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Lymphocyte PC-1 Activity in Patients on Maintenance Haemodialysis Treated With Human Erythropoietin and 1-[Alpha]-D3

Posted on: Friday, 25 February 2005, 03:00 CST

Abstract

Background Plasma cell differentiation antigen 1 (PC-1) is an inhibitor of insulin-receptor tyrosine-kinase. PC-1 content is elevated in muscle and adipose tissue from insulin-resistant subjects and its elevation correlates with in vivo insulin resistance. It is known that insulin resistance in uraemia may be improved with erythropoietin (EPO) and vitamin D therapy. Therefore, in this study the effects of human recombinant EPO and 1-alpha-D3 treatments on lymphocyte PC-1 expression in patients with end-stage renal failure on haemodialysis (HD) were investigated.

Methods Lymphocyte basal, concanavalin A (Con A), and phorbol-12- myristate-13-acetate (PMA)-stimulated PC-1 activity were investigated in HD patients before and after a two-month treatment with subcutaneous EPO (15 patients, 2000-3000 U thrice weekly) or oral 1-α-D3 (14 patients, 2 g thrice weekly). Twenty-nine patients (16 men and 13 women), aged 22-68 years (497 years), on HD from 13 to 112 months, and 30 healthy controls participated in the study. None was obese and all had normal fasting plasma glucose.

Results A two-month EPO treatment produced a 41% haematocrit increase, with a rise in haemoglobin from 6.510.18 g/dL to 9.690.14 g/dL. Basal lymphocyte PC-1 activity in HD patients was found to be significantly increased (P<0.005) over the level in healthy controls. Treatment of patients with EPO decreased unstimulated lymphocyte PC-1 activity to values significantly lower than before the treatment (P<0.001). Lymphocyte Con A and PMA-stimulated PC-1 activity in patients on HD was found to be slightly increased over the level in healthy controls, but significantly reduced (P<0.005 and 0.05, respectively) after the EPO treatment. A two-month pulse oral 1-α-D3 treatment increased haematocrit by 21% and raised haemoglobin from 7.110.32 g/dL to 8.800.39 g/dL. This treatment normalized serum alkaline phosphatase activity and slightly reduced serum parathyroid hormone concentration. PC-1 in unstimulated and PMA-stimulated lymphocytes was unchanged, but significantly decreased (P<0.05) in Con A-stimulated lymphocytes after 1-α- D3 treatment. Fasting plasma glucose was not changed by the treatment.

Conclusion An increased lymphocyte PC-1 activity over control was found in HD patients. A two-month EPO therapy significantly decreased PC-1 activity to the control values, suggesting that an effect on PC-1 expression could be implicated in the amelioration of insulin resistance in uraemic patients treated with EPO. Treatment with pulse oral 1-α-D3 had an effect only on PC-1 of Con A- transformed lymphocytes of haemodialysed patients and requires further investigation.

Ann Clin Biochem 2005; 42: 55-60

Introduction

Patients with end-stage renal disease suffer from a disorder of carbohydrate metabolism which is manifested mainly as the resistance of peripheral tissues (skeletal muscles and adipocytes) to the hypoglycaemic action of insulin.1 In some patients, hyperinsulinaemia occurs with augmented insulin response to the glucose overload, but without increased fasting glucose values. In other patients, insulin secretion is not adequate to compensate for the diminished uptake of glucose; therefore, the fasting hyperglycacmia and the abnormal glucose tolerance tests are used.1

Although widely explored, the true cause of insulin resistance in uraemic patients is not entirely elucidated yet. As the binding of insulin to its receptors was found to be normal, the resistance to insulin action is defined as a postreceptor's defect.2 Decreased sensitivity to the hypoglycaemic action of insulin is attributed either to anaemia or uraemic toxins or even to secondary hyperparathyroidism.

