A cross-sectional study of 727 stable KT recipients from 28 transplant clinics across Spain (95.4% single kidney deceased donor; 1.2% double kidney deceased donor, and 3.3% living donor) was performed. At the time of the study, a blood sample and lateral X-ray of the dorsal and lumbar spines were obtained and sent to a central laboratory and X-ray diagnosis unit, respectively. Demographic and clinical data were retrospectively recorded from each participant's chart.

The vast majority of patients were Caucasian (95%). Inclusion criteria were: age>18 years, date of transplantation between January 2008 to December 2010, and post-transplantation time ≥1 year. The exclusion criteria were: pregnancy, double transplant (except double kidney), cancer (except non-melanoma skin cancer), significant liver disease, and the use of drugs possibly affecting bone and mineral metabolism other than the usual post-transplant medications.

The number of patients included in each CKD-T stage was estimated according to the 95% confidence interval of the variance observed for phosphate levels in the UK Renal Registry.3 This required a total of 1106 patients to be included. Because 28 out of 34 centres participated, we were able to enrol 766 patients (68.7%). For logistical reasons, central laboratory determinations were unavailable for 39 patients. Consequently, a total of 727 patients were enrolled (Fig. 1): 512 stage 1–3T and 215 stage 4–5T. This sample size gave a precision >95% to estimate the phosphate levels of CKD-T stages I–III from the UK Registry. In order to avoid bias, the number of patients included per transplant centre was proportional to the number of kidney transplantations performed per year at that centre, ranging from 5 to 33. In addition, each centre included consecutive patients who fulfilled the inclusion criteria and attended the outpatient clinic at some point between April 2010 and February 2011.

Fig. 1.

Study flow chart.

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No significant differences were observed between the 39 recruited patients who did not have a central laboratory determination and the 727 patients who were ultimately included in this study (data not shown).

Demographic and clinical data were recorded from each participant, including a history of clinical fractures and parathyroidectomy. Past and current immunosuppression, as well as bone and mineral medications (calcium and/or vitamin D supplements, vitamin D receptor activators, phosphate binders, calcimimetics and bisphosphonates), were also recorded. Graft function was derived from the MDRD-IV formula as the eGFR. Proteinuria in a 24-hour sample or as the protein/creatinine ratio (mg/g creatinine) in a first morning voided sample was also measured using standard methods at each participating centre.

A blood sample was obtained from each participant, and serum albumin, total calcium, phosphate, total and bone-specific alkaline phosphatase, PTH, 25OHD3, and 1,25(OH)2D3 levels were evaluated at a central laboratory. Calcium levels were corrected with serum albumin.17 PTH and 25OHD3 (Liaison®) were measured by chemiluminescence (Immulite®); 1,25(OH)2D3 was measured by RIA (LKB Counter), and bone alkaline phosphatase was measured by IRMA (LKB Counter). Serum creatinine determination at each study Centre was calibrated to be traceable to IDMS. The seasons of the year when the samples were obtained were: spring, 43.7%; summer, 37.3%; autumn, 18%; and winter, 1%. New onset diabetes was defined as a fasting glucose ≥126mg/dl in the two most recent determinations or the use of anti-diabetic drugs. Ischaemic heart disease, cerebrovascular disease, peripheral vascular disease were defined by standard clinical criteria.18

At enrolment, lateral X-rays of the dorsal and lumbar spine were obtained. Vertebral morphometry was assessed using the semi-quantitative method of Genant.19 Briefly, a decrease in the anterior, mid-body, or posterior vertebral height of 20–25% was considered mild (grade 1), 25–40% moderate (grade 2), and >40% severe (grade 3) (Supplementary Figure 1). Similarly, calcifications of the abdominal aorta were assessed with the semi-quantitative method of Kaupila20 and scored from 0 to a maximum of 24. Both vertebral morphometry and abdominal aorta calcification were centrally semi-quantified by an expert radiologist or nephrologist, respectively, who lacked knowledge of the patient's clinical and biochemistry data. Valid data for vertebral deformities were obtained in 615 cases and for aortic calcification in 595 (Fig. 1). No significant differences were found in age, gender, time after transplantation, or mineral metabolism parameters between the recipients with or without a valid assessment of vertebral deformities or aortic calcification, with the exception of 25OHD3 levels which were slightly higher in those with a valid assessment of vertebral deformity (18.2±9.9 and 21.8±13.3ng/ml; p=0.04).

The Ethics Committee of the Hospital Universitario de Canarias approved the study, and each participant signed an informed consent form.

