Lymphocyte 5′-Nucleotidase and Aminopeptidase N Activity in Patients on Maintenance Hemodialysis Treated with Human Recombinant Erythropoietin and 1-Alpha-D3

Abstract

Background: Patients with end-stage renal disease (ESKD) present an immunodeficiency state paradoxically exacerbated by hemodialysis (HD) and associated with signs of T-cell activation. B cells are also activated in uremia, and this activation could be altered by erythropoietin therapy in HD patients. In this study, the effects of human recombinant erythropoietin (rHu-EPO) and 1-alpha-D₃ treatments on lymphocyte immunomodulatory enzymes, aminopeptidase N (APN), and 5′-nucleotidase activity in patients on HD were investigated in hemodialysis patients before and after two-month treatment with s.c. rHu-EPO (15 patients, 2000–3000 U three times weekly) or oral 1-alpha-D₃ (14 patients, 2 µg three times weekly). Results: A two-month EPO treatment of 15 HD patients produced a rise in hemoglobin from 6.51 ± 0.18 to 9.69 ± 0.14 g/dL. Basal lymphocyte APN activity in HD patients was not significantly different from the level in healthy controls. Treatment of patients with rHu-EPO increased unstimulated lymphocyte APN activity to values significantly higher than those before treatment (p < 0.05). A two-month pulse oral 1-alpha-D3 treatment of 14 HD patients increased hematocrit by 21% and raised hemoglobin from 7.11 ± 0.32 to 8.80 ± 0.39 g/dL. Unstimulated and Con A-stimulated lymphocyte APN activity after pulse oral 1-alpha-D₃ was significantly increased (p < 0.01 and p < 0.05, respectively) from the pretreatment levels. In HD patients lymphocyte basal, Con A-, and PMA-stimulated 5′-nucleotidase activity was significantly higher (p < 0.05) than it was in healthy controls. The two-month treatment with rHu-EPO or pulse oral 1-alpha D₃ did not change the level of lymphocyte 5′-nucleotidase in these patients. Conclusions: This study demonstrated that a two-month treatment of HD patients with rHu-EPO or pulse oral 1-alpha D₃ significantly increases activity of lymphocyte APN, important for cleavage of peptides and small proteins, which accumulate in the blood of ESKD patients. In HD patients lymphocyte ecto-5′-nucleotidase activity was significantly higher than that in healthy controls and was not changed after a two-month treatment with rHu-EPO or pulse oral 1-alpha D₃. We speculate that oxidative stress activates 5′-nucleotidase and production of adenosine by lymphocytes of HD patients.

• end-stage kidney disease
• hemodialysis
• anemia
• erythropoietin
• 1-Alpha D₃
• 5′-Nucleotidase
• aminopeptidase N
• lymphocytes

INTRODUCTION

Patients with end-stage kidney disease (ESKD) present an immunodeficiency state paradoxically exacerbated by hemodialysis (HD) and associated with signs of T cell activation.[1&2] Functional and phenotypic T- and B-cell abnormalities are observed in an early stage in the course of chronic renal failure, worsen with the progression of uremia, and are exacerbated by the dialysis procedure. However, the intrinsic function of T- and B-cells was normal when they are provided with normal signaling from antigen-presenting cells.[3] The progression of uremia is associated with a gradual increase in activation markers of T cells (soluble CD25), B cells (CD23), and monocytes (neopterin).[1] Serum soluble CD23 levels were significantly elevated in hemodialysis patients, and were significantly reduced by a four-month treatment with recombinant human erythropoietin (rHu-EPO).[4]

Lymphocyte 5′-nucleotidase and aminopeptidase N (APN) are immunomodulatory enzymes with an effect on adenosine production and splitting of small peptides with metabolic and endocrine functions, respectively.[5&6]

The aim of this work was to explore lymphocyte ectoenzymes of hemodialysis patients and the effect of achronic treatment with rHu-EPO or 1-alpha-D3 on lymphocyte 5′-nucleotidase and APN.