During the last decade it was found that EPO therapy, used for the correction of anaemia in patients with endstage renal failure, ameliorates insulin resistance. The mechanism of this effect is not understood and different opinions are presented.3-5 Long-term rHuEPO therapy significantly improves glucose metabolism in patients on maintenance haemodialysis (HD), mainly by reduction of insulin resistance. Neither anaemia correction nor a direct effect of rHuEPO on some metabolic steps seems to be responsible for these effects.5

Calcitriol, an active vitamin D metabolite, was found to improve anaemia and secondary hyperparathyroidism and to normalize insulin sensitivity. The mechanisms of the action of calcitriol on glucose intolerance, insulin resistance and insulin secretion in HD patients arc not yet resolved.6,7

The possible implication of alkaline phosphodicstcrase I (EC 3.1.3.1) - plasma cell differentiation antigen 1 (PC-1) in the origin of tissue resistance to insulin action was recently suggested. In insulin-resistant tissues, an increase of PC-I content was found, and a negative correlation between PC-I content and some parameters of tissue insulin sensitivity were demonstrated.8-10 A significant negative correlation between both muscle PC-1 content and in vivo insulin action as measured by the intravenous insulin- tolerance test and the sensitivity of in vitro insulin stimulation of insulin-receptor tyrosine-kinase activity was found.9 PC-1 content in cultured skin Hbroblasts from non-obese, non-diabetic, insulin-resistant subjects was significantly higher compared with cells from insulin-sensitive subjects, correlated with PC-1 content in muscle tissue and negatively correlated with both decreased in vivo insulin sensitivity and decreased in vitro insulin-receptor autophosphorylation.10 Moreover, in the transfection experiments it was shown that the overexpression of PC-1 in transfected cultured cells does not modify insulin binding but reduces the insulin- receptor tyrosine-kinase activity and also some other signs of insulin action.8 In a recent study, the increase of lymphocyte PC-I activity was found in patients with type 2 diabetes, and was abolished after three months of metformin therapy.11

The aim of this work was to explore the PC-1 status of HD patients and the effect of a chronic treatment with EPO or 1-alpha- D3 on lymphocyte PC-1 (alkaline phosphodiesterase activity).

Patients and methods

Patients

Twenty-nine haemodialysed patients (16 men and 13 women) participated in the study divided into two groups: one of 15 patients treated with EPO and the other one of 14 patients treated with 1-α-D3. Their ages ranged from 22-68 years (38 4 years). They had been on HD for 13-112 months and were dialysed thrice weekly for 12 h. None of the patients was obese, their mean body mass index (HMI) was 22.1 2.9 kg/m^sup 2^ (range 20.1-24.7). All patients had normal fasting venous plasma glucose (< 6.1 mmol/L; Table 1).

EPO was given to 15 patients subcutancously at each HD treatment. The epoetin α dose by the subcutaneous route was 98 units/kg/ wcck at the start of treatment, adjusted to 148 units/kg/wcek in the second month of treatment, fron supplements (oral, intravenous or both) were administered to maintain a serum ferritin of 100-500 ng/ mL. No intravenous iron was administered if the ferritin was more than 500 ng/mL.

Oral 1-α-D3 was given to 14 patients, 2 g twice weekly, for two months.

The control group was made up of 30 sex- and agematched volunteers. These individuals had no personal history of or first- degree relative with diabetes (type 1 or type 2), hypertension or dyslipidaemia. They had a normal BMI from 20.4 to 24.9, mean 21.9 3.2, not different from the BMI of HD patients. The kidney function of the control group, measured by creatinine clearance, was within the limits of reference range (from 92.7 to 140.5, mean 118.1 12.2 mL/min).

Laboratory studies

Fasting plasma glucose, calcium, phosphate and alkaline phosphatase activity were determined by standard laboratory methods. Fasting morning insulin was determined by commercial radioimmunoassay from TNEP, Zemun-Belgrade, Serbia. Insulin from the sample and ^sup 125^I-marked insulin are bound with specific anti- insulin antibodies, making immune complexes. The immune complexes are sedimented with a mixture of secondary antibody and polyethylene glycol. Standard samples of insulin are treated as serum samples and used to calculate insulin concentration in samples of the patient's serum. Intact parathyroid hormone (PTH) was determined by ELSA-PTH solid-phase twosite immunoradiometric assay kit from CIS Biointernational, Gif-sur-Yvette, France. Monoclonal antibodies coated on the RLSA solid phase are specific for the mid-region and C- terminal part 39-84 of the molecule. The radiolabelled antibodies are polyclonal antibodies (goat) recognizing the N-terminal part 1- 34 of the molecule. The molecules of PTH are sandwiched between the solid phase and the tracer. The radioactivity bound to the ELSA is proportional to the concentration of PTH present in the sample.

