Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1785 Influence of bone-associated and cardiovascular biomarkers on vascular events and mortality in relation to renal dysfunction PING-HSUN WU ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2021 ISSN 1651-6206 ISBN 978-91-513-1336-8 URN urn:nbn:se:uu:diva-456907
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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1785
Influence of bone-associated and cardiovascular biomarkers on vascular events and mortality in relation to renal dysfunction
PING-HSUN WU
ACTA UNIVERSITATIS
UPSALIENSIS UPPSALA
2021
ISSN 1651-6206 ISBN 978-91-513-1336-8 URN urn:nbn:se:uu:diva-456907
Dissertation presented at Uppsala University to be publicly examined in Enghoffsalen, Ing 50 bv, Akademiska sjukhuset, Uppsala, Wednesday, 15 December 2021 at 09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Associate Professor Jonas Spaak (Cardiology at the Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet).
Abstract Wu, P.-H. 2021. Influence of bone-associated and cardiovascular biomarkers on vascular events and mortality in relation to renal dysfunction. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1785. 63 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1336-8.
Biomarkers can help physicians identify subjects with an increased cardiovascular risk. Apart from the clinical factors, some biomarkers have been recognized as important predictors and risk factors for cardiovascular disease in renal disease. The applicability of biomarkers may be limited in patients with kidney disease due to the complex etiology of cardiovascular disease, which warrants separate evaluations, including established and novel biomarkers. The overall aim of the thesis was to investigate the association between bone-associated markers and cardiovascular proteins on death and vascular events in the elderly male population and patients with kidney disease.
Study I included 3,014 participants in Swedish multicenter prospective Osteoporotic Fractures in Men (MrOS) cohort and investigated the associations between Klotho single-nucleotide polymorphism and mortality. Two potentially damaging single-nucleotide polymorphisms (rs9536314 and rs9527025) in the Klotho gene were not associated with mortality.
Study II investigated the association between mineral bone markers and all-cause mortality / cardiovascular mortality. The composite evaluation of elevated fibroblast growth factor-23 levels, vitamin D deficiency, and renal impairment was associated with mortality.
Study III evaluated the bone-associated proteins and mortality/composite vascular events in the 331 Demark hemodialysis patients. Osteoprotegerin, as one of the most promising bone-related proteins, was associated with composite vascular events independent of cytokine.
Study IV investigated the association between 92 proteins measured by proximity extension assay and mortality/composite vascular events in hemodialysis patients. A higher level of Interleukin-8, T-cell immunoglobulin and mucin domain 1, C-C motif chemokine 20, and lower level of stem cell factor and galanin peptides were associated with poor outcomes.
This thesis addressed the issue of bone-vascular axis and cardiovascular disease. We evaluated from gene levels to circulating protein levels and from the general population to patients with kidney disease. Based on our research findings, more evidence was linked between bone and vascular complications. We also identified several cardiovascular proteins considered potentially important predictors for cardiovascular disease in patients with renal failure, especially hemodialysis patients.
Ping-Hsun Wu, Department of Medical Sciences, Endocrinology and mineral metabolism, Akademiska sjukhuset, Uppsala University, SE-751 85 Uppsala, Sweden.
in patients treated with hemodialysis. Nephrol Dial Transplant,
Jun 4;gfab192. doi: 10.1093/ndt/gfab192.
IV Ping-Hsun Wu*, Rie Io Glerup*, My Hanna Sofia Svensson,
Niclas Eriksson, Jeppe Hagstrup Christensen, Philip de Laval,
Inga Soveri, Magnus Westerlund, Torbjörn Linde, Östen Ljung-
gren, Bengt Fellström. Novel biomarkers detected by proteomics
predict death and cardiovascular events in hemodialysis patients.
(Submitted)
*Authors contributed equally to this work
Reprints were made with permission from the respective publishers.