PATIENTS AND METHODS

Patients

Twenty-nine hemodialyzed patients (16 males and 13 females) participated in the study. Their ages ranged from 22–68 years (38 ± 4). They had been on hemodialysis for 13–112 months and were dialyzed three times weekly for 12 hours, on single-use hemophane and polysulfone dialyzers. None was obese (body weight < 120% of ideal body weight). All patients had normal fasting venous plasma glucose ( < 6.1 mmol/L) (Table 1). rHu-EPO (Eprex^R) was given to 15 patients s.c. at each hemodialysis treatment, 2000 U during the first month, 3000 U during the second month of treatment. Oral 1-alpha-D3 was given to 14 patients, 2 µg, thrice weekly, for two months. The control group was made up of 16 sex and age matched volunteers. Informed consent was obtained before patients were included in the study.

Table 1. Characteristics of Patients on Hemodialysis Treated with rHu-EPO and 1-Alpha-D3.

Characteristic	Before EPO	After EPO	Before 1-alpha-D3	After 1-alpha-D3
Age (years)	38.3 ± 4.0 (27–68)		39.6 ± 3.8 (22–64)
Duration of HD (months)	49.4 ± 6.8 (13–72)		72.1 ± 9.9 (52–112)
Weight (kg)	68.7 ± 4.7		61.3 ± 5.1
Erythrocytes ( × 10^12/L)	2.33 ± 0.06	3.17 ± 0.05a	2.52 ± 0.12	3.09 ± 0.14b
Hematocrit (%)	22.0 ± 0.43	31.1 ± 0.59a	24.0 ± 1.0	29. 0 ± 1.2b
Hemoglobin (g/dL)	6.51 ± 0.18	9.69 ± 0.14a	7.11 ± 0.32	8.80 ± 0.39b
Glucose (mmol/L)	5.07 ± 0.26	5.19 ± 0.37	5.01 ± 0.21	5.10 ± 0.29
Creatinine (µmol/L)	972 ± 58	990 ± 55	998 ± 62	926 ± 56
Urea (mmol/L)	25.7 ± 2.2	27.1 ± 3.3	26.2 ± 1.5	24.2 ± 1.2
Calcium (mmol/L)	2.25 ± 0.07	2.27 ± 0.06	2.29 ± 0.05	2.48 ± 0.05b
Alkaline phosphatase	188 (61–545)	193 (66–552)	196 (65–571)	102 (51–422)b

Values are means ± SE, median for alkaline phosphatase, range in parentheses.

^avs. Before EPO p < 0.001.

^bvs. Before 1-alpha-D3 p < 0.05.

Isolation and Culture of Human PBMC

Peripheral blood mononuclear cells (PBMC) were isolated from 10 mL of freshly drawn heparinized (50 IU/mL) blood, layered over Ficoll-Hypaque (Lymphoprep, Nyegard, Oslo, Norway), washed in RPMI 1640 (Flow Laboratories, Irvine, UK) culture medium containing 25 mM HEPES, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL), and resuspended at aconcentration of 2 × 10⁶ /mL in the same medium supplemented with 10% fetal calf serum (FCS). The PBMC were incubated for 48 h at 37°C in an atmosphere of 95% air and 5% CO₂. Stimulation studies were performed for 48 h by adding Concanavalin A (Con A) or phorbol-12-myristate-13-acetate (PMA) (Sigma, St Louis, MO, USA), 25 µg/mL or 10 ng/mL, respectively, to the incubation medium.

Ectoenzymes of Lymphocytes

Basal, Con A-, and PMA-stimulated lymphocyte enzyme activities were determined. Nonadhering cells from the culture plates were transferred to centrifuge tubes after appropriate washing with saline, substrate was added, and enzyme activity determined as previously described.[5&6] The following substrates were used: adenosine-5′-monophosphate (5′-AMP) for 5′-nucleotidase, and alanine p-nitroaniline for Aminopeptidase N (APN).