Table 1. Laboratory characteristics of patients on haemodialysis treated with EPO and 1-α.-D3

Isolation and culture of human peripheral blood mononuclear cell

Peripheral blood mononuclear cells (PBMC) were isolated from 10 mL of freshly drawn heparini/ed (50 IIJ/mL) blood before HD, layered over Ficoll-Hypaque (Lymphoprep, Nyegard, Oslo, Norway), washed in RPMI 1640 (Flow Laboratories, Irvine, UK) culture medium containing 25 mmol/L HEPES, 2 mmol/L glutamine,100 U/mL penicillin and 100 g/ ml streptomycin, and resuspended at a concentration of 2 10^sup 6^/ rnl in the same medium supplemented with 10% fetal calf serum (FCS). PBMC were incubated for 48 h at 37C in an atmosphere of 95% air and 5% CO2. Stimulation studies were performed for 48 h by adding 25 g/ mL Concanavalin A (Con A) or 10 ng/mL phorbol-12-myristate-13- acetate (PMA) (Sigma, St Louis, MO, USA) to the incubation medium.

PC-1 of lymphocytes

Basal, Con A-, and PMA-stimulated lymphocyte PC-I activities were determined. Non-adhering 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.12 PC-I (determined as alkaline phosphodiesterase) was measured in 50 mmol/L Tris-HCl buffer, pH 8.0, 130 mmol/L NaCl, 1 mmol/L MgCl^sub 2^, with 3 mmol/L p- nitrophenyl thymidine 5'-phosphate as substrate. Incubation was carried out at 37C for 3-10 min with gentle agitation, under zero- order kinetic conditions. The enzyme reaction was stopped with lmol/ L sodium hydroxide. The p-nitrophenol formed was measured at 405 nm.

Statistical analysis

For statistical analysis, the Student's t-test and nonparametric analysis (Mann-Whitney) were used when appropriate. Correlations between two parameters were estimated by regression analysis. All analyses were performed using SPSS for Windows, version 8 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at P < 0.05.

Results

A two-month EPO treatment produced a 41 % haematocrit increase, with a rise in haemoglobin from 6.51 0.18 to 9.69 0.14 g/dL (Table 1). Prctreatment scrum alkaline phosphatase and PTH were over the normal limits and did not change upon EFO treatment.

Basal lymphocyte PC-I activity in HD patients was found to be significantly increased (P < 0.005) over the level in healthy controls. Treatment of patients with EPO decreased unstimulated lymphocyte PC-I activity to the values signilicantly lower than before the treatment (P < 0.001). Lymphocyte Con A and PMA- stimuIated PC-I activity in patients on HD increased slightly over the level in healthy controls, but significantly reduced (P< 0.005 and 0.05, respectively) after the EPO treatment (Table 2). The conlidcnce intervals are given in Table 4.

The two-month treatment with pulse oral l-alphaD3 significantly improved anaemia in HD patients; haemoglobin increased from 7.110.32 to 8.80 0.39 g/dL, and erythrocytes increased from 2.520.12 to 3.09 0.14 7times; 10^sup 12^/L (P< 0.05; Table 1). Serum alkaline phosphatase decreased significantly from 199 to 109 U/L (P < 0.01); however, the PTH moderately decreased (not statistically significant). Unstimulated and PMA-stimulated lymphocyte PC-I activity after pulse oral 1-α-D3 was not different from the pretreatment concentrations (Tables 3). Oral l-α-D3 treatment produced a significant decrease of Con A-stimulated lymphocyte PC-I activity from .17.5 to 8.73 nmol/min/ 10^sup 6^Ly (P < 0.05). The confidence intervals are given in Table 4.

Table 2. Lymphocyte PC-1 activity in haemodialysis patients treated with EPO

Table 3. Lymphocyte PC-1 activity in HD patients treated with 1- α-D3

Discussion

In this study an increase of PC-I activity in lymphocytes from uraemic patients was found, when compared with healthy controls. This increase was not as great as the increase demonstrated recently in insulinresistant patients with type 2 diabetes." Such an increase of PC-I could be expected in uraemic patients on maintenance HD, as it is known that dialysis treatment attenuates or even eliminates the insulin resistance in uraemia.1314 However, two months of EPO therapy produced a significant decrease of PC-I activity to normal values.