Contents
Introduction ................................................................................................... 11 Cardiovascular disease in patients with chronic kidney disease .............. 11 Chronic kidney disease and mineral bone disease ................................... 12 Biomarkers in patients with chronic kidney disease ................................ 12 Vascular calcification and cardiovascular disease ................................... 13 Bone-vascular axis ................................................................................... 13 Bone markers in patients with kidney disease .......................................... 15 Protein biomarkers and cardiac structure and function ............................ 15
Proteomics technology .................................................................................. 17 Brief introduction of proteomics .............................................................. 17 Biotechnology of clinical proteomics using proximity extension assays . 18
Aims .............................................................................................................. 19 General aim .............................................................................................. 19 Specific aims of the studies ...................................................................... 19
Study samples ............................................................................................... 21 The Swedish part of the Osteoporotic Fractures in Men Study (MrOS) .. 21 Denmark Århus Hemodialysis Cohort ..................................................... 21
Methods ........................................................................................................ 22 Study designs and methods ...................................................................... 22 Biomarker measurements ......................................................................... 27 Outcomes definitions and mortality assessment ....................................... 29 Statistical analysis .................................................................................... 30
Main results ................................................................................................... 33 Study I ...................................................................................................... 33 Study II ..................................................................................................... 33 Study III ................................................................................................... 35 Study IV ................................................................................................... 36
Additional Preliminary Study ....................................................................... 37 Method and Statistical analysis of the additional preliminary study ........ 37 Preliminary results of additional study ..................................................... 38
Klotho gene SNPs and all-cause or CV mortality .................................... 42 The mineral bone factors and all-cause or CV mortality ......................... 43 Bone-associated markers and mortality or CVE in hemodialysis
patients ..................................................................................................... 45 CV-associated markers for mortality or CVE in hemodialysis patients ... 46 Mortality differences among hemodialysis patients between Asian and
Caucasian ................................................................................................. 48 Strengths and limitations .......................................................................... 50
Conclusion and future perspectives .............................................................. 51
Summary in Swedish (sammanfattning på svenska) ..................................... 53
Figure 4. Kaplan–Meier curves for (A) all-cause mortality and (B) composite vascu-lar events in patients of Demark Århus hemodialysis cohort and Taiwan KMUH he-
modialysis cohort
Figure 5. Kaplan-Meier curves of hsTNT tertiles for all-cause mortality in patients of Demark Århus hemodialysis cohort, Taiwan KMUH hemodialysis cohort, and in
both cohorts combined.
Figure 6. Kaplan-Meier curves of hsTNT tertiles for CVEs in patients
of Demark Århus hemodialysis cohort, Taiwan KMUH hemodialysis cohort, and in both cohorts combined
41
Table 3. The comparison of all-cause mortality, composite vascular events, and single vascular event (myocardial infarction, ischemic stroke, peripheral artery disease) in hemodialysis patients between Taiwan KMUH cohort and Demark Århus cohort
Demark Århus cohort 1 [Reference] 1 [Reference] 1 [Reference] 1 [Reference] 1 [Reference] aModel 1: adjusted for age, sex, body mass index, systolic blood pressure, diastolic blood pressure, cause of ESKD, and hemodialysis vintage. bModel 2: model 1 with further adjusted for comorbidities (Diabetes mellitus, Myocardial infarction, Ischemic stroke). cModel 3: model 2 with further adjusted for clinical laboratory data (Hemoglobin, Albumin, Low-density lipoprotein, C-reactive protein, Total
Calcium, Phosphate, Parathyroid hormone), and propensity score. dModel 4: Confounders control using weighted by stabilized inverse probability of treatment weighting (stabilized IPTW)
42
Discussion
Klotho gene SNPs and all-cause or CV mortality
In study I, SNPs in the klotho gene were evaluated by computational pro-
grams and databases that use various methods to predict SNP damage. The
nonsynonymous SNPs rs9536314 and rs9527025 were classified as the most
damaging. In the clinical study, unadjusted logistic regression analysis using
an additive model found an association of all-cause mortality risk with the TT
genotype of rs9536282, the GG genotype of rs9536314, and the CC genotype
of rs9527025. However, no statistically significant associations between pol-
ymorphisms in klotho and mortality risk were observed in unadjusted Cox
regression or adjusted logistic regression models. Results from the dominant
and recessive models were similar. Subgroup analyses of rs9536314 and
rs9527025 stratified by eGFR or FGF23 also produced negative results for the
MrOS cohort.
