Uremia Retention Molecules and Clinical Outcomes

Abstract

Chronic kidney disease is characterized by the accumulation of organic compounds in the bloodstream that may exert a variety of toxic effects in the body. These compounds, collectively known as uremic toxins, may be classified according to their physicochemical properties as free water-soluble low molecular weight molecules, middle molecules or protein-bound uremic toxins. Most of these retention molecules, due to either their size and/or binding to protein, constitute a complex therapeutic challenge to the nephrologist, particularly in end-stage renal disease, because of their limited removal by conventional dialysis therapies. Therefore, we review in this article the current clinical evidences that have supported the important role of uremic toxins in uremia by contributing to the adverse outcomes related to chronic kidney disease, such as increased mortality and cardiovascular events, as well as renal impairment progression that cannot be solely explained by traditional risk factors. These observations have ultimately contributed to testing new therapeutic targets, such as the gut, and the development of modern dialysis strategies to manage chronic kidney disease patients.

Full text

Ronco C (ed): Expanded Hemodialysis – Innovative Clinical Approach in Dialysis.
Contrib Nephrol. Basel, Karger, 2017, vol 191, pp 18–31 ( DOI: 10.1159/000479253 )
Abstract
Chronic kidney disease is characterized by the accumulation of organic compounds in the
bloodstream that may exert a variety of toxic effects in the body. These compounds, col-
lectively known as uremic toxins, may be classified according to their physicochemical
properties as free water-soluble low molecular weight molecules, middle molecules or
protein-bound uremic toxins. Most of these retention molecules, due to either their size
and/or binding to protein, constitute a complex therapeutic challenge to the nephrolo-
gist, particularly in end-stage renal disease, because of their limited removal by conven-
tional dialysis therapies. Therefore, we review in this article the current clinical evidences
that have supported the important role of uremic toxins in uremia by contributing to the
adverse outcomes related to chronic kidney disease, such as increased mortality and car-
diovascular events, as well as renal impairment progression that cannot be solely ex-
plained by traditional risk factors. These observations have ultimately contributed to test-
ing new therapeutic targets, such as the gut, and the development of modern dialysis
strategies to manage chronic kidney disease patients. © 2017 S. Karger AG, Basel
Introduction
Chronic kidney disease (CKD) is associated with a high risk of mortality, par-
ticularly due to cardiovascular disease (CVD) that cannot be fully explained only
by traditional risk factors
[1] . Among the nontraditional factors, uremia reten-
tion molecules, also called uremic toxins, are of particular interest since they
represent new therapeutic targets for a better management of CKD patients.
Uremia Retention Molecules and
Clinical Outcomes
Fellype Carvalho Barreto a · Daniela Veit Barreto b ·
Maria Eugênia Fernandes Canziani
c
a Service of Nephrology, Department of Internal Medicine, Federal University of Paraná, and
b Hospital Marcelino Champagnat, Curitiba, and c Service of Nephrology, Department of Internal
Medicine, Federal University of São Paulo, São Paulo , Brazil
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Uremic Toxins and Outcomes 19
These molecules can be classified according to their physicochemical character-
istics and removal by dialysis into 3 classes: small water-soluble; middle mole-
cules, and protein-bound compounds ( Table1 ). Most of them, due to either
their size and/or binding to protein, constitute a therapeutic challenge to the
nephrologist, particularly in end-stage renal disease (ESRD) patients, because of
their limited removal by conventional dialysis therapies.
The importance of this concept is suggested by the fact that randomized con-
trolled trials have not been able to show any benefit of increasing Kt/V or, in
other words urea removal, on ESRD patient survival
[2] . Furthermore, it has
been suggested that estimated glomerular filtration rate (GFR) cannot be con-
sidered a good predictor for evaluating the accumulation of many solutes in the
course of CKD
[3] . Thus, investigating the contribution of different uremic
Table 1. Uremic toxins classification and characteristics
Classification Characteristics Examples
Small water-
soluble
molecules
Molecular weight <500 Da;
Easily removed by dialysis
Creatine
Creatinine*
Guanidine* (ADMA/SDMA)
Oxalate
Urea*
Uric acid
Trimethylamine
Middle
molecules
Molecular weight >500 Da;
Need dialysis membranes with large
pores large to be removed;
Many are peptides
AGES and AOPP
Complement factor D
Cystatin C
Cytokines
Endothelin
FGF23
Leptin*
β2-Microglobulin*
Protein-bound
molecules
Generally of low molecular weight;
Difficult to be removed by dialysis
CMPF