Regarding mechanisms by which the EPO therapy attenuates the insulin resistance in uraemia, divergent opinions were presented; however, the main emphasis was placed on the correction of anaemia by EPO therapy. It was suggested that the increase of insulin sensitivity is obtained by a better local tissue oxygenation, or especially that the correction of anaemia improved the exercise tolerance and so permitted more physical activity.3-5 Physical training increases insulin sensitivity, responsiveness of muscle glucose uptake and glycolytic utilization. The possible role of the eventual correction of malnutrition was also suggested/ In a recent study, insulin resistance improved significantly 10 days after the beginning of EPO treatment, before the rise of haematocrit. There was no further improvement of insulin resistance after the correction of haematocrit level, at least 8 weeks later.15 The results of this study suggest that beneficial effects of EPO treatment on insulin resistance in dialysis patients could be attributed to the EPO itself and not to the correction of anaemia.

Table 4. 95% Confidence Interval of lymphocyte PC-1 activity in HD patients treated with EPO and 1-α-D3

The improvement of anaemia in EfD patients with moderate to severe hyperparathyroidism by pulse oral l-α-D3 was previously demonstrated, but the relationship between improved tissue oxygenation and reduced insulin resistance was not studied.16 Intravenous 12-week vitamin D treatment of HD patients led to a significant reduction of PTH levels and to a complete normalization of insulin sensitivity.6 Since there was a significant reduction of PTH levels upon treatment, the authors could not differentiate between the actions of calcitriol per se and those of PTH reduction. The effect of four-week intravenous calcitriol therapy on insulin metabolism was therefore examined, during which time the PTH concentration did not change, so that the direct actions of calcitriol could be assessed independently. This treatment corrected glucose intolerance, insulin resistance, and hypoinsulinaemia in the absence of PTH suppression/ Treatment of metabolic acidosis in patients on HD increased both insulin sensitivity and insulin secretion in patients with uraemia. This was accompanied by an increase in circulating levels of 1,25(OH)2D3, but no change in those of PTH.17 1,25(OH)2D3 may provide supplementary calcium to the β cell by regulating intracellular signalling processes involving phospholipid metabolism, protein kinase C induction, Ca^sup 2+^ mobilization, and Ca^sup 2+^ entry by Ca^sup 2+^ channels.18 The effect of l-α-D3 treatment upon insulin resistance could be mediated by reducing secondary hyperparathyroidism, by improving anaemia and tissue oxygenetion, or acting per se. How this is related to the expression of PC-1 remains to be elucidated.

Patients in the present study were markedly anaemie and both EPO and 1 -α-D3 treatments corrected haemoglobin concentrations significantly. EPO therapy significantly improves glucose metabolism in maintenance HD patients mainly by reduction of insulin resistance.5 Since neither anaemia correction nor a direct effect of rHuEPO on some metabolic steps seem to be responsible for these effects, further studies are needed on EPO treatment for improvement of insulin resistance in HU patients. Although both EPO and pulse oral l-α-D3 treatments corrected anaemia of these patients signilicantly, only EPO produced a marked decrease in lymphocyte PC- I activity.

Patients in the present study had moderate to severe hyperparathyroidism. Treatment with EPO was without any effect; however, pulse oral l-α-D3 treatment corrected calcium concentration as well as produced a significant reduction in the alakaline phosphatase activity to normal values. Treatment with vitamin D metabolites not only controls secondary hyperparathyroidism but also improves insulin resistance and lipid abnormalities in HD patients/ Intravenous vitamin D treatment leads to a significant reduction of PTH concentrations and to a complete normalization of insulin sensitivity in HD patients.5 Thus, intravenous 1,25 dihydroxycholccalciferol improves insulin resistance in uraemic patients, acting per se or by reducing secondary hyperparathyroidism. Tn our study, treatment with pulse oral 1-α-D3 only had an effect on PC-1 of Con A-transformed lymphocytes of haemodialysed patients. It is possible that immune deficiency of uraemia changes the non-genomic effects of vitamin U. CaIcitriol plays a significant role in the regulation of both the monocyte and neutrophil functions in HU patients; however, the mitogen responses to Con A and PWM by lymphocytes were not significantly affected by this treatment.5

Further studies are needed to understand insulin resistance in uraemia and the effects of EPO and vitamin D treatments, as well as the role of PC-I in insulin resistance.

