72 mm tubes were labeled for each sample (1 tube for extraction and the other for neutralization). 2. 100 UL of sample was pipetted into the extraction tube. 3. 400 UL of extraction solution was added to each sample, vortexed, and incubated for 30 minutes at room temperature (25°C). 4. The samples were placed in a refrigerated centrifuge and centrifuged at 5000 rpm for 30 minutes at 4°C. 5. Without disturbing the pellet, 100 UL of clear supernatant was transferred into the neutralizing tube. The pipet tips were prewetted by pipetting. 6. 500 UL of the neutralizing solution was then added to the second tube (containing the extracted supernatant). This is the neutralized sample extract which was used in the assay. The neutralized sample was vortexed gently to avoid foaming. Assay Procedure All reagents were brought to room temperature and mixed thoroughly before use. Standard and controls were reconstituted and mixed thoroughly, avoiding foaming. Standards, controls, and unknowns were assayed in duplicate. 1. Two plain (uncoated) tubes were labeled for total counts. Anti-IGF coated tubes were labeled in duplicate. 2. 50 UL of the reconstituted standards, controls, and extracted unknowns were added to the appropriate tubes and pipetted to the bottom of the tubes. 3. Immediately after, 200 UL of antiIGF-1 (1–125) reagent was added to all tubes. 4. Tubes were mixed by shaking the test rack gently and all tubes were incubated at room temperature for 3 hours on a shaker set at 180 rpm. 5. All tubes were decanted, except the total count tubes, by simultaneous inversion with a sponge rack into a radioactive waste receptacle. The tubes were shaken sharply on absorbent material for 1–2 minutes. The tubes were blotted to remove any droplets adhering to the rim before returning them to an upright position. Failure œ 30 renal transplant recipients without PTE (i.e., with hematocrit levels that never exceeded 49%). These patients were closely matched with those of the first group regarding age, sex, source of transplant (living related, or living unrelated), and duration since transplantation, as much as possible. Again, the third group of 15 normal individuals was obtained to ascertain the influence of transplantation in this study. The only treatment modality used to treat the PTE was phlebotomy, and no patient in the study was receiving drugs inactivating the renin-angiotensin system (RAS) such as an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin II type 1 receptor blocker (ARB). The exclusion criteria was as follows: patients treated with ACE inhibitors or ARBs or patients who had a second transplant. Post-Transplant Erythrocytosis 6. 7. 8. 9. to blot tubes adequately may result in poor replication and spurious values. 6. 3 mL of deionized water was added to each tube, except total count tubes. Step 6 was repeated twice more for a total of 3 washes. All tubes were counted for 1 minute, using a computerized Wallac 1282 Reagents Compugamma counter. Results were calculated automatically in ng/mL. Estimation of Serum IGFBP-3 Serum IGFBP-3 was measured by DSL6600 Active, IGFBP-3 coated tube IRMA kit supplied by DSL. 2. 3. 4. 5. 6. 7. Principle The procedure employs a 2 site immunoradiometric assay (IRMA) principle.5 The IRMA is a non-competitive assay in which the analyte to be measured is sandwiched between 2 antibodies. The first antibody is immobilized to the inside walls of the tubes. The other antibody is radio-labeled for detection. The analyte present is bound by both of the antibodies to form a “sandwich” complex. Unbound reagents are removed by decanting and washing the tubes. Materials Supplied and Initial Preparation 1. IGFBP-3 sample diluent (standard A). 2. 5 vials of IGFBP-3 standards B through F. 3. Anti-IGFBP-3 coated tubes. 4. Anti-IGFBP-3 (1–125) reagent. 5. IGFBP-3 control level I. 6. IGFBP-3 control level II. 8. 9. 10. 11. 12. the controls were not multiplied by sample dilution factor. Two plain (uncoated) tubes were labeled for total counts. Anti-IGFBP coated tubes were labeled and arranged in duplicate. 