The APN was measured in calcium-free phosphate-buffered saline containing 130 mM NaCl, 1 mM MgCl₂, 9 mM Na₂HPO₄, 9 mM NaH₂PO₄, 1.5 mM KH₂PO₄, 3 mM KCl, and 3 mM alanine p-nitroanilid, at pH 7.4. Incubation was carried out at 37°C for 5–20 min with gentle agitation, under zero-order kinetic conditions. The enzyme reaction was stopped with 0.1 mL of 10% (w/v) trichloroacetic acid, and the amount of p-nitroaniline formed was measured at 405 nm.

5-Nucleotidase activity was determined in 30 mM Tris-HCI buffer, pH 7.4, 130 mM NaCl, and 5 mM MgCl₂, by addition of 3 mM 5′-AMP as substrate. Incubation was performed at 37°C for 10–20 min with gentle agitation, under zero-order kinetic conditions. The enzyme reaction was stopped with 0.1 mL of 10% (w/v) trichloroacetic acid and the inorganic phosphate liberated determined in the supernatant.

Statistical Analysis

For statistical analysis Student's t-test and non-parametric analysis (Mann-Whitney) were used when appropriate. Differences were considered significant at p < 0.05.

RESULTS

A two-month rHu-EPO treatment produced a 41% hematocrit increase, with a rise in hemoglobin from 6.51 ± 0.18 to 9.69 ± 0.14 g/dL (Table 1). Basal lymphocyte APN activity in hemodialysis patients was not significantly different from the level in healthy controls. Treatment of patients with rHu-EPO increased unstimulated lymphocyte APN activity to values significantly higher than those before the treatment (p < 0.05). Lymphocyte Con-A and PMA-stimulated APN activity in patients on hemodialysis did not differ from the level in healthy controls, and did not change after the rHu-EPO treatment (Table 2).

Table 2. Lymphocyte APN Activity in Hemodialysis Patients Treated with rHu-EPO.

Group	n	APN activity (nmol/min/10^6 Ly)
		Unstimulated	Con A-stimulated	PMA-stimulated
Control	16	91.3 ± 8.4	53.5 ± 6.3	23.4 ± 2.9
		90.5 (35.1–160.0)	58.3 (8.8–84.1)	24.5 (7.4–42.4)
HD-before EPO	15	112.9 ± 12.1	53.8 ± 9.3	18.1 ± 2.2
		113.1 (64.7–167.1)	57. 7 (20.7–89.1)	18.3 (9.3–26.1)
HD-after EPO	15	196.0 ± 24.4	82.1 ± 15.6	22.1 ± 2.5
		188.4 (111.0–296.7)a,b	66.9 (43.0–159.8)	21.1 (11.8–29.9)

Values are means ± SE, medians, range in parentheses.

^avs. Control p < 0.001.

^bvs. HD-before EPO p < 0.05.

The two-month treatment with pulse oral 1-alpha D3 significantly improved anemia in hemodialysis patients; hemoglobin increased from 7.11 ± 0.32 to 8.80 ± 0.39 g/dL, and erythrocytes increased from 2.52 ± 0.12 to 3.09 ± 0.14 × 10¹²/L (p < 0.05) (Table 1). Serum alkaline phosphatase decreased significantly from 199 to 109 U/L (p < 0.01); however, immunoreactive parathyroid hormone (PTH) was moderately decreased, without statistical significance (data not presented). Unstimulated and Con A-stimulated lymphocyte APN activity after pulse oral 1-alpha-D3 was significantly increased (p < 0.01 and p < 0.05, respectively) from the pretreatment levels (Table 3).

Table 3. Lymphocyte APN Activity in Hemodialysis Patients Treated with 1α-D₃.

Group	n	APN activity (nmol/min/10^6 Ly)
		Unstimulated	Con A-stimulated	PMA-stimulated
Control	16	91.3 ± 9.4	53.5 ± 6.3	23.4 ± 2.9
		90.5 (35.1–160.0)	58.3 (8.8–84.1)	24.5 (7.4–42.4)
HD-Before 1α-D3	14	112.9 ± 13.0	57.3 ± 8.2	28.6 ± 5.3
		112.5 (39.6–208.3)	47.7 (26.4–110.3)	23.0 (11.0–77.3)
HD-After 1α-D3	14	183.3 ± 18.3	93.8 ± 12.6	26.5 ± 2.7
		174.6 (87.3–265.5)a	82.9 (54.3–173.0)b	26.6 (14.5–41.0)

Values are means ± SE, medians, range in parentheses.

