Influence of secondary hyperparathyroidism in management of anemia in patients on regular hemodialysis

Background/Aim. Anemia is a common complication in hemodialysis patients. Treatment of anemia is affected by iron deficiency, insufficient dose of erythropoietin, microinflammation, vitamin D deficiency, increased intact parathyroid hormone concentration and inadequate hemodialysis. The aim of the study was to determine the prevalence of vitamin D deficiency and its impact on hemoglobin con-cen-tration, iron status, microinflammation, malnutrition, dialysis adequacy and erythropoietin dose in patients on regular hemodialysis. Methods. The study involved 120 patients divided into three groups: severely deficient of vitamin D: 25-hydroxyvitamin D  25(OH)D   10 ng/mL; deficient – 25(OH)D within range of 10–20 ng/mL, and insufficient – 25(OH)D  20 ng/mL. For statistical analysis Kolmogorov-Smirnov test, the single-factor parametric analysis of variance – ANOVA and Kruskal-Wallis test were used. Results. The prevalence of vitamin D deficiency in patients on regular hemodialysis was 75.83%, while the prevalence of severe vitamin D deficiency was 24.7%. Patients with severe vitamin D deficiency had lower blood concentration of hemoglobin, hematocrit, serum concentration of total proteins and albumin, and dialysis indices were also lower compared to the other two groups of patients. The level of C-reactive protein was significantly higher in the group of patients with severe vitamin D deficiency than in the two rest groups. Conclusion. Hemodialysis patients with severe vitamin D deficiency have lower hemoglobin, lower dialysis adequacy, significant microinflammation, malnutrition, bone meta-bo-lism disorders and need higher dose of erythropoietin than patients whose vitamin D was higher than 10 ng/mL. Vitamin D is important risk factor for development of anemia in hemodialysis patients and important factor that can affect treatment of


Introduction
Chronic kidney disease (CKD) is a progressive disease and loss of renal function is followed by various complications of which anemia, secondary hyperparathyroidism (SHPTH) and cardiovascular diseases are the most important 1,2 . Ninety percent of patients starting with hemodialysis are diagnosed with anemia caused by lack of endogen erythropoietin (EPO) which stimulates erythropoiesis in the bone marrow 3 . Among other causes of anemia the most important is hemorrhage 3 . Screening for anemia should be performed when gromelular filtration rate (GFR) is less than 60 mL/min/1.73 m 2 . Measured parameters include hemoglobin concentration (Hb), hematocrit (Hct), red blood cell indices, serum iron (Fe 2+ ) and ferritin concentration (FER), transferrin saturation (TSAT) and serum concentration of C-reactive protein (CRP) [1][2][3] .
SHPTH is a common and significant complication in hemodialysis patients. The lack of vitamin D, the reduced production of the active metabolite of vitamin D [1,25(OH)D], hypocalcaemia and hyperphosphatemia are the main causes of the development of SHPTH in these patients 22 .The risk factors that reduce the production of vitamin D in hemodialysis patients include: reduced vitamin D synthesis in the skin, reduced filtration and reabsorption of vitamin D in epithelial cells of proximal renal tubules, reduced intake of vitamin D-rich foods, reduced absorption of vitamin D from the gastrointestinal tract 22 . The main clinical consequences of vitamin D deficiency are: development of SHPTH, reduced bone density and increased risk of fractures, reduced iron availability for Hb synthesis in erythrocytes ("functional" iron deficiency), reduced response to EPO, atherosclerosis, hypertrophy of the left heart ventricle, vascular calcification, cognitive impairment, progressive loss of residual renal function and increased mortality rate 22 . The potential mechanisms of the impact of vitamin D deficiency on the development of anemia include increased production of proinflammatory mediators in the cells of the immune system: interleukin (IL)-1, IL-6, interferon-, tumor necrosis factor alpha (TNFα). Proinflammatory mediators block proliferation and differentiation of erythrocyte precursor cells in the bone marrow, and IL-6 stimulates formation of hepcidin in liver, which has been proved to cause a "functional" iron deficiency 22,23 . SHPTH causes anemia through both direct and indirect ways. The direct effects of intact parathyroid hormone (iPTH) include: blocking the formation of endogenous EPO, blocking the proliferation and differentiation of erythroid progenitors (EP) and the shortened erythrocyte life span. Indirect effects of PTH have been mainly based on the inducing revival of the bone marrow (the loss of erythrocyte precursor cells) 22,23 .
The aim of this study was to determine the prevalence of anemia, disorders of the metabolism of minerals, vitamin D and PTH, as well as to examine the effect of vitamin D deficiency and enhanced secretion of iPTH on the blood concentration of Hb, the status of iron, microinflammation, malnutrition, the adequacy of hemodialysis and EPO dose in patients on regular hemodialysis.