50 UL of the reconstituted standards, controls, and diluted unknowns were added to the appropriate tubes and pipetted to the bottom of the tubes. Immediately after, 200 UL of antiIGFBP-3 (1–125) reagent was added to all tubes. All tubes were vortexed gently. All tubes were incubated at room temperature (⫺25°C) overnight (18– 24 h) on a shaker at 180 rpm. All tubes were decanted, except the total count tubes, by simultaneous inversion with a sponge rack into a radioactive waste receptacle. The tubes were shaken sharply on absorbent material for 1–2 minutes. The tubes were blotted to remove any droplets adhering to the rim before returning them to an upright position. Failure to blot tubes adequately may result in poor replication and spurious values. All tubes were washed, except total count tubes, by adding 3 mL of deionized water. Tubes were decanted and step 8 was repeated twice for a total of 3 washing. All tubes were counted for 1 minute, using a computerized Wallac 1282 Compugamma counter. All sample results were multiplied by the dilution factor. Results were calculated automatically in ng/mL. Assay Procedure All reagents were brought to room temperature and mixed thoroughly before use. Standards and controls were reconstituted and mixed thoroughly, avoiding foaming. Standards, controls, and unknowns were assayed in duplicate. 1. Human serum samples were diluted 1:100 with IGFBP-3 sample diluent prior to assay. Samples with very low expected values were diluted 1:50 with IGFBP-3 sample diluent prior to assay. Control was not diluted prior to assay and hence the results for Duplex Ultrasonography for Diagnosis of Renal Artery Stenosis Color coded Doppler-ultrasonographic examination was performed using Acuson computed sonography 128 X/10C (Mountain View, Calif.) for patients and the control groups. Subjects were prepared by fasting overnight and having a morning rectal enema. They were placed in a supine position starting with the transducer in the right iliac area, using a 2.5 or 3 MHz transducer to visualize the renal artery of the transplanted kidney to measure their systolic and diastolic velocities in cm/sec, then the transducer was placed using a cronal section to get the internal vessels. The systolic and diastolic flow velocities were measured in the internal arteries using proper angulation to get the resistivity and pulsatility indices. The following duplex parameters were studied: • Maximum, mean, and minimum flow velocities in the main renal arteries as well as the internal arteries. • Pulsatility and resistivity indices in the main renal arteries as well as their internal branches. Pulsatility index ⫽ Systolic velocity⫺Diastolic velocity Mean velocity Resistivity index ⫽ Systolic velocity⫺Diastolic velocity Systolic velocity Statistical Methodology SPSS software was used for analysis of this data as follows: description of quantitative variables in the form of mean, standard deviation, and range; description of qualitative variables in the form of frequency and percentage; Student’s t-test was used to compare quantitative variables; a ␹2 test used to compare qualitative variables; an analysis of variance test was used to test the significant differences between more than 2 groups. Results Demographic Data A total of 60 patients were included in this tria1, 15 patients with post-transplantation erythrocytosis (2 females, 13 males), 30 patients without post-transplantation erythrocytosis, (5 females, 25 males), as well as 15 healthy subjects matched with the cases as a control group (2 females, 13 males). There was no statistically significant difference among the study groups in regard to age and sex (p > 0.05). A total of 28 patients (22 male, 6 female) out of 120 (78 male, 42 female) kidney-transplanted consecutive patients who retained a functional graft for at May 2009 Dialysis & Transplantation 3 FIGURE 2. Comparison of IGF-1 levels among studied groups. FIGURE 3. Comparison of IGFBP-3 levels among studied groups. FIGURE 4. Gender specific rate in transplanted patients. 4 Dialysis & Transplantation May 2009 œ FIGURE 1. Incidence of PTE. least 3 months were determined to have erythrocytosis with incidence of PTE (23.3% as shown in Figure 1). The onset of PTE in the majority of the patients occurred in the first 6 months after transplantation (8 out of 15 patients), while for 5 of these 15 patients, the onset was between 6 and 12 months while for the remaining 2 patients onset of PTE occurred between 12 and 24 months. There was a significant statistical difference among the study groups in regard to mean IGF-1 levels (Table I and Figure 2). The difference appeared when comparing patients with PTE (group I) or patients without PTE (group II) with the normal control (group III), IGF-1 (ng/mL) was 309.4 ⫾ 72.9, 301.6 ⫾ 67.5, and 232.5 ⫾ 95.3 respectively (p < 0.01), but there was no significant difference between group I and group II in regard to IGF-1, (p ⫽ 0.749). There was significant statistical difference among the study groups in regard to mean IGFBP-3 levels (Table I and Figure 3). The difference appeared when comparing patients with PTE (group I) or patients without PTE (group II) with the normal control (group III), IGFBP-3 (ng/mL) was 10,088.6 ⫾ 1,118.8, 5,123.4 ⫾ 906.8, and 7,851 ⫾ 727.7 respectively (p < 0.01); also this significant difference persisted between group I and group II in regard to IGFBP-3 (p < 0.01). There was no significant statistical difference among the transplant groups (groups I and II) in regard to history of smoking, hepatitis, cytomegalovirus infections, native kidney nephrectomy, age at transplant, duration of pre-transplant dialysis, underlying renal disease, number of pre-transplant transfusions, donor age, donor sex, source of transplantation (living related or unrelated), duration since transplantation, onset and duration of PTE, and average follow-up period (p > 0.05). A total of 22 out of the 28 with PTE were men (78.5%) and the gender specific rate for males was 23.3% compared with the female specific rate of 11.4% (p < 0.01; Figure 4). Also, there was no significant statistical difference among the transplant groups (groups I and II) in regard to incidence of hypertension (13/15 [86.7%], 21/30 [70%], respectively; p > 0.05) and transplant renal artery steno- Post-Transplant Erythrocytosis TABLE I. Comparison of IGF-1 and IGFBP-3 levels among studied groups. Variable IGF-1 (ng/mL) IGFBP-3 (ng/mL) Group I Group II Group III (PTE) (without PTE) (Normal Control) (n ⫽ 15) (n ⫽ 30) (n ⫽ 15) 309.4 ⫾ 72.9 301.6 ⫾ 67.5 232.5 ⫾ 95.3 10,088.6 ⫾ 1,118.8 5,123.4 ⫾ 906.8 7,851 ⫾ 727.7 PTE, post-transplant erythrocytosis sis (1/15 [6.7%], 3/30 [10%], respectively; p > 0.05). In regard to renal function, there was no significant statistical difference among the transplant groups (groups I and II) in regard to graft function (serum creatinine: 1.44 ⫾ 0.36, 1.46 ⫾ 0.33 mg/dL, respectively; p > 0.05), incidence of acute tubular necrosis (2/15 [13.3%], 8/30 [26.7%], respectively; p > 0.05), incidence of acute rejection (6/15 [40%], 16/30 [53.3%], respectively; p > 0.05), or incidence of chronic rejection (1/15 [6.7%], 3/30 [10%], respectively; p > 0.05), but there was significant statistical difference in regard to free rejection course (7/15 [46%], 2/30 [6.7%], respectively; p ⫽ 0.003). Also, there was significant statistical difference among the transplant groups (group I and II) in regard to pre-transplant treatment with recombinant human erythropoietin (7/15 [46.7%], 24/30 [80%], respectively; p ⫽ 0.039). Discussion Erythrocytosis is a common complication of renal transplantation with an incidence of up to 17%. It is associated with an increased risk of complications due to thromboembolic events and has traditionally been treated by intermittent venesection. Recently, inactivation of RAS by an ACE inhibitor, or an ARB represents the most effective, safe, and well-tolerated therapeutic modality.8 In our study, 28 patients out of 120 consecutive kidney transplanted patients who retained a functional graft had a 23.3% incidence of PTE. The only treatment modality used to treat PTE was phlebotomy, and no patient in the study was receiving ACE inhibitors or an ARB. Studying the pathogenesis and the possible risk factors of PTE and its impact on graft function was the aim of many prior studies. Post-transplant erythrocytosis results from the combined trophic effect of multiple and interrelated erythropoietic factors, excess production of erythropoietin (EPO), either by the native or transplanted kidneys, enhanced sensitivity of red cell precursors to EPO, or altered regulation of the hematocrit-EPO feedback system have been proposed to play a role.9 However, erythropoietin levels in most PTE patients still remain within the “normal range” indicating that erythrocytosis finally ensues by the contributory action of additional growth factors on erythroid progenitors.1 But the hematocrit-erythropoietin feedback system was not studied in our study. Insulin-like growth factor-1 has previously been shown to be an important regulator of erythropoiesis in vitro10 and recent studies have revealed the importance of IGF-1 as a regulator