Worsening calcification propensity precedes all-cause and cardiovascular mortality in haemodialyzed patients

A novel in-vitro test (T50-test) assesses ex-vivo serum calcification propensity which predicts mortality in HD patients. The association of longitudinal changes of T50 with all-cause and cardiovascular mortality has not been investigated. We assessed T50 in paired sera collected at baseline and at 24 months in 188 prevalent European HD patients from the ISAR cohort, most of whom were Caucasians. Patients were followed for another 19 [interquartile range: 11–37] months. Serum T50 exhibited a significant decline between baseline and 24 months (246 ± 64 to 190 ± 68 minutes; p < 0.001). With serum Δ-phosphate showing the strongest independent association with declining T50 (r = −0.39; p < 0.001) in multivariable linear regression. The rate of decline of T50 over 24 months was a significant predictor of all-cause (HR = 1.51 per 1SD decline, 95% CI: 1.04 to 2.2; p = 0.03) and cardiovascular mortality (HR = 2.15; 95% CI: 1.15 to 3.97; p = 0.02) in Kaplan Meier and multivariable Cox-regression analysis, while cross-sectional T50 at inclusion and 24 months were not. Worsening serum calcification propensity was an independent predictor of mortality in this small cohort of prevalent HD patients. Prospective larger scaled studies are needed to assess the value of calcification propensity as a longitudinal parameter for risk stratification and monitoring of therapeutic interventions.


Results
This study was performed in a subgroup of 188 European haemodialysis patients of the ISAR cohort who were still alive and active in the study after the first follow up (FU) period of 24 months with a maximum spread of ± 3.2 months and for whom sera were available at both baseline and FU. Patients for whom timely 24 [23][24][25] months FU had been missed were excluded a priori (Fig. 1). Most patients were of Caucasian ethnicity with only four black (2%) and two Chinese (1%) participants.
In comparison with the total cohort, the study population did not significantly differ with regard to demographic parameters, body mass index (BMI) and dialysis modality. Included patients were, by nature of the study design, enriched for survivors (22.9% versus 35.8% all-cause mortality in the total cohort; p < 0.001). Additionally, history of myocardial infarction (14.4% versus 19.8%; p = 0.02) and use of central venous catheters (2.7% versus 7.3%; p = 0.001) at baseline were less frequent among included patients. Median albumin levels were slightly higher in the study population (41 g/l versus 40 g/l, p = 0.04). For details please see supplementary Table 1.

Change
). Table 1 displays the characteristics of the study population stratified by median T 50 Change (above or below −22.7%) or by median absolute T 50 Follow up (above or below 192.5 min) at time of 24 months FU. Median age of the entire   (Table 1). Whereas in the total study population absolute phosphate levels tended to decline between baseline (1.7 ± 0.5 mmol/l) and FU (1.6 ± 0.5 mmol/l; p = 0.003), patients with declining T 50 values (T 50 Change < −5.1%) presented with stable high phosphate levels (1.7 ± 0.5 mmol/l; p paired t-test = 0.31). Subjects presenting with stable or increasing T 50 at baseline and 24 months FU showed declining phosphate levels between baseline and 24 months FU (1.8 versus 1.5 ± 0.4 mmol/l, respectively; p < 0.001). Both groups did not significantly differ in phosphate levels measured at baseline (p = 0.43) but exhibited significantly different phosphate at 24 months FU (Fig. 3A). Δ phosphate was significantly correlated with T 50 Change (r = −0.39; p < 0.001; Fig. 3B), suggesting a permanently elevated or increasing phosphate associates with declining T 50 . Δ phosphate remained the strongest regressor of T 50 Change in multivariable linear regression (β = −8.4; 95%CI: −13.1 to−3.6; p = 0.001). R² of the model created was 0.24 -"(enter method)". Additionally, Kt/V was a significant regressor of T 50 Change (β = 5.4; 95% CI: 0.8 to 9.9; p = 0.001; Table 2). Of note, lower absolute T50 at FU was associated with higher parathyroid hormone and IL-6 levels (p = 0.01 and 0.03 respectively, Table 1).
