Abstract
Background and objectives Serum alkaline phosphatase (AP) is associated with vascular calcification and mortality in hemodialysis patients, but AP derives from various tissues of origin. The aim of this study was to assess the effect of bone-specific AP (BAP) on morbidity and mortality in dialysis patients.
Design, setting, participants, & measurements From a prospective cohort study of incident dialysis patients in The Netherlands, all patients with measured BAP at 12 months after the start of dialysis (baseline) were included in the analysis (n = 800; mean age, 59 ± 15 years; mean BAP = 18 ± 13 U/L). By Cox regression analyses, we assessed the impact of BAP levels on short-term mortality (6 months) and longer-term mortality (4-year follow-up).
Results High levels of BAP strongly affected short-term mortality. After adjustment for confounders, patients in the highest BAP tertile had a 5.7-fold increased risk of death within 6 months compared with patients in the lowest tertile. The effect applied to both cardiovascular and noncardiovascular mortality. Furthermore, high levels of BAP were associated with increased cardiovascular mortality in the longer term. In comparison with total AP, the effect sizes related to clinical outcomes were much higher for BAP.
Conclusions High levels of BAP were strongly associated with short-term mortality in dialysis patients, pointing out the important impact of bone turnover. Longitudinal assessments of BAP may be useful for the treatment monitoring in clinical practice in dialysis patients.
Introduction
Vascular calcification importantly contributes to the morbidity and excess mortality of dialysis patients (1). Numerous factors influence the development of vascular calcification, among which the presence of diabetes mellitus, uremia, and a disturbed mineral metabolism (2,3) is of major importance. The latter is a hallmark of patients with chronic renal failure, being highly prevalent and characterized as chronic kidney disease (CKD)–mineral and bone disorder. Renal osteodystrophy, secondary hyperparathyroidism, and vascular disease are all parts of the syndrome. Interestingly, alkaline phosphatase (AP) has been suggested to play a role as a useful indicator of bone health (4).
APs are enzymes that remove phosphate from proteins and nucleotides, functioning optimally at alkaline pH (4). Recent in vitro studies have shown that AP is an essential component of serum calcification activity (5). Inactivation of AP prevents serum collagen calcification. AP was furthermore reported to activate a serum nucleator of apatite crystal formation (6). In line with this, clinical studies have found serum AP to be associated with coronary artery calcification and all-cause mortality in patients with CKD and on hemodialysis (7–9). In humans, the enzyme AP is found throughout the body in the form of isoenzymes, deriving from not only the liver and bone, but also intestines, placenta, kidneys, and leukocytes. By now, it is not clear whether the observations made for serum AP in renal patients mainly reflect changes in bone and mineral metabolism or other systemic processes.
Specific isoenzymes to identify the tissue source can be determined after fractionation and heat inactivation (4). As such, bone-specific AP (BAP) has been found to have a higher sensitivity and specificity than AP to reflect histologically proven alterations in bone mineral metabolism (10,11). The aim of this study was to assess the effect of BAP on morbidity and mortality in dialysis patients. Second, we aimed to compare the impact of BAP on clinical outcomes with that of total AP, analyzing data of a prospective multicenter cohort study of incident dialysis patients in The Netherlands.
Materials and Methods
Study Design
The Netherlands Cooperative Study on the Adequacy of Dialysis (NECOSAD) is an observational prospective follow-up study in which incident dialysis patients have been enrolled in 38 participating dialysis centers since 1997 in The Netherlands. Study visits took place at the start of dialysis, at 3 months, 6 months, and subsequently at 6-month intervals until the date of loss to follow-up (death, kidney transplantation, or transfer to a nonparticipating dialysis center) or the end of the follow-up at January 1, 2009. Demographic and clinical data, as well as blood and 24-hour urine samples, were obtained at the start of long-term dialysis treatment and at subsequent study visits.
For this analysis, baseline is defined as 12 months after the start of dialysis treatment, when the patients' fluid and metabolic conditions had stabilized and when adequate amounts of serum material for laboratory measurements were available.