Methods
This study included patients of the Center for Nephrology and Dialysis, Clinic for Urology, Nephrology and Dialysis, Clinical Center Kragujevac, Kragujevac, Serbia. The study was in compliance with the principles of the Declaration of Helsinki and was approved by The Ethics Comitee of the Clinical Center Kragujevac. All patients involved in the study signed informed consent prior to enrollment. All examined patients were treated using bicarbonate hemodialysis 12 h per week for period longer than three months on hemodialysis machines, the type Fresenius and the type Gambro. Ultrapure dialysis fluid and high-flux as well as low-flux polysulfone dialysis membranes were used. Patients with active proved infections were not included in the study.
In order to evaluate impact of SHPTH in management of anemia in hemodialysis patients the following parameters were measured: Hb, Hct, FER, total iron binding capacity (TIBC), unsaturate iron binding capacity (UIBC), TSAT, calcium (Ca 2+ ), inorganic phosphate (PO 4 3-), alkaline phosphatase (ALP), vitamin D and iPTH. Parameters of hemodialysis adequacy were also considered. Serum samples from patients were collected prior to hemodialysis and prior to heparin administration. Every laboratory parameter was assigned with the value that was the mean of two measuring in two succesive months.
Total Hb was measured using colorimetric method. The target Hb level in patients on dialysis was 100-120 g/L.
Nutritional status of the patients was assessed by measuring total protein (TP) and albumin (Alb) concentrations in the serum, as well as by calculating the body mass index (BMI) and normalized protein catabolic rate (nPCR).
Serum iron was determined by photometric method using TPTZ [2,4,6-Tri-(2-pyridyl)-5-triazine] as the chromogen. Serum iron reference range is 6.6-26.0 μmol/L. TIBC was done indirectly by the Unsaturated Iron Binding Capacity (UIBC) method. TIBC reference range is 48-56 μmol/L. TSAT was calculated using formula TSAT = Fe/TIBC × 100%. Reference range for TSAT in hemodialysis patients is 20-40%. UIBC was measured using spectrophotometric method. Reference range for UIBC is 28-54 μmol/L. A method for FER was turbidimetric one. FER reference range in the patients underwent regular hemodialysis is 100-500 pg/mL. CRP level in the serum was determined by the turbidimetric method. Normal CRP level in the serum is ≤ 5 mg/L. Microinflammation is defined as level of CRP in the serum higher than 5 mg/L. Ca 2+ concentration in the serum was determined by a photometric test. Normal Ca 2+ level in the serum is 2.20-2.65 mmol/L. PO 4 3in the serum was determined by a photometric test. The normal PO 4 3in the serum is 0.80-1.60 mmol/L. Level of vitamin D in the serum was determined by a method of electrochemiluminescence, on the Cobase 411 analyser. Normal level of vitamin D in the serum is 20-40 ng/mL. In hemodialysis patients, normal vitamin D level is ≥ 30 ng/mL (30-80 ng/mL). A severe deficit is defined as the level of vitamin D < 10 ng/mL, vitamin D deficiency exists if level is 10-20 ng/mL, and the insufficiency is defined as the level of vitamin D in the serum of 20-30 ng/mL. The level of iPTH in serum was determined by an immunoradiometric method (IRMA), on the gamma counter WALLAC WIZARD 1470. Normal level of iPTH in the serum is 11.8-64.5 pg/mL. In patients with hemodialysis the upper normal limit is 500 pg/mL.
The adequacy of hemodialysis was assessed on the basis of the single-pool index of adequacy of hemodialysis (Kt/Vsp) calculated according to the Daugridas second-generation formula: spKt/V = -ln(C 2 /C 1 -0.008 × T) + (4-3.5 × C 2 /C 1 ) × UF/W, with: C1 -the value of urea before dialysis, C2 -the value of urea after dialysis (mmol/L), T -duration of hemodialysis (h), UF -interdialysis yield (l), W -BW after hemodialysis (kg). According to K/DOQI guidelines, hemodialysis is adequate if single-pool adequacy of hemodialysis index (spKt/V)  1.2. The degree of reducing urea (URR) index was calculated using following formula: URR = (1-R) × 100%, where: R is the ratio of urea concentration in the serum after and before the hemodialysis treatment. Hemodialysis is adequate if the URR index = 65%-70%.
Depending on the level of vitamin D [25(OH)D]in the serum the patients were divided into three groups. The first group involved the patients with the level of 25(OH)D lower than 10 ng/mL, the second group constituted patients with 25(OH)D levels within range of 10-20ng/mL, while the third group constituted of the patients with 25(OH)D levels higher than 20 ng/mL. Depending on the serum level of iPTH, the patients were divided into three groups. The first group consisted of the patients who had the serum level of iPTH lower than 500 pg/mL, the second group constituted patients with iPTH levels within range 150-500 pg/mL, while the third group constituted of the patients with the iPTH levels higher than 500 pg/mL.
The statistical analysis was performed using the Kolmogorov-Smirnov test, the single-factor parametric analysis of variance (ANOVA) and Kruskal-Wallis test. The threshold of significance was the probability of 0.05 and 0.01.