^avs. HD-before 1α-D₃ p < 0.01.

^bvs. HD-after 1α-D₃ p < 0.05.

In hemodialysis patients lymphocyte basal, Con A-, and PMA-stimulated 5′-nucleotidase activity was significantly higher (p < 0.05) than found in healthy controls. The two-month treatment with rHu-EPO did not change the level of lymphocyte 5′-nucleotidase in these patients (Table 4). Similarly, the two-month treatment with pulse oral 1-alpha D3 did not change the level of lymphocyte 5′-nucleotidase in hemodialysis patients (Table 5).

Table 4. Lymphocyte 5′-Nucleotidase Activity in Hemodialysis Patients Treated With rHu-EPO.

Group	n	5′-Nucleatidase activity (nmol/min/10^6 Ly)
		Unstimulated	Con A-stimulated	PMA-stimulated
Control	16	0.95 ± 0.07	0.91 ± 0.04	1.30 ± 0.09
		0.94 (0.78–1.20)	0.92 (0.78–1.02)	1.36 (0.95–1.46)
HD-before EPO	15	1.48 ± 0.13	1.39 ± 0.14	1.71 ± 0.14
		1.57 (0.74–1.85)a	1.44 (0.65–1.86)a	1.79 (0.93–2.09)a
HD-after EPO	15	1.66 ± 0.06	1.50 ± 0.10	1.44 ± 0.11
		1.74 (1.44–1.80)b	1.49 (1.21–1.88)b	1.40 (1.07–1.88)

Values are means ± SE, medians, range in parentheses.

^avs. Control p < 0.05.

^bvs. Control p < 0.005.

Table 5. Lymphocyte 5′-Nucleotidase Activity in Hemodialysis Patients Treated with 1α-D₃.

Group	n	5′-Nucleatidase activity (nmol/min/10^6 Ly)
		Unstimulated	Con A-stimulated	PMA-stimulated
Control	16	0.95 ± 0.07	0.91 ± 0.04	1.30 ± 0.09
		0.94 (0.78–1.20)	0.92 (0.78–1.02)	1.36 (0.95–1.46)
HD-before 1α-D3	14	1.45 ± 0.12	1.28 ± 0.12	1.51 ± 0.12
		1.50 (0.77–1.81)a	1.39 (0.81–1.79)a	1.75 (0.91–2.04)a
HD-after 1α-D3	14	1.53 ± 0.09	1.48 ± 0.05	1.78 ± 0.10
		1.44 (1.23–2.09)a	1.48 (1.26–1.79)a	1.70 (1.28–2.22)a

Values are means ± SE, medians, range in parentheses.

^avs. Control p < 0.05.

DISCUSSION

Lymphocyte ecto-APN is important for cleavage of peptides and small proteins with important biological functions, which accumulate in the blood of ESKD patients. In this study it was demonstrated that two-month treatment of hemodialysis patients with rHu-EPO or pulse oral 1-alpha D₃ significantly increases activity of lymphocyte APN. The effect of 1-alpha-D3 treatment upon APN activity could be mediated by reducing secondary hyperparathyroidism, by improving anemia and tissue oxygenation, or acting per se.[7] How this is related to the APN expression remains to be elucidated. Regarding mechanisms by which the EPO therapy increases lymphocyte APN expression, the correction of anemia by EPO therapy and a better local oxygenation could play the major role; however, this effect could be attributed to the EPO itself and not to the correction of anemia.[8] Aminopeptidase N is an integral membrane protein, which cleaves biologically active oligopeptide with N-terminal preferentially neutral amino acid. The APN was detected primarily on the brush border membranes of kidney and small intestine, and different cell types including lymphocytes. This enzyme has broad substrate specificity cleaving peptides such as enkephalin, endorphin, somatostatin, thymopentin, and f-Met-Leu-Phe.[9]