of erythropoiesis in vivo.2 One of these studies has reported that polycythemia vera, a clonal disorder of erythrocytosis, represents an abnormality of the IGF-1/BP profile. Earlier studies have suggested that the receptor for IGF1 in mononuclear cells in polycythemia vera was constitutively phosphorylated and hypersensitive11 and Cottea et al12 demonstrated that production of an erythroid colony-forming unit under serum-free conditions was better correlated with the presence of IGF-1 than EPO. Also the same group has reported increased levels of IGFBP-1 in polycythemia vera and their stimulatory effect on in vitro culture of erythroid progenitors. Moreover, IGF1 was identified as the major circulating factor supporting erythropoiesis in anephric dialysis patients with no measurable erythropoietin levels.13 Plasma IGF-1, but not erythropoietin, levels are positively correlated with hematocrit values in uremic subjects and renal transplant recipients14 and all of this data points to an important question, whether IGF-1 has a role in the pathogenesis of erythrocytosis. In this study, the serum levels of IGF-1 was measured in renal transplant recipients with and without erythrocytosis and was compared to the serum levels found in a group of apparently healthy age- and sexmatched controls, We compared the results of renal transplant recipients without erythrocytosis to those with PTE, to determine how the role of renal transplantation might influence these results. Because IGF-1 levels are influenced by the presence of binding proteins, we also measured the levels of IGFBP-3, one of the major regulators of IGF-1 function.15,16 In order to understand the role of IGF-1 and IGFBPs and because of its inactivation of RAS by an ACE inhibitor or ARB, in our present study we chose to study only those patients not receiving these medications, therefore eliminating the confounding influence of drugs used to treat this disorder. The clinical and demographic characteristics were similar in terms of age, gender, and serum creatinine in the erythrocytosis and non-erythrocytosis groups. On comparing mean serum level of IGF-1 in the 3 studied groups, it was found that the mean serum levels of IGF-1 in transplant recipients with erythrocytosis (309.4 ⫾ 72.9) was not significantly different from transplant recipients without erythrocytosis (301.6 ⫾ 67.5). However, the mean serum levels of IGF-1 in both groups were significantly higher when compared to mean levels in the control group (232.5 ⫾ 95.4; p < 0.05). But regarding the mean serum ICFBP-3 level, it was significantly higher in the transplant recipients with erythrocytosis (10,088.6 ⫾ 1,118.8) when compared with its mean level in the transplant recipients without erythrocytosis (5,123.4 ⫾ 906.8; p < 0.05), and when compared with its mean level in the control group (7,851 ⫾ 727.7; p < 0.05). In accordance with these results, Brox et al2 found that patients with PTE, when compared to normal individuals, have a significant increase in the serum level of May 2009 Dialysis & Transplantation 5 Post-Transplant Erythrocytosis 6 Dialysis & Transplantation May 2009 risk factors of PTE. In our study, among the 120 renal recipients, 22 out of the 28 renal recipients who developed erythrocytosis after transplantation were men (78.5%), and male specific rate of 23.3% compared with the female specific rate of 11.4% which point to male gender as 1 of possible predisposing factors for PTE; meanwhile PTE was independent of recipient age, donor race, and type. In accordance with these results, Sumrani et al18 found that PTE occurred more frequently in men (12.8%) than women (1.6%) and Kessler et al19 identified male gender as an important predisposing factor for PTE. Although men comprised 61% of the renal graft recipients in the United States in 1996,20 more than 80% of PTE patients reported in American studies in the mid-1990s were men. In the past, there were several case reports suggesting that erythrocytosis may affect graft function and link post-transplant erythrocytosis to acute or chronic rejection. One of these studies was done by Abbrecht and Greene,21 in which they serially measured serum erythropoietin levels by bioassay in 7 patients before and after transplantation and noted erythropoietin elevation during rejection episodes. Also, in a study done by Swales and Evans,22 they noted that only 2 out of 7 patients with post-transplant