A nonsignificant trend of association was observed for the nonsynonymous
SNPs rs9536314 and rs9527025 with all-cause mortality, but not CV mortal-
ity. The SNPs rs9536314 (F352V) and rs9527025 (C370S) have been reported
to be related to CV diseases.67, 68 However, a meta-analysis of rs9536314
(F352V) and rs9527025 (C370S) with respect to CV disease failed to indicate
any statistically significant association in the additive genetic model, the dom-
inant genetic model, or the recessive genetic model.69 The minor allele fre-
quency differences for rs9536314 and rs9527025 in Sweden’s MrOs data and
other databases (the 1000 Genomes EUR Population, the HapMap CEU Pop-
ulation, and the Genome Aggregation Database European Population) might
partly explain the outcomes difference. Although computational prediction
signaled a “damaging” effect of the klotho KL-VS genotype in our study, no
statistically significant differences were observed in the cohort, possibly be-
cause the cohorts were different in age, sex, ethnicity, and comorbidities. The
functional klotho KL-VS variant results in amino acid substitutions that alter
secretion, catalytic activity, and functionality of the αkl protein.70, 71 However,
a study has also reported that substitution SNPs rs9536314 (F352V) and
rs9527025 (C370S) alter amino acid-related shedding and trafficking. Still,
the effects are small, and intragenic complementation could further minimize
the effects in vivo.72
43
Another klotho gene synonymous variant, rs564481 (C1818T), located in the
fourth exon, showed no association with all-cause or CV mortality in our
study, according to earlier studies that showed no CVD correlation for this
polymorphism.68, 69, 73 Although rs564481 is a synonymous variant, it is likely
to be functionally relevant, given that reports have associated it with CV risk
factors (blood pressure, glucose metabolism, and lipid levels)74, 75 and CAD76
in Asian populations. The SNP rs577912 was previously related to higher
mortality risk in patients receiving hemodialysis77 but our clinical study re-
sulted in negative findings. Interestingly, the computational tool FATHMM
predicts that the rs577912 genotype could be a damaging intron non-coding
region; however, no association with mortality outcomes was found in our
study.
The mineral bone factors and all-cause or CV mortality
In Study II, we demonstrated that high FGF23 was associated with all-cause
and CV mortality, especially in individuals with moderate to severe CKD
(CKD 3B or worse). Moreoever, in an RF analysis, compared with vitamin D
or PTH, FGF23 was found to be the mineral metabolic factor most important
to all-cause mortality and CV mortality. However, that association was atten-
uated after controlling for renal function. Conversely, vitamin D deficiency
was independently associated with all-cause mortality, but not CV mortality.
High and low PTH showed no relationship with mortality in our study. In a
combined evaluation, as the values for FGF23, vitamin D, and renal function
became more abnormal, all-cause and CV mortality were found to rise. Be-
cause compensation effects between these mineral bone factors are known,
FGF23 excess, vitamin D deficiency, and renal impairment together imply
loss of the compensating effect and might predict poor outcomes. Our findings
tease out the complexity of these mineral metabolic factors, renal function,
and mortality relationships.
In our study, the association of FGF23, but not vitamin D, with the risk for
all-cause mortality attenuated after adjustment for eGFR, indicating that renal
function remains the central factor. In contrast, the association of vitamin D
deficiency with mortality risk was independent of known mineral metabolism
and renal function. As reported, vitamin D deficiency was found in several
meta-analytic studies to be associated with mortality.78-80 However, vitamin D
deficiency can result from comorbidities and nutrient deficiencies, which
could themselves explain the observed links with mortality.81, 82 Currently, no
conclusive evidence shows that vitamin D supplementation provides a sur-
vival benefit in adults.83 In fact, vitamin D supplementation increases FGF23
concentrations84, which might counter the benefits of vitamin D supplements.