Table 1 shows the demographic, clinical, and biochemical data as a function of the CKD-T stage at the time of the study. Additionally, the percentage of patients with grade ≥2 vertebral deformity and an aortic calcification score >0 is shown. Age, female gender, time since transplantation, a history of acute rejection, patients on steroids, and proteinuria significantly increased with advancing CKD-T stage. The proportion of patients receiving oral calcium (as supplement or as a phosphate binder), as well as vitamin D receptor activators, increased with advancing CKD-T stages. Serum PO4 and PTH levels increased while serum calcitriol decreased with advancing CKD-T stages. Hypercalcemia (>10.3mg/dl) was observed in 6.5% of recipients and hypophosphatemia (<2.5mg/dl) in 6.2%. PTH levels were above the normal (>70pg/ml) in 76.9% of patients. Vitamin D insufficiency (15–30ng/ml) or deficiency (<15ng/ml) was observed in 50.8% and 32.5% of patients, respectively. Therefore, only 16.7% of patients had normal serum vitamin D levels.

The prevalence of compression fractures (grade ≥2) was 14.6%, which was not significantly different across CKD-T stages. Overall, 67.2% of patients had some degree of abdominal aortic calcification; however, this proportion increased at CKD-T stages III–V compared with stages I/II. Immunosuppression and the percentage of patients on statins (57%) or ACEI/ARA (56%) were not different among CKD-T stages.

Table 2 shows a comparison of male vs. female recipients. Estimated GFR, 25OHD3 and 1,25(OH)2D3 levels were significantly lower in female recipients. Serum calcium and phosphate levels were significantly higher in female recipients, and no significant differences were observed in the circulating levels of PTH or bone alkaline phosphatase. A significantly higher proportion of women were receiving oral calcium (as a supplement or as a binder), vitamin D supplements, and bisphosphonates. Although a history of clinical fractures after transplantation was more common in female recipients (5.9 vs. 1.2%, respectively), prevalent vertebral fractures with vertebral deformity grade ≥2at the time of study were less common in women (10.9% vs. 17.1%).

To avoid the confounding effect of eGFR differences, a comparison of the mineral metabolism parameters by gender in each CKD-T stage is provided in Supplementary Table 1. The eGFR was similar in each CKD-T stage. As shown in Fig. 2a, serum calcium was significantly higher in women in CKD-T I/II. As a result, more women were receiving a calcimimetic in this stage (16.7% vs. 4.3%; p=0.03). Serum calcium and phosphate levels were significantly higher in CKD-T stage III women (Fig. 2a and b), and a higher proportion of women were treated with vitamin D derivatives (35.9% vs. 26%; p=0.045) and oral calcium supplements (24.6% vs. 16.5%; p=0.06) compared with men. Serum PTH and 1,25(OH)2D3 levels were similar between men and women at each CKD-T stage (Fig. 2c and d).

Fig. 2.

Albumin-corrected total serum calcium (a), serum phosphate (b), PTH (c), and 1,25(OH)2D3 (d) by CKD-T stages as a function of gender. Female vs male gender: (*) p<0.001; (**) p=0.02. Mean±SE.

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Overall, 25OHD3 levels were not significantly different between genders at each CKD-T stage; however, the proportion of women with vitamin D deficiency (<15ng/ml) was higher than in men in CKD-T stages I–III (36.8% vs. 26.9%; p=0.02) but not in CKD-T stages IV (36.6% vs. 35.2%) or V (50% vs. 60%). After excluding patients receiving vitamin D supplementation, gender differences in vitamin D deficiency at CKD-T I–III increased (44.4% vs. 29.6%; p=0.003) (Fig. 3a); in addition, serum 25OHD3 levels were significantly lower in women compared with men at CKD-T stages I–III (Supplementary Table 1; Fig. 3b, p=0.003), which was not the case for stages IV and V. These differences in vitamin D status by gender were not explained by the season of the year when the blood samples were collected (males: 52.8% in summer or autumn, 46% in spring, and 1.1% in winter; females: 59% in summer or autumn, 40.3% in spring, and 0.7% in winter).

Fig. 3.

Prevalence of Vitamin D deficiency (25OHD3 <15ng/ml) by the CKT-T stage as a function of gender (a). For those recipients who were not treated with vitamin D, 25OHD3 by CKD-T stage serum levels (b) and correlation of log transformed 25OHD3 with log transformed PTH levels are shown as a function of gender (slope was steeper in women; p=0.01). For all recipients, the correlation between the log transformed 25OHD3 and log transformed 1,25(OH)2D3 is shown (r=−0.35; p<0.001) (d). (*) p=0.003 for the comparison of males vs females in CKD-T Stages I through III; for CKD-T stages IV–V no significant differences between males vs. females.