References

1 De Fronzo RA, Smith D, Alvestrand A. Insulin action in uraemia. Kidney Int 1983; 6(Suppl 24): S102-14

2 Smith D, DeFronzo RA. Insulin resistance in uraemia mediated by post-binding defects. Kidney lnt 1982; 22: 54-62

3 Borissova AM, Djambazova A, Todorov K, Dakovska L, Tankova T, Kirilov G. Effect of erythropoietin on the metabolic state and peripheral insulin sensitivity in diabetic patients on haemodialysis. Nephrol Dial Transplant 1993; 8: 93-5

4 Mak RHK. Correction of anaemia by erythropoietin reverses insulin resistance and hyperinsulinemia in uraemia. Am J Physiol 1996; 270: F839-S4

5 Allegra V, Mengozzi G, Martimbianco L, Vasile A. Early and late effects of erythropoietin on glucose metabolism in maintenance haemodialysis patients. Am J Nephrol 1996; 16: 304-8

6 Kautzky-Willer A, Pacini G, Barnas U, et al. Intavenous calcitriol normalizes insulin sensitivity in uraemic patients. Kidney lnt 1995; 47: 200-6

7 Mak RHK. 1,25-Dihydroxyvitamin D3 corrects insulin and lipid abnormalities in uraemia. Kidney lnt 1998; 53: 1353-7

8 Maddux BA, Sbraccia P, Kumakura S, ef a/. Membrane glycoprotein PC-1 and insulin resistance in non-insulin-dependent diabetes mellitus. Nature 1995; 373: \148-51

9 Frittitta L, Youngren J, Vigneri R, Maddux BA, Trischitta V, Goldfine ID. PC-1 content in skeletal muscle of non-obese, nondiabetic subjects: relationship to insulin receptor tyrosine kinase and whole body insulin sensitivity. Diabetologia 1996; 39: 1190-5

10 Frittitta L, Spampinato D, Solini A, et al. Elevated PC-1 content in cultured skin fibroblasts correlates with decreased in vivo and in vitro insulin action in non-diabetic subjects. Evidence that PC-1 may be an intrinsic factor in impaired insulin receptor signaling. D/aoefes1998; 47: 1095-100

11 Stefanovic V, Antic S, Mitic-Zlatkovic M, Vlahovic P. Reversal of increased lymphocyte PC-1 activity in patients with type 2 diabetes treated with metformin. Diabetes Metab Res Rev 1999; 15: 4OCM

12 Stefanovic V, Golubovic E, Vlahovic P, Mitic-Zlatkovic M. Lymphocyte ectoenzymes in childhood idiopathic nephrotic syndrome. Pediatr Nephrol 1998; 12: 755-60

13 Foss MC, Gouveia LM, Moyses Neto M, Paccola GM, Piccinato CE. Effect of haemodialysis on peripheral glucose metabolism of patients with chronic renal failure. Nephron 1996; 73: 48-53

14 Kobayashi S, Maejima S, lkeda T, Nagase M. Impact of dialysis therapy on insulin resistance in end-stage renal disease: comparison of haemodialysis and continuous ambulatory peritoneal dialysis. Nephrol Dial Transplant 2000; 15: 65-70

15 Spaia S, Pangalos M, Askepidis N, et al. Effect of short-term rHuEPO treatment on insulin resistance in haemodialysis patients. Nephron 2000; 84: 320-5

16 Djordjevic V, Radivojevic J, Stefanovic V. Improvement of anaemia in haemodialysis patients after pulse oral 1-α-D3 treatment. Clin Nephrol 2002; 57: 487-8

17 Mak RHK. Effect of metabolic acidosis on insulin action and secretion in uraemia. Kidney lnt 1998; 54: 603-7

18 Billaudel BJ, Bourlon PM, Sutter BC, Faure-Dussert AG. Regulatory efect of 1,25-dihydroxyvitamin D3 on insulin release and calcium handling via the phospholipid patway inislets from vitamin D- deficient rats. J Endocrinol Invest 1995; 18: 673-82

19 Tokuda N, Kano M, Meiri H, Nomoto K, Naito S. Calcitriol therapy modulates the cellular immune responses in haemodialysis patients. Am J Nephrol 2000; 20: 129-37

Accepted for publication 11 November 2004

Addresses

Institute of Nephrology and Haemodialysis, Faculty of Medicine, 18000 Nis, Serbia

Correspondence:

Professor Vladisav Stefanovic

E-mail: stefa@ni.ac.yu

Copyright Royal Society of Medicine Press Ltd. Jan 2005

Source: Annals of Clinical Biochemistry

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