44
FGF23 can suppress the production of active vitamin D metabolites. Any det-
rimental effect of FGF23 might be influenced by reduced vitamin D activity.85
In our study, vitamin D deficiency was found to be a factor independently as-
sociated with all-cause mortality beyond FGF23. Other reports support our
finding that the highest risk for all-cause mortality was found in hemodialysis
patients with a low vitamin D level and with a high FGF23 level.19
High serum FGF23 has been observed as a physiologic reaction to impaired
renal function when renal phosphate excretion is compromised.86 The health
impact of these mineral bone factors should therefore be evaluated simultane-
ously. During the early stages of CKD, expression of the renal FGF23 co-
receptor, αkl, progressively declines in response to kidney damage and de-
clines along with the loss of functional nephrons are lost, promoting partial
resistance to FGF23’s physiologic actions.87-89 As a compensatory mecha-
nism, FGF23 rises above normal values by a factor of 1000 to maintain a neu-
tral phosphate distribution.87, 90, 91 The compensatory increase in FGF23 pro-
motes the suppression of 1-25-dihydroxyvitamin D production, which in turn
promotes the elevation of PTH, causing secondary hyperparathyroidism.92
Thus, renal dysfunction is the major contributing factor to increased circulat-
ing FGF23. As demonstrated in our study, high FGF23 was not associated
with risk for all-cause mortality after adjustment for eGFR. Thus, high FGF23
is a consequence, but not a cause, of risk for mortality.
FGF23 has been associated with non-CV causes of death,93 reflecting a lack
of specificity between a rise in FGF23 concentration and disease risk. In our
study, a higher level of FGF23 was not associated with increased CV mortality
after adjustment for diabetes, hypertension, stroke, and CAD. Theoretically,
FGF23 rises before any other marker of mineral bone disorder, and so it tem-
porally mirrors the rise in CV risk as kidney disease progresses. Many condi-
tions can cause that rise, and its presence and severity generally reflect disease
activity. Nevertheless, elevated FGF23 has still been associated with mortality
across a spectrum of CKD.91, 92 In our study, lower CKD status, particularly
eGFR less than 45 ml/min, produced higher mortality spline plots. Because
the kidney is the main source of systemic αKlotho, any reduction in nephron
numbers and in eGFR is followed by a progressive decline in circulating
αKlotho.94 That the protection conferred by αkl is greater than the deleterious
effect of FGF23 has been demonstrated in clinical95 and animal studies.96 This
raises the possibility that αKlotho deficiency may be the primary alteration in
MBD.97 Thus, we found that renal function is the crucial factor contributing
to the risk association between FGF23 and mortality.
45
Bone-associated markers and mortality or CVE in hemodialysis patients
In Study III, we evaluated the relationships of 9 bone-associated biomarkers
with all-cause and CV mortality, and CVEs in 331 patients receiving HD. Us-
ing an RF approach, the importance of the bone biomarkers with respect to
outcomes was evaluated, and the top biomarkers were TRAIL-R2 for all-cause
death, OPG for CV death, and both OPG and TRAIL-R2 for CVEs. However,
only OPG was independently associated with CVEs in a cytokine adjustment
model. OPG could potentially provide clinical information beyond that cur-
rently available for the prediction of CVEs. Relationships of the bone-associ-
ated proteins, such as OPG, TRAIL-R2, CTSD, and CTSL1, with CV disease
have previously been reported.98-105 Our study also showed a relationship be-
tween TRAIL-R2 and OPG106 with higher CV risk. However, in a departure
from previous reports,104, 107 we found no correlation between low TRAIL and
poor outcomes.
In our study, OPG was the strongest predictor of CVEs. Elevated OPG has
previously been associated with aortic stiffness and coronary artery calcifica-
tion,108, 109 as well as CV outcomes.110 Interestingly, the RF approach was re-
cently used to show an association of OPG with CVEs in non-dialysis CKD.
Forne et al. investigated 19 biomarkers in a subcohort of the NEFRONA study
(Observatorio Nacional de Atherosclerosis en NEFrologia).111 They used var-
iable importance from the RF analysis for CV risk in the first step and then
evaluated the most promising variables for CVE in a Fine and Gray competing
risks model. OPG was a potential predictor of CV risk in the RF analysis and
also a statistically significant predictor of increased CVEs in the competing
risks model.111 Elevated OPG had already been associated with increased CV
mortality112-114 in patients treated with hemodialysis. Compared with our
study, the study by Sigrist et al. enrolled patients receiving hemodialysis who
were younger and had lower mortality rates109 ; patients receiving hemodialy-
sis who were enrolled in other studies by Morena et al.112 and Winther et al.114
had a higher prevalence of baseline CV diseases. In contrast to these previous
studies,112-114 OPG was not an independent risk marker for all-cause mortality
in our study. The results of discrepancies may exist because of different con-
founders' adjustments across studies.