Results
The cross-sectional study was conducted at the the Clinic for Urology, Nephrology and Dialysis, Clinical Center Kragujevac, including the patients treated with regular hemodialysis in a period longer than three months. We examined 120 patients (75 men and 45 women), average age being 63.15 ± 10.39 years, the average length of treatment with hemodialysis 6.18 ± 5.95 years, and the average spKt/V 1.01 ± 12.27. General patient data are shown in Table 1. Patients were treated with short-acting and long-acting EPO with a parenteral iron composition, and their average monthly doses are shown in Table 2.
The average values of parameters of the standard laboratory tests are shown in Table 3.
The prevalence of anemia (Hb < 100 g/L) in the examined patients was 44.17% (53 patients). The average blood concentration of Hb, and average monthly single dose of EPO with short and long effects are shown in Table 2.
The prevalence of absolute iron deficiency in the examined patients was 4.17% (5 patients), and the prevalence of functional iron deficiency was also 4.17% (5 patients). One hundred and ten patients (91.66%) had the normal status of iron in the body. Twenty patients with normal level of iron and normal TSAT, had level of FER higher than 1,000 µg/L. The examined patients were given parenteral iron. The average monthly single dose of parenteral iron was 155.83 ± 180.76 mg.    4 3binding agent, 54 (45%) of patients were treated with active vitamin D metabolites and 1 (0.83%) patient was treated with vitamin D. Parenteral form of paricalcitol was given to 14 (11.67%) of patients, and average monthly dose was 30.00 ± 15.20 µg.
The patients with 25(OH)D level in the serum of less than 10 ng/mL had a highly statistically significant (p < 0.01) lower concentration of Hb TP, Alb and TSAT and statistically significantly lower Hct and CRP (p < 0.05) compared to the patients with 25(OH)D level in the serum of 10-20 ng/mL and higher than 20 ng/mL (Table 4). There was no statistically significant difference between the second and third group of patients in concentrations of Hb, Hct and CRP (p > 0.05) ( Table 4). There was no statistically significant difference in average monthly dose of short-acting and longacting EPO among the examined groups of patients (Table 5).
There was no statistically significant difference between the second and third group of patients in the level of TP and Alb and TIBC (Table 4).