Patients
Patients with ESRD who were at least 18 years old and started long-term dialysis therapy for the first time were invited to participate in NECOSAD.
In this analysis, all patients in whom the amount of collected blood was sufficient to measure BAP at 12 months after initiation of dialysis (the baseline of our study) were included. The medical ethical committees of the participating centers approved the study, and all patients gave their written informed consent before inclusion.
Data Collection
Demographic and clinical data included age, gender, ethnicity, smoking habits, primary kidney disease, and comorbidity. Primary kidney diseases and causes of death were classified according to the coding system of the European Renal Association–European Dialysis and Transplant Association. Diagnoses of comorbid conditions were reported by the patients' nephrologists and used to calculate the comorbidity score according to Khan et al. (12). Renal function expressed as GFR was calculated as the mean of creatinine and urea clearance, corrected for body surface area (ml/min per 1.73 m2). The clinical and comorbidity data that were collected at 12 months after initiation of dialysis were used for this study.
Biochemical measures including cholesterol, hemoglobin, total serum calcium, phosphorus, intact parathyroid hormone (PTH), total AP, and albumin were measured by standard laboratory techniques in the different centers at 12 months after start of dialysis. Measurements of BAP and 25-hydroxyvitamin D [25(OH)D] levels were performed centrally at the University Hospital of the RWTH Aachen in blood samples collected at 12 months after the start of dialysis according to standard techniques and stored at −80°C until their analysis. 25(OH)D was measured by chemiluminescence immunoassay on the Liaison autoanalyzer (DiaSorin, Saluggia, Italy). Serum concentrations of BAP were determined by the MicroVue BAP immunoassay (Quidel, San Diego, CA). This assay uses a monoclonal anti-BAP antibody, which has a selective, high affinity for the BAP isoform. In contrast, it shows a low cross-reactivity to the liver form of AP and negligible binding of intestinal and placental isoenzymes.
Definition of Endpoints
Cardiovascular mortality was defined as death caused by the following: myocardial ischemia and infarction, hyperkalemia, hypokalemia, cardiac arrest, (hypertensive) cardiac failure, fluid overload, cerebrovascular accident, hemorrhage from ruptured vascular aneurysm, mesenteric infarction, or cause of death uncertain/unknown. All other causes of death were designated as noncardiovascular mortality.
Statistical Analyses
Mean values with SD were calculated for continuous variables, and median values with interquartile range as appropriate. Categorical variables were expressed as proportions.
We performed correlation analyses to examine associations between BAP, PTH, and total AP. Survival analyses were performed to assess associations of BAP levels with all-cause mortality, the specific outcomes of death from cardiovascular causes, and death from noncardiovascular causes. Because of the lack of recommendations for clinical thresholds of BAP and AP, the patients were categorized into tertiles according to BAP and AP levels.
Cumulative mortality curves were calculated using Kaplan-Meier analysis for all-cause mortality. This method is known to profoundly overestimate the cumulative mortality when analyzing competing endpoints (13). Separate analysis of cardiovascular mortality and noncardiovascular mortality is a clear example of competing endpoints. For that reason, we calculated the cumulative mortality curves for cardiovascular mortality and noncardiovascular mortality using competing risk analysis, taking into account that patients dying of cardiovascular causes are no longer at risk to die of noncardiovascular causes and vice versa (13,14).
By Cox regression analyses, we calculated hazard ratios (HRs) with 95% confidence intervals (95% CIs) for subsequent short-term (6 months) and longer-term (4 years) periods, according to BAP levels at baseline. The lowest category of BAP was thereby used as the reference group. All analyses were adjusted for potential confounders including age, gender, dialysis modality, primary kidney disease, diabetes mellitus, cardiovascular disease, Khan comorbidity index, BP, body mass index, levels of serum albumin, calcium, phosphate, PTH, and 25(OH)D. Furthermore, we investigated BAP in combination with PTH levels in association with mortality. We formed six groups according to the median levels of BAP (15 U/L) and the cut-offs for PTH as suggested by the Kidney Disease Outcomes Quality Initiative (KDOQI) guideline at that time (2003; PTH ≤150, 150 to 300, and >300 pg/ml).