Although a positive association between baseline OPG concentration and risk
for CV disease has been shown, some analytic issues should be discussed. No
study has evaluated the independent association between OPG and CVEs in a
model adjusted for inflammatory cytokines.108, 109, 112, 113, 115 Inflammation is
well known to be a significant risk factor for mortality and CV events in pa-
tients with CKD.116 Inflammatory cytokines influence the production of OPG,
suggesting that the OPG/RANKL/RANK system plays a modulatory role in
46
vascular injury and inflammation.117 One study suggested that OPG levels are
significantly correlated with inflammatory markers, such as IL-6.118 As
demonstrated in our study, OPG was positively associated with inflammatory
cytokines, such as IL-6, IL-8, IL-16, IL-18, IL-27, IL-1RA, and IL-6RA.
However, we found no significant correlation between OPG and CRP, in line
with previous studies in patients with CKD and those receiving hemodialy-
sis.109, 112 It has been suggested that OPG alone or in combination with elevated
CRP is associated with worse outcomes in such patients.109, 112 In addition,
after adjustment for confounders (including CRP), OPG was found to be a
significant independent predictor of PAD in patients receiving peritoneal di-
alysis.119 In our adjusted multivariable model, we also demonstrated that OPG
predicts CVEs even after adjustment for CRP. Furthermore, we demonstrated
that the association between OPG and CVEs was independent of cytokine ac-
tivity or calcium/phosphate/PTH parameters. More studies are needed to con-
firm our findings.
CV-associated markers for mortality or CVE in hemodialysis patients
In Study IV, a recent PEA-based proteomics study in the Mapping of Inflam-
matory Markers in Chronic Kidney disease (MIMICK) cohort, TIM-1, matrix
TRAIL-R2, spondin-1, and FGF23 showed significant associations with CV
death after adjustment for age and sex in 183 patients receiving HD.120 Several
of the proteins reported in the MIMICK study were also correlated with CVEs
in our study—specifically, TIM-1, MMP-7, IL-6, brain natriuretic peptide,
HGF, and TRAIL-R2. However, some proteins associated with CV death in
our study (e.g., tissue plasminogen activator [tPA], CCL20, GAL, IL-8, and
SCF) were not associated with CV death in the MIMICK cohort. Importantly,
TIM-1, also known as kidney injury molecule-1, was the most important risk
marker for CV death in the MIMICK cohort,120 a result confirmed in our study.
Importantly, TIM-1, also known as kidney injury molecule-1, was the most
important risk marker for CV death in the MIMICK cohort.120 There could be
several reasons for the differences in the two studies, including a smaller sam-
ple size in MIMICK (n=183), the longer dialysis vintage in our study, and
adjustment for different confounders. In addition, the definition of CV death
was validated by a physician in our study, while the MIMICK cohort used
International Classification of Diseases diagnostic codes. Moreover, in a dis-
covery-validation study of individuals with CKD not receiving dialysis,
MMP-12 was significantly associated with an increased risk of major adverse
CV events (fatal or nonfatal myocardial infarction or fatal and nonfatal
47
stroke).121 That research also agrees with our finding that MMP-12 is an inde-
pendent biomarker predicting CVEs in patients receiving HD.
Chronic inflammation is an essential CV risk factor in patients with kidney
disease, characterized by enhanced production of CRP and other inflamma-
tory mediators, including IL-6 and IL-8. Those cytokines and chemokines
were previously associated with all-cause and CV mortality in patients receiv-
ing hemodialysis.122-125 In line with previous studies in such patients,126 EN-
RAGE, another inflammatory biomarker, was also positively associated with
CVEs. Furthermore, we found that TRAIL-R2 (member protein of the TNF-
receptor superfamily) and OPG, reflecting the bone-vascular axis, were asso-
ciated with a higher risk of CVEs. OPG has previously been described as a
CV marker for all-cause death in hemodialysis patients,112-114 and TRAIL-R2
is present in human atherosclerosis lesions, with higher expression in vulner-
able plaques than in stable ones.106
Potential mechanisms and clinical studies of novel biomarkers
Several novel biomarkers, such as HGF, CCL20, tPA, GAL, and SCF, were
related to death or CV outcomes.