Declining T 50 predicts all-cause and cardiovascular mortality in haemodialysis patients. After 24 months of observation, patients were followed up for all-cause (n = 43) and CV mortality (n = 17) for a median of 19  months. Three patients underwent renal transplantation and 12 were lost to FU (Fig. 1). In Kaplan T 50 non-decliners T 50 decliners Change values. Patients displaying a decline in T 50 (defined by T 50 Change < −5.1%; 5,1% was the inter-assay coefficient for T 50 standards at 260 min in the EVOLVE cohort) were stained red. Absolute number of patients and (percent of total) are reported with dashed rectangles. The same colour code was used to stain the histogram in B.
In contrast, stratification of patients by median of T 50 Follow up didn't significantly predict all-cause mortality in our cohort (Fig. 4B). Likewise, stratification of the study population by T 50 Baseline was not significantly associated with all-cause mortality (Supplementary Figure 2), likely due to the preselection described above.
By contrast, neither absolute T 50 Follow up nor T 50 Baseline remained significant predictors of all-cause mortality in the final adjusted model (supplementary Table 2 and supplementary Table 3, respectively) and were relevantly associated with CV mortality in our study (not shown).

Discussion
The main findings of this subgroup analysis in chronic haemodialysis patients, were, firstly, that serum calcification propensity T 50 declined between baseline and FU. Secondly, that a decline in T 50 was accompanied by increasing phosphate levels at baseline and 24 months FU. Thirdly, that the intra-individual decline of calcification resistance predicts all-cause and cardiovascular mortality in HD patients. These findings suggest that observing change of T 50 might add prognostic value to absolute T 50 values in HD patients.
The development of the T 50 -test was an important step ahead for a more comprehensive assessment of colloidal chemical interactions within the complex field of "calcification and calcium x phosphate nanocrystal formation" 5,10 . Elevated serum phosphate, calcium, low magnesium and fetuin-A levels have long been linked to vascular calcification and mortality in ESRD and HD patients [14][15][16][17][18][19][20][21][22][23][24][25] . The colloidal chemical nature of the crystallization process per se suggests strong interaction effects amongst these players, which is in line with findings from cohort studies 16,[26][27][28] . In fact, mortality is especially high for patients with both excessively high phosphate and calcium serum levels 21,28 . On the contrary, magnesium appears to dampen the detrimental effects of elevated phosphate levels in HD patients 16 . The strength of the T 50 -test is that it takes these interactions into account by assessing the distal tract of the calcification cascade, i.e. the interaction between calcium and phosphate, which takes place in the CPP-forming biological matrix of human serum 6 . Elevated serum phosphate, calcium and lower serum albumin have already been associated with lower absolute T 50 at a single time point in CKD and HD patients 7,10 . Our data add further plausibility to this concept by indicating, that high or increasing phosphate, parallels declining T 50 . We did not, however, observe a similar relation for Δ albumin and Δ calcium, Metric variables were centred on their mean before entering the model. All variables were included at once. Patients characteristics at 24 months FU were used for modelling. Δ phosphate, Δ calcium and Δ albumin were calculated as value Follow up −value Baseline Abbreviations: Body mass index (BMI); Vitamin D receptor activators (VDRA); coronary heart disease (CHD); peripheral arterial occlusive disease (PAOD); Interleukin-6 (IL-6).
which might in part be due to a lower variance of these parameters between baseline and 24 months FU in our cohort. Nevertheless, our data underscore the importance of controlling serum phosphate levels in HD patients, as high or rising phosphate levels may well weaken the natural resistance against the formation of secondary CPP. However, given the moderate fit of our linear regression model, besides Δ phosphate or Kt/V other factors will also relate to declining T 50 . Magnesium is such a candidate indicated by our data. Unfortunately, we were unable to investigate Δ ionized magnesium levels over time, due to an incomplete magnesium-dataset at baseline.