To compare the outcome effects of total AP with those of BAP, we performed survival analyses in a similar manner as for BAP. All P values are reported two-sided. Analyses were performed using SPSS version 16.0.
Results
Patients
A total of 1753 patients with ESRD who started long-term dialysis were included and still participated in NECOSAD at 12 months after the initiation of dialysis therapy (the baseline of this study). Of those, BAP was measured in 800 patients, in whom the amount of collected blood was sufficient for the measurement of BAP. These patients were included in the present analyses. Of note, the included patients were not different from the remaining study cohort (n = 953). Both patient groups were similar with regard to demographic and clinical characteristics, including comorbidites and levels of routine laboratory markers (data not shown).
In the study population (n = 800), patients had a mean age of 59 ± 15 years. Mean levels of BAP were 18 ± 13 U/L, and mean levels of total AP were 78 ± 53 U/L.
With higher BAP levels at baseline, more patients had diabetes mellitus either as the primary kidney disease or as a comorbidity. Levels of PTH were higher in the patients with higher BAP levels, and the percentage of male patients was smaller (Table 1). To investigate whether practice patterns changed over the years during conduction of the NECOSAD study, we assessed median PTH and BAP levels of patients included before and after the 2003 KDOQI guideline publication. The PTH levels in patients included before publication of the guideline were 12.6 pmol/L, and they were 13.0 pmol/L in the patients included after publication of the guideline. Similarly, median BAP levels were not meaningfully different (15.0 and 17.0 U/L, respectively), supporting similar practices of patient care over time. During the 4-year follow-up period, 277 patients died, of whom 152 patients died of cardiovascular causes, and 125 patients died of noncardiovascular causes. Of all deaths, 55 occurred in the short term, i.e., within 6 months after baseline. These included 33 cardiovascular and 22 noncardiovascular deaths.
Baseline characteristics of the study population, according to tertiles of BAP
BAP and Clinical Outcomes
BAP was significantly correlated with PTH (Pearson correlation coefficient, r = 0.34; P < 0.001). Furthermore, there was a strong relationship between BAP and total AP (r = 0.55, P < 0.001), whereas the correlation of BAP with 25(OH)D was low (r = −0.07, P = 0.043).
We investigated short-term and longer-term mortality according to BAP levels. The respective follow-up intervals were 6 months and 4 years after the measurements of BAP. High levels of BAP strongly affected short-term mortality (Figure 1A). Patients of the highest BAP tertile had a greater than four-fold increased risk of death within 6 months (HR, 4.8; 95% CI, 1.8 to 12.7) compared with patients of the lowest tertile. This association became even stronger in multivariate analyses adjusting for confounders (HR, 5.7; 95% CI, 2.0 to 15.9). The effects applied to both cardiovascular (HR, 5.0; 95% CI, 1.3 to 18.9; Figure 1B) and noncardiovascular mortality (HR, 6.2; 95% CI, 1.3 to 29.2). Furthermore, high levels of BAP were, by trend, associated with increased risks of adverse outcomes in the longer term (Figure 1, C and D). The HRs comparing patients of the third to those of the first BAP tertile were 1.3 (95% CI 0.9 to 1.8) for all-cause mortality and 1.7 (95% CI 1.0 to 2.8) for cardiovascular mortality, respectively. These effects of BAP in the longer term were, however, much smaller compared with the effects in the short term (Table 2). In additional subgroup analyses, the results were similar for hemodialysis and peritoneal dialysis patients, indicating a higher mortality risk at higher BAP levels, especially in the short term. Similarly, higher BAP levels were associated with an increased mortality both in patients with diabetes and patients without diabetes (data not shown).