In hemodialysis patients lymphocyte 5′-nucleotidase activity was found to be significantly higher than that in healthy controls. The two-month treatment with rHu-EPO and pulse oral 1-alpha D3 did not change the level oflymphocyte 5′-nucleotidase in these patients. The increased 5′-nucleotidase activity could be related to the increased oxidative stress in patients on hemodialysis.[10] Lymphocyte 5′-nucleotidase is sensitive to superoxide anion, and is an indicator of oxidative stress in humans.[11]

There is a high prevalence of inflammation and oxidative stress in the maintenance hemodialysis population.[12] It appears that the patients undergoing peritoneal dialysis are affected less than are patients with hemodialysis. The continuous peritoneal dialysis (CAPD) patients have raised malondealdehyde (MDA) levels, but a lower level than do hemodialysis patients.[13] Three possible causes of oxidant stress have been suggested: the uremic state, the dialyzer membrane, and bacterial contaminants from the dialysate.[14] Oxidant stress occurs in uremia before dialysis therapy is initiated, as evidenced by increased production of reactive oxygen species, increased levels of oxidized plasma proteins and lipids, and decreased antioxidant defenses. It has been proposed that increased production of reactive oxygen species during hemodialysis is also an important contributor to oxidant stress. Hemodialysis is associated with a transient increase in production of reactive oxygen species, particularly with cellulose membranes. Neutrophil oxygen radical production normalizes during high-flux dialysis, despite a transient increase early in dialysis. This decrease in oxygen radical production is associated with an improvement in some, but not all, measures of protein oxidation.[15] Hemodialysis continued for more than 2 years aggravates oxidant stress. Decreased potential for oxygen-radical-scavenger activity becomes pronounced after 7 years of hemodialysis treatment.[16] In addition, surveys have shown widespread contamination of dialysate by endotoxin, which may cross membranes and prime production of reactive oxygen species by phagocytic cells. Even a dialysate containing 50 EU/L or less endotoxin may stimulate monocytes and cause oxidative stress; even low levels of endotoxin may aggravate arteriosclerosis in dialysis patients. Thus, in order to prevent cardiovascular events in dialysis patients, it is necessary to purify the dialysate.[17] Taken together, these data suggest that uremia, per se, is the most important cause of oxidant stress in hemodialysis patients. Dialysate quality may also contribute to oxidant stress, but evidence that the dialyzer membrane plays a role is weak.

Patients with end-stage renal failure have increased oxidative stress and show elevated cardiovascular mortality. Whether increased cardiovascular events can be prevented by the administration of antioxidants is still unknown. The effects of acetylcysteine, a thiol-containing antioxidant, on cardiovascular events in patients undergoing hemodialysis were evaluated in a prospective, randomized, placebo-controlled trial. In hemodialysis patients, treatment with acetylcysteine (600 mg BID) reduces composite cardiovascular end points.[18] The EPO influences plasma antioxidants and, to an extent, lipid peroxidation processes. However, even in patients treated with low rHuEPO doses, red blood cell survival close to normal and sufficient correction of anemia is achieved only when appropriate alpha-tocopherol levels are reached.[19] Vitamin E-bonded hemodialyzer is known to improve oxidative stress in patients with hemodialysis, and it is useful for improving atherosclerosis.[20] Kidney transplantation seems to restore a nearly normal level of glycoxidative stress markers, but a complete remission is only possible when the renal function is normal.[21]

During the last decade it was found that rHu-EPO therapy, used for correction of anemia in patients with end stage renal failure, ameliorates insulin resistance, corrects lipid abnormalities, and reduces oxidative stress.[22&23] rHu-EPO therapy activates vascular endothelium in patients receiving maintenance hemodialysis, and this effect may influence cardiovascular risk.[24] Increased lymphocyte ecto-5′-nucleotidase activity in hemodialysis patients, found in the present study, and not affected by rHuEPO treatment, is in support of that risk.

ACKNOWLEDGMENT

This work was supported by a grant No. 1724 from the Ministry for Science, Technology and Development of Serbia.