Given that a short T 50 time represents lower resistance to secondary CPP formation 5,6 , a decline of T 50 , as observed in our study, implies the loss of anti-calcific capacities of biologic fluids. In our cohort, a decline of T 50 occurred over the course of dialysis dependency and was independently associated with all-cause mortality and remained associated with cardiovascular mortality after adjustment for potential confounders. As such, the per cent decline of T 50 between baseline and 24-months reassessment showed a considerably stronger association with mortality than absolute T 50 Follow up . Since absolute T 50 values are to a large extent genetically determined in the general population 13 , it appears plausible that the dynamics of T 50 predict future risk more accurately in HD   patient, which face longitudinal weakening of their "natural" calcification resistance. By analogy, prognosis of e.g. calcific aortic stenosis is not only determined by the absolute reduction of valve area but also the rapidity of stenosis progression which is especially high in HD patients 29,30 . Based on this reasoning, it is tempting to speculate that preventing a decline of T 50 in HD patients or restoring T 50 in those who present with declining T 50 might favourably affect the future clinical course. Importantly, T 50 represented the only modifiable risk factor in our adjusted Cox model for mortality. In fact, first interventional pilot studies suggest that increasing dialysate magnesium and bicarbonate act concordantly to prolong T 50 time in HD patients 31 . Similarly, phosphate binder therapy has the potential to elevate T 50 32 .
Our study has several limitations. Although this is the first study to assess individual dynamics of serum calcification propensity in HD patients it doesn't fulfil all criteria for studying change, since T 50 was only assessed from sera collected at two not three different time points. In addition, the study design required exclusion of patients who did not survive the first 24 months FU period or who missed timely FU. Therefore, the study population was significantly enriched for survivors, which limits the generalizability of our results and explains the lack of predictive potential of T 50 Baseline . Due to the relatively low number of cardiovascular deaths we were unable to fully adjust the Cox model for cardiovascular mortality. The study design and post hoc character of the study preclude conclusions regarding causality and therefore, the pathophysiologic mechanism linking declining T 50 to excess mortality remains elusive. We could not consider dietary calcium or phosphorus intake since these parameters were not available in the dataset. Lastly, although these data provide a fascinating new perspective on intra-individual serum calcification resistance and the interpretation of the T 50 test, larger scaled studies are needed to replicate and develop these results.
In conclusion, longitudinally monitoring changes in T 50 -time in HD patients might offer a suitable tool to identify patients at risk for adverse outcome and open the conceptual possibility of basing multimodal and personalized therapeutic interventions on longitudinal changes of T 50 5 . Larger scaled prospective interventional studies are needed to put this concept to a test.

Methods
Subjects/Study population. This study was undertaken in a subset of patients of the original "rISk strAtification in end stage Renal disease" -(ISAR)-cohort, a multicentre, prospective longitudinal observatory cohort study 33 . Between 2010 and 2013, 519 stable HD patients from 17 dialysis centres in Munich, Germany and the surrounding area were included. Out of these patients the present study included 188 patients, which were still alive and active in the study after the first FU period of 24 months with a maximum spread of ±3.2 months and for whom sera were available at both baseline and follow-up. Patients for whom timely 24 [23][24][25] months FU had been missed were excluded a priori (Fig. 1). Patients were ≥18 years of age, had an HD vintage of least 90 days and gave written and informed consent. The ISAR study was approved by the ethics committees of the Klinikum rechts der Isar, Technical University Munich and of the Bavarian State Board of Physicians 33 . It was carried out in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants. The study was registered under ClinicalTrials.gov identifier: NCT01152892 prior to its start. For the derived study, no additional ethics committee approval was sought. For more details, we refer to the study protocol 33 . Clinical data assessment. Patients' age, comorbidities and medication at FU were assessed using medical records from the contributing dialysis centres. Comorbidities were recorded following an adapted version of the Charlson Comorbidity Index (CCI) for ESRD as introduced by Liu et al. 34 . BMI at FU was calculated as body weight/height 2 [kg/m 2 ]. Access type (fistula or permanent catheter) was determined at time of FU and considered constant for the consecutive observation period. Information on dialysis prescription (Ultrafiltration, session duration, Kt/V as a measure of dialysis efficiency, HD/HDF, anticoagulation) was provided by the contributing centres. All patients underwent regular bicarbonate dialysis with synthetic membranes. For details, we refer to the study protocol 33 .  Endpoints. The primary outcome parameters for the ISAR-trial and this project were all-cause and CV mortality. Observation for all-cause and cause-specific mortality started the day after the 24 month FU visit. In the absence of a final medical report stating the cause of death, attending physicians and relatives were contacted to confirm death and gather available information, based on which the ISAR-study physician board assigned each case an underlying cause of death. CV mortality (n = 17) was due to sudden cardiac death (n = 6), myocardial infarction (n = 4), heart failure (n = 1), cardiac surgical procedure (n = 2), pulmonary embolism (n = 1), major stroke (n = 2) and rupture of aortic aneurysm (n = 1). Non-CV lethal events (n = 26) comprised infectious events, gastrointestinal bleeding, non-CV related surgery, withdrawal from treatment, chronic pulmonary disease, malignant diseases and unknown causes of death.