(A–D) Cumulative mortality curves for (A) all-cause mortality within 6 months, (B) cardiovascular (CV) mortality within 6 months, (C) all-cause mortality within 4 years, and (D) cardiovascular mortality within 4 years according to tertiles of bone alkaline phosphatase levels at baseline.
Hazard ratios with 95% confidence intervals for all-cause, cardiovascular, and noncardiovascular mortality according to tertiles of BAP
To gain deeper insight into potential dose–response relationships, we performed further analyses using sixtiles of BAP (levels of BAP in the respective groups: 1 [<9], 2 [9 to 12 U/L], 3 [12 to 15 U/L], 4 [15 to 18 U/L], 5 [18 to 23.5 U/L], 6 [>23.5 U/L]). Compared with patients with the lowest BAP levels (sixtile 1, reference), the adjusted short-term mortality risks of patients in the other groups were as follows: 4.9 (0.5 to 45.7) in sixtile 2, 10.0 (1.2 to 80.8) in sixtile 3, 11.9 (1.5 to 96.1) in sixtile 4, 8.1 (0.9 to 69.8) in sixtile 5, and 30.0 (3.7 to 243.1) in sixtile 6, respectively.
In additional analyses using combinations of BAP and PTH, six groups were formed according to the median levels of BAP (15 U/L) and the cut-offs for PTH as suggested by the KDOQI guideline at that time (2003; PTH ≤150, 150 to 300, and >300 pg/ml). We found that patients with low BAP and low or moderate PTH had the best survival, both during 4 years and in the short term during 6 months. Compared with those (reference group), patients with low BAP and high PTH did not significantly differ regarding survival, whereas the group of patients with high BAP and low PTH had an adjusted 2.8-fold increased risk of death within 6 months (Table 3).
HRs with 95% CIs for all-cause mortality according to groups defined by levels of BAP and PTH
Total AP and Clinical Outcomes
Similarly to the bone-specific isoenzyme BAP, we investigated total AP in its association with clinical outcomes. High levels of total AP were associated with increased rates of death: short-term mortality was almost two-fold higher in patients of the highest tertile of total AP compared with those of the lowest tertile (HR, 2.4; 95% CI, 1.1 to 5.1). The associations were slightly attenuated after adjustment for confounders (Table 4). In the longer term, total AP did not meaningfully affect mortality. Using sixtiles of total AP, short-term death rates were lowest in the patients of the first sixtile (AP <46 U/L; reference) and highest in those of the highest sixtile (AP >102 U/L; HR, 5.1; 95% CI, 1.4 to 18.1). When the associations of BAP and total AP with mortality and specific fatal events were compared, the bone-specific isoenzyme BAP showed much higher effect sizes than total AP.
HRs with 95% CIs for all-cause, cardiovascular, and noncardiovascular mortality according to tertiles of total AP
Discussion
The major finding of our study is that BAP is a strong risk factor for all-cause, cardiovascular, and noncardiovascular mortality in dialysis patients, particularly in the short term. Patients in the highest tertile of BAP levels had a markedly six-fold increased risk of death within 6 months compared with patients in the lowest tertile. The effects applied to both cardiovascular (HR, 5.0; 95% CI, 1.3 to 19.0) and noncardiovascular (HR, 6.2; 95% CI 1.3 to 29.2 mortality). Furthermore, high levels of BAP were associated with increased risks of cardiovascular and, by trend, with all-cause death also in the longer term. Compared with BAP, the effects of total AP were less pronounced. Patients in the highest tertile of AP levels had a two-fold increased risk of death within 6 months compared with patients in the lowest tertile. The associations were independent of common known risk factors because we adjusted for age, gender, dialysis modality, primary kidney disease, diabetes mellitus, cardiovascular disease, Khan comorbidity index, body mass index, systolic BP, levels of albumin, calcium, phosphate, PTH, and 25(OH)D.