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The log transformed serum PTH levels correlated inversely with the eGFR (r=−0.33; p<0.001), albumin corrected serum calcium (r=−0.14; p<0.001) and log transformed 25OHD3 levels (r=−0.23; p<0.001). After adjusting for eGFR by partial correlation analysis, the inverse correlation between PTH and 25OHD3 levels remained significant (r=−0.22; p<0.001). In patients who were not treated with vitamin D supplements, the slope of the regression line between 25OHD3 and PTH levels was steeper in women than in men (−0.25±0.08 vs. −0.62±0.13; p=0.01; Fig. 3c). The log transformed serum calcitriol levels correlated directly with eGFR (r=0.44; p<0.001) and, as shown, the log transformed 25OHD3 levels (r=−0.35; p<0.001) (Fig. 3d). The correlation between 25OHD3 and calcitriol levels remained significant after adjusting for eGFR (r=−0.36; p<0.001).

The prevalence of asymptomatic vertebral deformity grade ≥1 was 26%. Compression fractures that were grade 2 (deformity >25%) or higher were found in 15% of the patients. Table 3 shows the univariate comparison of patients with and without a prevalent asymptomatic vertebral fracture, defined as a deformity ≥grade 2. Patients with a prevalent vertebral fracture were older, and vertebral fractures were more common in men than in women. A postmenopausal status was more prevalent among women with vertebral fractures. A history of previous cardiovascular disease was more common among patients with vertebral fractures. Immunosuppression was different between the groups, and CsA use was more common among patients with vertebral fractures than those without a vertebral fracture (22.2% vs. 10.9%, respectively; p=0.006). Indeed, 26% of patients treated with CsA and 13% of those treated with tacrolimus or mTOR inhibitors had a vertebral fracture (p=0.005). Using backward conditional logistic regression analysis, we investigated the factors independently related to prevalent vertebral fractures. We included age, gender, BMI, time since transplantation, corticosteroid treatment (dichotomic), CsA treatment (dichotomic), eGFR, serum 25OHD3, and PTH levels as independent variables. Age, gender, and treatment with CsA were independently associated with prevalent vertebral fractures (Table 4). We also explored the interactions between gender and different mineral metabolism variables. The only significant interaction was gender*PTH (p=0.016). Therefore, it was logical to proceed with separate analyses of men (n=368) and women (n=235).

Men with vertebral fractures were older (60±12 vs. 54±14 years; p=0.001) and more commonly received CsA as the maintenance immunosuppressant (27% vs. 12.5%; p=0.006) than those without fractures. No other significant difference was observed between the two groups (data not shown). Using backward conditional logistic regression analysis, we investigated the factors independently related to prevalent vertebral fractures in men. We included age, BMI, time since transplantation, corticosteroid treatment, CsA treatment, eGFR, serum 25OHD3, and PTH levels as independent variables. Age and CsA treatment, but not time since transplantation, were independent factors associated with vertebral fractures (Table 4). Additionally, time since transplantation was not statistically significant when excluding immunosuppression therapy from the model (p=0.7).

Women with vertebral fractures were older (63.4±9 vs. 54.2±13 years; p<0.001) and had higher serum PTH [216 (92–412) vs. 114 (71.8–189.8) pg/ml, p=0.007] and lower 25OHD3 levels [14.8 (10.4–20.9) vs. 18.4 (12.3–26.3) ng/ml; p=0.049) than women without fractures (no other significant difference was observed between the groups; data not shown). By backward logistic regression analysis, age and serum PTH levels were significantly related to vertebral fractures after adjusting for the eGFR and 25OHD3 level. Interestingly, and unlike male recipients, immunosuppression was not related to vertebral fractures in women.

Compared to patients receiving tacrolimus or mTORi at the time of the study, those who were treated with CsA had a longer post-transplantation time. This difference was similar in men (85±26.6 vs. 66.1±30.5 months; p<0.001) and women (93.9±26.3 vs. 67.7±30.7 months; p<0.001). However, the prevalence rates of vertebral fractures in patients who were treated with CsA differed by gender, 31.5% vs. 14.6% (p=0.005) in men and 10.7% vs. 13% (p=0.8) in women.

A Kaupila's score ≥1 was observed in 67.2% of the patients. Table 5 shows the univariate comparison of patients as a function of their tertile of calcification score. Age, time since transplantation, pulse pressure, and proportion of patients with prior ischaemic heart disease increased with the severity of abdominal aorta calcification (Table 5). Conversely, eGFR decreased with increasing calcification. Mineral metabolism parameters were not significantly different among the calcification tertiles.

Using backward logistic regression analysis, age [OR 1.03 (1.02–1.05)], time since transplantation [OR 1.01 (1.001–1.02)], and history of ischaemic heart disease [2.4 (1.07–5.7)] were independent predictors of the highest calcification score (third tertile) compared with patients who lacked calcification (first tertile). No mineral metabolism parameters were related to aortic calcification.