Pro-inflammatory cytokines and bone fractures in CKD patients. An exploratory single centre study

The study protocol was approved by the Ethics Committee of the Azienda Ospedaliera “Bianchi-Melacrino-Morelli” di Reggio Calabria. All patients provided informed consent.

All prevalent patients in January 1995 and incident patients in 1996–1997 [66 on haemodialysis (HD) and 34 on continuous ambulatory peritoneal dialysis (CAPD), 63 males and 37 females] belonging to a single renal Unit, who had been on regular dialysis treatment (RDT) for at least 6 months and without inter-current clinical problems requiring hospitalization were recruited for the study. Patients mean age was 61 ± 15 years and the median duration of dialysis treatment was 43 months (inter-quartile range 18–99 months). Further clinical details about the study population are given in Table 1. Hemodialysis patients were being treated thrice weekly with standard bicarbonate dialysis (Na 138, HCO₃ 35, K1.5, Ca 1.25, Mg 0.75 mmol/L) and 1.1-1.7 m² dialysers (89% cuprophan, 11% semi-synthetic membranes). The average fractional urea clearance (Kt/V) in these patients was 1.28 ± 0.31. Dialysis fluid was produced by a reverse osmosis system and Aluminium never exceeded 5 μg/L which is well below the safety limit recommended by the European Council. Patients on CAPD were all on 4 exchanges/day schedule with standard dialysis bags containing 1.75 mmol/L calcium. The average weekly Kt/V in these patients was 1.67 ± 0.30. Sixteen patients were diabetics and 48 were habitual smokers. Forty-nine patients were on treatment with erythropoietin and 60 were taking various anti-hypertensive drugs (42 on mono-therapy with ACE inhibitors, calcium channel blockers or beta blockers and the remaining 18 on double or triple therapy with various combinations of these drugs). Eighteen patients out of 60 on anti-hypertensive therapy were on treatment with beta blockers (alone or in combination with other drugs). Eighty-six patients were assuming calcium chelating agents (either calcium carbonate or calcium acetate). Forty-seven patients were being treated with calcitriol. None of patients who took part in the study had undergone parathyroidectomy.

Fasting blood sampling was performed during a midweek non-dialysis day for HD patients. Samples were stored in prechilled vacutainers containing edetic acid, placed immediately on ice, and centrifuged within 30 min at 4 °C; plasma was stored at −80 °C until required. Serum calcium, serum phosphate, haemoglobin and alkaline phosphatase measurements were made using standard methods in the routine clinical laboratory. Intact PTH molecule measurement was made by a specific immuno-radiometric assay (Scantibody Laboratory Inc, Santee, CA, USA). Bone skeletal phosphatase was measured by an immunoradiometric method (Tandem-Ostase, Hybritech, USA).

C-Reactive Protein was determined by a standard immunonephelometric method (Behring, Scoppito, L’Aquila, Italy) with normal values <5 mg/L. ELISA by commercially available kits (R&D Systems Inc, Minneapolis, USA) was used to determine IL-6 (normal values < 3.13 pg/ml; coefficient of variation: intra-assay = 2.6%, inter-assay = 4.5%) and TNF- α (normal values <4.1 pg/ml; coefficient of variation: intra-assay = 4.7%, inter-assay = 5.8%) [16].

Fractures were defined as non-traumatic events documented by imaging techniques (i.e. radiography, computerized axial tomography or nuclear magnetic resonance). We considered fractures of the femoral neck (intertrochanteric, or subtrochanteric fractures), fractures of other parts of the femur (condyle, supracondylar, and epiphysis), vertebral fractures and fractures interesting other skeletal segments. Vertebral fractures were diagnosed using a semi-quantitative approach [17].

After enrollment patients were followed-up until the first fracture, those who died or underwent transplant or were free of fractures at the end of study were censored no patient was lost to follow-up. Median follow-up was 74 months (range 0.5-84.0 months).

Data are reported as mean ± SD, median and inter-quartile range or as prevalence rate and differences between groups were analyzed by the T-test, the Mann–Whitney test or the Chi-Squared Test, as appropriate. The association between incident fractures with serum PTH, total and bone alkaline phosphatase, calcium, phosphate, TNF- α, CRP, Il-6 and others potential risk factors was preliminarily analyzed by dividing patients into two groups (patients with and without incident fractures) and by testing the differences between them. To identify patients with low and high bone turn-over we used intact PTH thresholds suggested by European Best Practice Group [18]. The predictive value of biomarkers of inflammation, mineral and bone disorder and other potential risk factors was analyzed by univariate Cox’s proportional hazards method. In the Cox’s analysis the proportional hazard assumption was tested by the analysis of Schoenfeld residuals and no violation was found. Due to the small number of fractures to assess the idependent link between TNF-α and fractures we entered this risk factor in(restricted) bivariate models considering other risk factors one at a time. Furthermore, we computed a risk score [19] in each patient by summing up the individual profile of 5 risk factors for fractures, each dichotomized as following : sex female = 1 male = 0, previous fractures = 1 no previous fractures = 0, previous transplants = 1 no previous transplants = 0, intact PTH > median value = 1 intact PTH < median value = 0, age > median value = 1 age < median value = 0. The potential confounding effect of this score, reflecting the combined effect of major risk factors for fractures, was then tested in an additional bivariate model.

Data are expressed as hazard ratio (HR) and 95% confidence interval (CI). All calculations were done using a standard statistical package (SPSS for Windows).