These findings are in line with previous study results from Regidor et al. (8) examining the effects of total AP on mortality in a large American cohort of dialysis patients (DaVita study). The authors showed an HR of 1.25 (95% CI 1.21 to 1.29) for mortality within 3 years of follow-up in patients with AP levels >120 U/L. Although our study comprised fewer patients and did not examine repetitive laboratory measurements, our results expand the previous findings in several important aspects. We studied a well-characterized cohort of Caucasian patients, which also included a significant proportion of peritoneal dialysis patients. Importantly, the baseline data came from a very homogenous starting point regarding dialysis vintage (all patients 12 months after initiation of dialysis). In contrast, patients of the DaVita study showed a wide range of dialysis vintage (8). This might be of special importance, because our data indicate a striking short-term, versus no or only a moderate long-term, effect of AP and BAP on mortality.
In the outcome analyses, 25(OH)D was furthermore considered as potential confounder. It has been shown to impact on mortality in dialysis patients (15–17) and, moreover, might directly affect BAP or AP levels (18) in cases of renal bone disease with mineralization defects or overt osteomalacia. The most important extension of our study, however, is that the bone isoenzyme BAP was studied in parallel with total AP.
Low levels of both AP and BAP were associated with an improved survival, warranting potential explanations and mechanisms. An important question arises as to whether this association may allow the conclusion that low bone turnover is associated with better outcome or whether low BAP levels clearly reflect low bone turnover. The role of BAP as a biomarker of renal osteodystrophy has previously been investigated in several bone biopsy studies in patients with ESRD (altogether >400 patients) (10,11,19–22), but results are variable. Most patients underwent bone biopsy after tetracycline labeling, allowing the measurement of dynamic bone parameters such as bone formation rate. Bervoets et al. (10) and Urena et al. (11) found that BAP was a better predictor for bone metabolism than total AP. Depending on the laboratory methods used, applied cut-off levels, and the cohorts investigated, low BAP levels showed positive predictive values for adynamic bone disease between 0.89 to 1.0 (10,11,21). However, Youden indices for the diagnosis of renal osteodystrophy vary between 0.49 and 0.93 (10,19–21). Our own data with receiver operating characteristic analyses in 57 patients with advanced chronic kidney disease/ESRD indicated that the best BAP cut-off level for discriminating low turnover from high turnover bone disease was 15.8 U/L. However, sensitivity (0.75) and specificity (0.59) were moderate (V. Brandenburg and G. Lehmann, unpublished data). In summary, BAP at a single time point provides a hint, but not proof, of low turnover. The exact role of BAP in the assessment of bone turnover is currently addressed by an ongoing large bone biopsy trial performed by the Kidney Disease: Improving Global Outcomes (KDIGO) initiative.
Considering potential negative effects that oversuppression of bone metabolism has on bone and vascular health (23), the finding of low BAP levels being associated with improved outcome appears surprising. Potential explanations need to be acknowledged. First, low BAP levels as assessed at one single time point after initiation of dialysis may indicate that serious attention was paid to the development and correction of secondary hyperparathyroidism during predialysis care by the attending physicians. Low BAP may thus additionally represent a surrogate parameter of the general quality of care before and during ESRD. In particular, low BAP levels may partially reflect the amount of active vitamin D administered, because the application of active vitamin D or vitamin D receptor activators can lower BAP by more than one third within several weeks of treatment and uncouple serum BAP levels from bone turnover (24,25). In this context, low BAP as a potential sign for good vitamin supplementation also had a beneficial impact on noncardiovascular mortality. Our strong association between low BAP levels and survival may potentially differ when long-term dialysis patients develop oversuppression of bone turnover. Unfortunately our data do not allow us to look into BAP developments over time that might reflect therapeutic interventions and changes in bone turnover. Future studies are needed to address this topic in further detail, investigating whether BAP reflects more than bone turnover status. Such an association of BAP with conditions other than bone disease is supported by further hints. First, we found a striking association between high BAP and noncardiovascular short-term mortality. Second, our combined BAP + PTH survival analyses indicated that low BAP levels may help overcome the overall reduced survival associated with low PTH levels as currently seen with the Analysing Data Recognising Excellence Optimising Outcomes in CKD (ARO) study (26). Interestingly, the combined analyses showed that, compared with the group with low BAP and low PTH (reference), the group of patients with high BAP and low PTH had an adjusted 2.8-fold increased risk of death within 6 months, which was highly significant (Table 4). To further identify these patients, we evaluated the clinical characteristics and found that this patient group may have the poorest nutrition. Compared with the other patient groups, the individuals with high BAP and low PTH showed lower levels of phosphate, creatinine, total cholesterol, and albumin, as well as body mass index, potentially representative of wasting or malnourishment. Furthermore, residual GFR was lower and the percentage of diabetes higher in this group, potentially additionally supportive of the notion that wasting may be present in these patients (data not shown).