CRP -C-reactive protein; Ca 2+ × PO 4 3--solubility product; ALP -alkaline phosphatase; 25(OH)D -vitamin D; iPTH -parathyroid hormone.
Patients with severe vitamin D deficiency had statisticaly significant (p < 0.005) lower Kt/V as well as URR compared to other groups of patient ( Table 5). The two rest groups had no statistically significant difference in the parameters of hemodialysis adequacy (Table 5).
Patients with levels of iPTH > 500 pg/mL had statistically significantly higher level of ALP, a higher PO 4 3level, and a solubility product compared to patients with the serum levels of iPTH lower than 150 pg/mL (Tables 6 and 7).

Discussion
Among the patients with the end-stage kidney disease who begun regular hemodialysis, 90% suffer from anemia. Main clinical consequences of CKD are: progressive loss of residual renal function, cardiovascular complications, cognitive impairment and reduced quality of life of hemodialysis patients 24 .
Regardless of appropriate treatment of anemia, which includes parenteral administration of iron and EPO, anemia is still a common complication in the population of patients treated with regular hemodialysis. Anemia, defined as blood Hb concentration lower than 100 g/L, had high prevalence (44.17%) in the examined patients with CKD. The most important risk factor that affect the treatment of anemia in patients on dialysis include: iron deficiency, insufficient dose of EPO, inflammation, infection, SHPTH, increased serum iPTH levels, lack of vitamin D in the serum, malnutrition and inadequate hemodialysis 24 . Table 4 The

influence of vitamin D deficiency on the concentration of hemoglobin (Hb), C-reactive protein (CRP), parameters of nutritive status and metabolism of minerals and bone tissue in hemodialysis patients
Patients' groups according to 25       Results of recent clinical studies show that vitamin D deficiency plays an important role in the development of anemia in patients treated with regular hemodialysis. Vitamin D deficiency is defined as the serum level of 25(OH)D  20 ng/mL, while severe vitamin D deficiency is defined as the serum level of 25(OH)D  10 ng/mL 24 . Vitamin D insufficiency is defined as the serum level of 25(OH)D within range of 20-30 ng/mL. Normal serum level of 25(OH)D is ≥ 30 ng/mL. Target level in the serum of 25(OH)D in patients treated with regular hemodialysis is higher than 30ng/mL (30-80 ng/mL), while the level higher than 80 ng/mL can lead to toxic effects 24 . Prevalence of severe vitamin D deficiency in our study group was 24.17%. Vitamin D deficiency was present in 75.83% patients, vitamin D insufficiency in 15.83% (19 patients), while normal serum level of vitamin D had 8.33% patients. These results are similar with those demonstrated by former studies that showed vitamin D deficiency prevalence in hemodialysis patients of about 80% 24 .
Our results point to a difference in Hb concentration among groups of patients with different vitamin D levels; patients with 25(OH)D level lower than 10 ng/mL had lower blood Hb concentration than the group with serum 25(OH)D level of 10-20 ng/mL or the group with serum 25(OH)D level higher than 20 ng/mL. These two groups of patients had no difference regarding average dose of short-term and long-term EPO indicating that patients with severe vitamin D deficiency require higher dose of EPO for the treatment of anemia. Other authors have also demonstrated that vitamin D deficient hemodialysis patients have significantly lower blood Hb concentration than patients without vitamin D deficiency and require a significantly higher dose of EPO than patients having target level of 25(OH)D [24][25][26] . Prevalence of SHPTH in our study was 14.17%. The examined groups of patients did not differ in Hb concentrations as well as in average monthly dose of short-term and long-term EPO. Patients with high levels of serum iPTH had higher level of PO4 3and ALP in the serum, and also higher Ca 2+ × PO 4 3-. Former studies had similar findings -patients with SHPTH had higher Ca 2+ × PO 4 3and increased risk of vascular and valvular calcifications [27][28][29][30] .

Conclusion
Prevalence of vitamin D deficiency in hemodialysis patients was high -75.83%. Severe vitamin D deficiency was present in 24.17% of patinets, while normal vitamin D level had 8.33% of patients. Patients with vitamin D deficiency level lower than 10 ng/mL had significantly lower Hb concentration and adequacy of hemodialysis indices, as well as microinflamation, malnutrition and bone metabolism disorders present and needed higher dose of EPO compared to the patients with the serum 25(OH)D level higher than 10 ng/mL. Accordingly, vitamin D could be a significant risk factor for development of anemia in hemodialysis patients.