HGF is a pleiotropic cytokine involved in regulating several biologic pro-
cesses, such as cardiometabolic activity, inflammation, angiogenesis, and tis-
sue repair.127 HGF is associated with CAD128 and ischemic stroke129 in patients
without CKD. In hemodialysis patients, higher levels of HGF are associated
with concentric left ventricular geometry130 and cerebral infarction.131 Those
associations might be related to leukocyte activation during hemodialysis.132
Our study is the first to demonstrate that HGF is associated with an increased
risk of both all-cause mortality and CVEs in hemodialysis patients.
Previous studies have shown that CCL20 excretion is increased in patients
with stage 5 CKD compared with healthy individuals and patients with
stages 1–3 CKD.133 Furthermore, patients with ischemic heart disease have
higher levels of circulating CCL20,134 and CCL20 is expressed in atheroscle-
rotic plaques.135 A recent study demonstrated that CCL20 is associated with
increased CVEs in patients with stage 3-5 CKD.121 CCL20 contributes to vas-
cular endothelial inflammation136 and triggers pathways similar to those acti-
vated by low-density lipoprotein cholesterol.135 The IL-6, IL-8, and CCL20
cytokines or chemokines have been associated with the Th17 CD4 lympho-
cyte.137 Several studies have shown that Th17 cells play a critical role in arte-
riosclerosis development.138 Genetic deletion of CCL20 receptors in Apoe−/−
mice decreases atherogenesis and endothelial inflammation.139 We, therefore,
speculate that the accumulation of CCL20 might significantly affect the risk
for CV disease in patients receiving hemodialysis.
48
The tPA glycoprotein is involved in coronary plaque rupture140 and is consid-
ered a marker for regulating endogenous fibrinolysis.141 In the general popu-
lation without kidney disease, there is an association between elevated circu-
lating levels of tPA and subsequent coronary heart disease.141 In general, cir-
culating levels of tPA are elevated in patients receiving dialysis compared
with healthy individuals,142 and circulating levels of tPA in hemodialysis pa-
tients are positively associated with several CV risk factors, such as age,
smoking, blood pressure, and CRP.142
GAL is an endocrine hormone of the central and peripheral nervous systems
involved in central CV regulation, affecting heart rate and blood pressure.143
However, functional properties of the galaninergic system are not fully eluci-
dated for cardiac diseases. In an animal model, GAL can limit myocardial in-
farction size and improve post-ischemic cardiac function recovery.144 It also
suppresses myocardial apoptosis and mitochondrial oxidative stress in cardiac
hypertrophic remodeling.145 These basic studies could support our results of
the negative association between GAL and CV mortality in patients receiving
hemodialysis.
The association between high levels of SCF and lower all-cause or CV mor-
tality was a novel finding of our study. Similar results were described in the
general population (Malmö Diet and Cancer study)146 and in patients with sta-
ble CAD.147 SCF is involved in vasculogenesis and cardiac repair by stimulat-
ing the recruitment and activation of bone marrow-derived stem cells and tis-
sue-resident progenitors.148 An increase in SCF expression occurs naturally in
response to myocardial infarction, which mediates the migration of c-kit+ car-
diac and bone marrow cells to the injured area for cardiac remodeling.149 We
found that circulating SCF was positively correlated with albumin. Albumin
is well known to be a predictive marker in patients receiving hemodialysis.
We speculate that SCF might play a protective role in vascular injury and
could reflect the clinical nutrition status in patients receiving hemodialysis.
Mortality differences among hemodialysis patients between Asian and Caucasian
In the additional preliminary study, we found that patients in Demark’s År-
hus HD cohort had a higher risk for all-cause mortality and CVEs than patients
in Taiwan’s KMUH HD cohort. Interestingly, racial and ethnic differences in
mortality for patients receiving HD have been reported in several studies.150-
153 Some reports suggested a 25% to 35% lower mortality rate in Asian pa-
tients than in White patients.154, 155 The causes of these ethnic disparities re-
main largely unknown. Factors that might explain the mortality differences
49
include genetic and environmental factor differences151 and racial disparities
in diet, nutrition, and inflammation.156 This recent comparison between hemo-
dialysis patients in the two nations will be developed further into a full manu-
script and hopefully published in the near future.