erythrocytosis had clinical evidence of rejection. In this present study, post-transplant erythrocytosis did not seem to be a major etiologic factor of allograft rejection as the PTE patients had excellent allograft function and prognosis. On comparing the group that developed the PTE with the transplant control we found 46.7% rejection-free recipients within the PTE group compared with only 6.7% rejectionfree recipients found in the transplant control group (p < 0.05) which may suggests the rejection free course as 1 of the predisposing factors of PTE (odds ratio [OR]: 12.2) The odds of having been a recipient with a history of acute rejection are more than 12 times greater for renal recipients without PTE compared with renal recipients with PTE. In our study, regarding renal artery stenosis as a possible risk factor for PTE, it has occurred with equal frequency in both populations with no significant difference between the renal recipients with PTE and renal recipients without PTE suggesting that it did not play a major etiologic role in the pathogenesis of PTE. In previous studies, renal artery stenosis was one of the suggesting risk factors of PTE because of the renal ischemia that theoretically could increase the erythropoietin production,3 but subsequent studies failed to confirm the association.4 Hypertension, due to increased plasma renin activity, small vessel disease in the allograft, or because of an associated plasma volume contraction, theoretically could lead to erythrocytosis.23 In our study, hypertension is a common post-transplant complication with an incidence rate of 75.6% in the 120 renal recipients and it occurred with very close frequency in both populations suggesting that it does not play a major etiologic role in patients developing elevated hematocrit values. Dagher et al24 has also postulated that erythropoietin production by native kidneys leads to erythrocytosis. In our study we found that native kidneys do not seem to be necessary for the development of erythrocytosis as 3 out of 15 patients in our study had pre-transplant bilateral nephrectomies, which is compatible with reports by Wickre et al4 that stated that an increased venous effluent concentration of erythropoietin in the face of poor renal blood flow accompanying end-stage disease is not indicative of increased absolute production of erythropoietin by the native kidney. In addition, we found that patients requiring erythropoietin treatment with recombinant human erythropoietin prior to transplantation are less likely to develop PTE (OR: 4.5). The odds of having been not treated with recombinant human erythropoietin prior to transplantation are more than 4 times greater for renal recipients with PTE then for renal recipients without PTE. In accordance with these results, Kessler et al19 found that renal dialysis patients with poor endogenous erythropoietic capacity appear “protected” from developing PTE after they had kidney transplantation. Finally, the nature and dose of immunosuppressive agents have varied across studies. Perazella et al25 suggested that PTE is more frequent when graft recipi- œ IGF-1 and IGFBP-3, and was the first to suggest that IGF-1 and its binding proteins may account for this secondary form of erythrocytosis. Previous studies have reported a different mechanism that affects the final IGF-1 pool and its biological effect, 1 of them is the modulation of the IGFBP-3. Baxter and Martin16 agreed that IGFBP-3 was involved in the storage of IGF-1 and prevents IGF-1 degradation in its bound state, which suggests the mechanism of IGFBP-3 increases with the concomitant increase of IGF found in the renal recipients with erythrocytosis. A study made by Drop et al15 found that IGFBP-3 may function as a facilitator between the IGF-1 receptor and its ligand. Concerning our study, we found a significant difference in IGF-1 and IGFBP levels between normal individuals and those with PTE, however, when we compared matched renal transplant recipients without erythrocytosis controls to those with PTE, the levels of IGF-1 was similar in both groups and only the level of IGFBP-3 was significantly higher in those patients with PTE. Because IGF-1 function is tightly regulated by the presence of IGFBP any modification in their circulating levels would be expected to result in an appreciable effect on IGF-1s erythropoietic action. Our results strongly suggest that there is an