Blood specimen collection and Laboratory methods. Serum was collected prior to a midweek dialysis session at time of inclusion and at FU. Serum was centrifuged after 30 min of resting at room temperature, aliquoted and frozen at −20 °C, transferred to our laboratory on dry ice and stored at −80 °C for later analysis. Routine laboratory analysis was performed by ISO certified laboratories. IL-6 was determined using BD Flex-sets on a FACS Canto II and BD Diva software following the manufacturer's instructions. BD FCAP Array software 3.0 was used for analysis. Ionized magnesium levels were determined from frozen sera using the Nova 8 Analyzer (Nova Biomedical, Waltham, MA, US). T 50 was determined at previously described by Pasch et al. 6,10 . Sera had never been thawed prior to analysis and were sent to Bern on dry ice and analyzed in a blinded manner. Median storage duration was 48 [42-70] or 24  months for samples collected at baseline or 24 months follow, respectively.
Statistical Analysis. Statistical analysis was performed using IBM SPSS Statistics 23 and R version 3.3.2 software. We present mean ± standard deviation (SD), median and [interquartile-range] or counts and (% of superset) for normally distributed data, non-normally distributed metric and ordinal variables or nominal data, respectively. IL-6 levels were natural logarithm-(ln)-transformed to adjust for skewness of distribution.
Paired t-test was used to compare differences between absolute T 50 -values at baseline and 24 months FU. , respectively for description of baseline characteristics at time of 24 months FU. Group differences were tested using unpaired t-test, Mann-Whitney-U, χ²-test and paired t-test as appropriate. Additionally, we assessed associations of continuous variables with T 50 Change and T 50 Follow up using Pearson correlation. Median T 50 Follow up and median T 50 Change were also used to stratify patients for univariate Kaplan-Meier survival analysis of all-cause and CV mortality. Cumulative incidence functions were estimated for CV and non-CV deaths to account for competing risks. Log-rank tests were performed to compare (cause specific) hazard rates between relevant groups. Patients were censored at time of transplantation or at time of loss to FU.
A multivariable linear regression model was built including age, sex, BMI, coronary heart disease (CHD), peripheral arterial occlusive disease (PAOD), diabetes, hypertension, intake of calcium containing phosphate binders, Vitamin D3 supplementation and laboratory parameter (calcium, magnesium, albumin, phosphate, intra-individual Δ-phosphate (=phosphate Follow up −phosphate Baseline [mmol/l]) and Δ-calcium, Δ-albumin to identify factors that were associated with T 50 Change . Coefficients of regression are reported per 1 SD increase of the regressor. Variance of inflation factors were below 2 for all entered variables. Cox proportional hazard models were fit to the data to characterize the association of T 50 Change with all-cause mortality. Proportionality of hazard rates was investigated by the use of Schoenfeld residuals and a statistical test for proportional hazard proposed by Grambsch and Therneau 35 .
The models were adjusted for demographic factors (age, gender, BMI), comorbidities (following the adapted CCI as proposed by Liu et al. 34 ), albumin and ln-transformed IL-6. Hazard ratios (HR) and 95 % confidence interval (95% CI) are reported per 1SD of T 50 Change (≈30.5 % change in T 50 ) during the 24 months FU, 2 of 188 cases were censored before the earliest event had occurred. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.