50
Strengths and limitations
Strengths of Studies I and II include the large sample size of the MrOS cohort
of community-dwelling men with nearly complete follow-up of surviving co-
hort participants, and outcome measures that were well-validated because of
high-quality national registers. We used 14 different computational tools in a
bioinformatics approach, plus a clinical study, to perform a complete evalua-
tion of klotho gene polymorphism. However, several limitations must still be
addressed. First, self-reported questionnaires were used at baseline visits, and
we cannot exclude the possibility of underestimation of smoking or disease
prevalence. Second, the study was observational, and so causality was not pos-
sible to cannot be determined. Third, the generalizability of our study is lim-
ited to healthy community-dwelling Swedish males. Fourth, the minor allele
frequency of rs9536314 homozygotes could be different in elderly and young
subjects.70 The latter limitation might also affect our study result because of
the enrolled elderly population in the MrOS cohort (Study I). Fifth, FGF23,
vitamin D, and PTH were measured at a single point, so we could not evaluate
those mineral metabolic factors as time-varying covariates (Study II). Sixth,
residual and unmeasured confounders might remain despite the attempt to ad-
just for potential confounders such as genetics and other CV biomarkers
(hsTNT) (Study II). Lastly, we could not evaluate other FGF23 regulators or
mediators (i.e., iron metabolism,157, 158 inflammatory proteins,158, 159 or 1-25-
dihydroxyvitamin D158) because of unavailable data in the Sweden MrOs co-
hort (Study II).
In Studies III and IV, the strengths of our investigation include a longitudinal
study design, proteomics chip for simultaneous measurement of multiple
bone-associated proteins, and exploration analysis using the RF approach.
Limitations include the observational design, including prevalent patients with
varying disease durations and low statistical power in some of our analyses.
The study focused mainly on White patients receiving HD therapy in Scandi-
navia, so extrapolations to non-White patients receiving HD or patients with
CKD not yet on dialysis should be made cautiously. Furthermore, the scale
for PEA-based protein levels is not an absolute concentration, so the perfor-
mance of the protein biomarkers and definition of risk thresholds in a clinical
setting warrants further study or the use of methods yielding absolute values,
possibly by calibrating the PEA values. The study also used single assess-
ments of the proteomic assay, so misclassification and short-term variability
are potential issues. Finally, the number of statistical tests performed for this
manuscript increases the risk of spurious findings; hence, novel findings not
previously published have to be replicated in an independent cohort of pa-
tients.
51
Conclusion and future perspectives
This thesis contributes to the identification of biomarkers related to all-cause
mortality, CV mortality, and CVEs in patients in general and in those with
ESKD, but especially markers targeting the bone–vascular axis. In Sweden’s
MrOs elderly community cohort, two potentially damaging SNPs (rs9536314
and rs9527025) in the klotho gene were not associated with all-cause mortality
or CV mortality (Study I). However, composite evaluation of elevated
FGF23, vitamin D deficiency, and renal impairment was associated with all-
cause and CV mortality. Among the bone-related factors, elevated FGF23 was
the most important marker for predicting mortality, but it was clearly influ-
enced by renal function. Long-term follow-up data on the outcome will be
analyzed prior to submission of the study (Study II). In Denmark Århus co-
hort of individuals receiving hemodialysis, one of the most promising bone-
related proteins, OPG, was associated with CV events independent of cytokine
activity (Study III). Using the PEA proteomics approach, we identified
higher IL-8, TIM-1, and CCL20, as well as lower SCF and GAL, as being
associated with poor outcomes (Study IV). We addressed the issue of the
bone–vascular axis and CVD. We evaluated from gene levels to circulating
protein levels and from the general population to patients with CKD. Based
on our research findings, more evidence was generated to link bone and vas-
cular complications. We also identified several CV proteins considered to be
potential biomarkers in patients receiving HD. Because we had 2 independent
prospective HD cohorts (Demark Århus cohort and Taiwan KMUH cohort), a
direct comparison of mortality and CVEs between the two geographic regions
was intriguing. We found a higher risk for all-cause mortality and CVEs in the
patients of the Århus cohort than in the patients of the KMUH cohort. Inter-
estingly, hsTNT can adequately predict outcomes in both cohorts. This will
all be analyzed further and developed into a separate communication (Addi-
tional preliminary study).