Further explanations of the protective effects of low BAP and AP levels include the mechanisms via pyrophosphate. Experimental data link high APs to the development of coronary artery calcification (27) with their ability to hydrolyze inorganic pyrophosphate (28). Pyrophosphate is a potent inhibitor of vascular calcification, and its biologic action is reduced by phosphatases.
In the context of BAP relating to clinical outcomes, our study impressively showed that high levels of BAP were associated with mortality and specific fatal events, particularly in the short term. Considering that BAP levels were determined only once in single measurements, potential changes in BAP status over time may contribute to explain the time differentiating effects. In comparison with total AP, the effect sizes related to clinical outcomes were much higher for BAP. Apart from the pathophysiologic characteristics differentiating both enzymes, BAP may furthermore provide additional value for the risk stratification of dialysis patients. In this context, measuring BAP may help identify people at risk for all-cause and cardiovascular mortality. In additional analyses, BAP increased the C-statistic of the full model from 0.75 to 0.76 in long-term analyses and from 0.78 to 0.79 in 1-year mortality analyses. C-statistics of short-term models were not possible, however, because of the low number of events and may be promising targets for further studies predominantly designed for prediction modeling. An intriguing perspective emerges from the fact that high BAP levels were also associated with a high mortality in patients with low PTH. This makes us hypothesize about the role of BAP in identifying those patients in whom vitamin D therapy with consecutive lowering of BAP might decrease mortality despite low PTH levels.
Potential limitations need to be acknowledged. Our data indicate a strong association for both AP and BAP with all-cause and cardiovascular mortality in stable ESRD patients; however, we cannot extrapolate our findings toward ESRD patients starting on dialysis. Furthermore, the PTH measurements were performed using various first-generation immunometric PTH assays, dependent on the different participating centers. We are aware that these methods may yield different results, and we cannot report about potential changes in assay use over time. Another limitation of our study is the missing data on active vitamin D treatment. Finally, potential influences by prolonged storage on BAP measurements cannot completely be ruled out. We investigated and compared the median levels of BAP in older and younger samples, which were not meaningfully different. Therefore, potential influences caused by storage time are likely to be small, and the samples used for BAP measurements did not undergo repetitive freeze-thaw cycles.
In summary, high levels of BAP were strongly associated with short-term mortality in dialysis patients, suggesting BAP as an important biomarker of bone and mineral disorder in dialysis patients. Longitudinal assessments of BAP may be useful for the treatment monitoring in clinical practice in patients with ESRD.
Disclosures
None.
Acknowledgments
We thank the investigators and study nurses of the participating dialysis centers and the data managers of the NECOSAD study for collection and management of the data. C.D. is grateful to the Deutsche Forschungsgemeinschaft (DR 804/1-1) and to the Medical Faculty of the University of Wuerzburg for the support with a research fellowship. An abstract representing this work has been presented in an oral presentation at ASN Renal week 2010 in Denver, Colorado.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
- Received November 15, 2010.
- Accepted March 6, 2011.
- Copyright © 2011 by the American Society of Nephrology