increased amount of bioavailable IGF-1 as a cause of the erythrocytosis seen after renal transplantation, which again corroborates the accumulating evidence supporting an in vivo role for IGF-1 and IGFBP-3 in human erythropoiesis as confirmed by Brox et al2 and Mirza et al11 and suggests that the IGF-1/BP profile can be reset after kidney transplantation.17 Because erythrocytosis is a relatively common phenomenon following renal transplantation and it is not only associated with special clinical conditions,8 we also studied possible risk factors of erythrocytosis in our renal-transplant patients. There were many previous studies that tried to define the differences between transplant recipients with erythrocytosis and without erythrocytosis including age, sex, duration of transplantation, duration on dialysis, diagnosis of original renal disease, and transplantation medication in an attempt to clarify these possible Post-Transplant Erythrocytosis ents are treated with cyclosporine A than with azathioprine. By contrast, Koziak et al26 failed to demonstrate any differences in hematocrit or erythropoietin levels among subgroups of their general renal transplant population who were chronically and contemporaneously treated with prednisone and azathioprine; prednisone and cyclosporine A; or prednisone, azathioprine, and cyclosporine A. In our study, all of the transplanted patients were treated with the same immunosuppression protocol, so we cannot know the role of the immunosuppression therapy as an etiologic factor for PTE. Although the previously suggested causes of erythrocytosis did not play significant roles in this study such as smoking and diabetes,4 several other risk factors were identified including absence of acute rejection episodes, and to a lesser extent, history of recombinant human erythropoietin prior to transplantation. Interestingly, no other clinical risk factor was correlated significantly with erythrocytosis, which we can explain by the small numbers of patients in the various subgroups that may have prevented differences from reaching statistical significance, or due to the possibility that some suggested clinical risk factors acted synergistically. Conclusion The present study showed that PTE developed with an incidence rate of 23.3%. Posttransplant erythrocytosis occurred more frequently in men than women with a specific rate of 28.2% and 11.4% respectively. The serum of IGF-1 and IGFBP-3 were altered in patients with PTE, the serum IGF-1 was found to be similar in the transplant recipients with and without erythrocytosis; however, the group with erythrocytosis had significantly elevated IGFBP-3. The risk factors for the development of erythrocytosis were found to be the history of rejection free course and renal recipients with less history of erythropoietin treatment with recombinant human erythropoietin prior to transplantation. No other significant differences were found between the groups. In conclusion, we could speculate that post-transplant erythrocytosis represents an anomaly of IGF-1 and its major binding proteins in which they altered erythropoietin homeostatic mechanisms in the denervated transplant kidney. This altered erythropoietin homeostatic mechanisms is superimposed on a setting of multiple risk factors and leads to the state of erythrocytosis that is seen after kidney transplantation. D&T References 1. Demetrios V, Vlahako S, Katerina P, et al. Post transplant erythrocytosis. Kidney Int. 2003;63:11871194. 2. Brox AG, Mangel G, Fafard J, et al. Erythrocytosis after renal transplantation represents an abnormality of IGF-1 and its binding proteins. Transplantation. 1998;66:1053-1058. function. Prog. Growth Factor Res. 1989;1:49. 17. Hussenet F, Dousset B, Gervais P. Serum insulin like growth factor-I and binding proteins concentrations after renal transplantation in adults. Transplant Proc. 1996;28:3615. 18. Sumrani NB, Daskalakis P, Miles AM, et al. Erythrocytosis after renal transplantation: a prospective analysis. ASAIO J. 1993;39:51-55. 19. Kessler M, Hestin D, Mayeux D. Factors predisposing to post-renal transplant erythrocytosis. A prospective matched-pair control study. Clin Nephrol. 1996;45:83-89. 20. Bethesda. US Renal Data System: USRDS National Institute of Diabetes and Digestive and Kidney Diseases,National Institute of Health: 1997. 21. Abbrecht PH, Greene JA. 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