For the future, a simultaneous evaluation of PEA-based CV proteomics in the
Århus and KMUH cohorts will be fascinating. Given the risk for death or
CVEs triggered by uremic toxins in patients with kidney disease, an investi-
gation of molecules associated or interacting with uremic toxins and CV pro-
teomics could illuminate the pathophysiology of uremic toxins with respect to
the CV system. Given that the gut microbiota produces many uremic toxins, a
study to link the gut microbiome, uremic toxins, CV protein biomarkers, and
52
clinical CV phenotype would be reasonable. A CV risk prediction model
based on different omics data could be constructed to improve risk stratifica-
tion in patients with kidney disease. Based on the principles of precision med-
icine, we expect an early diagnosis and early treatment to overcome CVD in
patients with CKD.
53
Summary in Swedish (sammanfattning på svenska)
Denna avhandling bidrar till att identifiera biomarkörer som är relaterade till
allmän dödlighet, kardiovaskulär (CV) dödlighet, samt kardiovaskulära hän-
delser hos patienter med eller utan njursvikt. Ett särskilt fokus har varit på
biomarkörer involverade i ben-kärl-axeln.
Bland äldre män i Sverige (Sweden MrOs kohort) var två potentiellt skadliga
SNPs (rs9536314 och rs9527025) i Klotho-genen inte associerade till allmän
eller specifik CV-dödlighet (studie I). En sammansatt bedömning av förhöjda
FGF23-nivåer, D-vitaminbrist och njurfunktionsnedsättning var dock associ-
erad med dödlighet i allmänhet och även specifikt CV-dödlighet. Bland ben-
relaterade faktorer var förhöjda nivåer av FGF23 den viktigaste markören för
att förutsäga mortalitet, men denna faktor var tydligt relaterad till njurfunkt-
ionsstatus (studie II). Hos hemodialys (HD) patienter i en Dansk kohort var
osteoprotegrin det benrelaterade protein som var starkast relaterat till CV-hän-
delser oberoende av annan cytokinaktivitet (studie III). Med hjälp av PEA-
baserad metod för proteomik kunde vi slå fast att högre nivåer av IL-8, TIM-
1 och CCL20, samt lägre nivåer av SCF och GAL, var förknippade med ökad
mortalitetsrisk och även risk för CV händelser (studie IV).
Fokus i studierna har varit på proteinmarkörer, speciellt inom ben-kärl-axeln,
och kardiovaskulär sjuklighet. Vi har utvärderat sambanden både på gennivå
och vad gäller cirkulerande proteinnivåer. Studierna utfördes både inom den
allmänna befolkningen och i patienter med njursjukdom. Baserat på dessa
forskningsresultat föreligger det ett tydligt samband mellan benomsättning
och vaskulära komplikationer. Forskningsprojektet har även identifierat ett
flertal proteiner som kan betraktas som potentiella biomarkörer för hjärtkärl-
sjukdom hos HD-patienter.
54
Acknowledgments
There are many I would like to thank. This thesis is the result of many collab-
orations and the support and effort of many people. I am most grateful to
Östen Ljunggren, my main supervisor, has introduced me to the concept of
mineral bone disease. I appreciate your positive attitude to help me with the
research work and allow me to take lots of different courses.
Bengt Fellström, my co-supervisor and the senior mentor, your engagement,
determination, and enthusiasm for science have been invaluable for my devel-
opment as a researcher. You have always been supportive when I struggled
and encountered difficulties. Thank you for sharing your experience with car-
diovascular disease research in nephrology. You allow me to join this fantastic
field.
Torbjörn Linde (my co-supervisor) and My Hanna Sofia Svensson (my co-
author for study III and study IV), your wise advice in scientific writing and
commentaries on our research works were inspiring.
Rie Io Glerup and Per-Anton Westerberg, your data sharing and detailed in-
troduction for the prospective cohort helped me a lot.
I want to thank all colleagues in Kaohsiung Medical University Hospital, Tai-
wan. Mei-Chuan Kuo, my main supervisor in Taiwan, I must express my grat-
itude to you for guiding me in the thinking process, clinical experience, and
knowledge of nephrology. Yi-Wen Chiu, you are brilliant, knowledgeable,
and the best leader to support my research work in Taiwan. I appreciate your
patience and guidance.
A special thanks to my family for supporting me to study abroad and helping
me to take care of our baby. It’s such a blessing that I have you in my life.
55
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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1785
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)