Hippuric acid
Homocysteine
Indole-3-acetate
Indoxyl sulfate*
p-Cresilsulfato*
Spermidine/Spermine
* Indicates the molecules considered as the prototypes of each group.
AGES, advanced glycation end-products; AOPP, advanced oxidation protein products;
ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; CMPF, 3-car-
boxy-4-methyl-5-propyl-2-furan-propanoic acid.
Ronco C (ed): Expanded Hemodialysis – Innovative Clinical Approach in Dialysis.
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20 Barreto · Barreto · Canziani
toxins for the uremic syndrome is crucial for the better understanding of CKD
and, above all, to guide the development and implementation of novel therapeu-
tic strategies.
In the subsequent sections, we will present the current evidences derived
from clinical observational studies that support the key role of uremic toxins in
the uremia-related poor outcome, such as cardiovascular events, increased mor-
tality, and progression of CKD.
Small Water-Soluble Molecules
Symmetric Dimethylarginine and Asymmetric Dimethylarginine
Symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine
(ADMA), compounds from the group of guanidines, are derived from the me-
tabolism of L-arginine. Serum levels of methylarginines increase in CKD due to
reduced renal clearance, increased synthesis, and reduced catabolism. Despite
being structurally similar to urea, methylguanidines have a greater distribution
volume, resulting in lower removal efficiency by dialysis procedures in com-
parison to urea
[4] . Higher dialysis dose or longer sessions seem to be incapable
of further lowering ADMA or SDMA concentrations
[5] .
ADMA is an inhibitor of nitric oxide synthase, causing vasoconstriction, ar-
terial stiffness, and hypertension. Several studies have reported a strong and in-
dependent association of higher serum levels of ADMA and mortality and car-
diovascular events in general, predialysis and dialysis populations
[6–8] . Re-
cently, in a post-hoc analysis of the Hemodialysis (HEMO) study, Shafi et al.
[5]
have found an association between ADMA levels and higher risk of cardiac
death (HR 1.83; 95% CI 1.29–2.58), sudden cardiac death (HR 1.79; 95% CI
1.19–2.69), first cardiovascular event (HR 1.50; 95% CI 1.20–1.87), and any-
cause death (HR 1.44; 95% CI 1.13–1.83). Interestingly, they were also able to
demonstrate an association between SDMA levels and the risk for cardiac death
(HR 1.40; 95% CI 1.03–1.92), though it was no longer significant after adjusting
for ADMA. Studies have also reported a similar association between SDMA and
cardiovascular and cerebrovascular outcomes
[6, 9] . Taken together, these find-
ings corroborate to the current concept that SDMA is not an inert compound.
Actually, it has been demonstrated that SDMA has pro-oxidant and pro-inflam-
matory effects, and inhibits nitric oxide production
[10, 11] . Finally, a recent
meta-analysis found that higher levels of ADMA and SDMA were associated
with increased risk of all-cause mortality (RR 1.52, 95% CI 1.37–1.68; RR 1.31,
95% CI 1.18–1.46; respectively) and CVD (RR 1.33, 95% CI 1.22–1.45; RR 1.36,
95% CI 1.10–1.68; respectively)
[12] .
Ronco C (ed): Expanded Hemodialysis – Innovative Clinical Approach in Dialysis.
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Uremic Toxins and Outcomes 21
Phosphate
Hyperphosphatemia usually appears in CKD patients when GFR <30 mL/
min/1.73 m
2 , being particularly common among ESRD patients due to the low
clearance of phosphate by conventional dialysis. Until the late 1990s, it was con-
ceived that the deleterious effects of hyperphosphatemia were restricted to bone
and mineral metabolism. Actually, hyperphosphatemia is closely associated
with the development of secondary hyperparathyroidism (sHPT) through direct
and indirect mechanisms that may stimulate the synthesis and secretion of para-
thyroid hormone and parathyroid cell growth. Hyperphosphatemia is also an
important stimulus for the secretion of the phosphaturic hormone, fibroblast
growth factor-23 (FGF23).
Several observational studies have reported that hyperphosphatemia is asso-
ciated with an increased risk of all-cause and cardiovascular mortalities in dialy-
sis and pre-dialysis CKD patients
[13, 14] . Hyperphosphatemia has also been
associated with vascular calcification in CKD patients as well as among people
with relatively preserved renal function
[15, 16] . Notably, in vitro experiments
have shown that phosphate may contribute to CVD by inducing endothelial dys-
function and/or by promoting vascular smooth muscle cell transdifferentiation
to osteoblast-like cells that participate in the pathogenesis of vascular calcifica-
tion. It has also been noted that a significant association exists between serum
phosphate levels near the upper limit of the normal range and the risk of all-
cause death (HR 1.27; 95% CI 1.02–1.58) and cardiovascular event, such as new
heart failure and myocardial infarction, in subjects with prior myocardial infarc-
tion
[17] . Finally, a recent study expanded the relevance of hyperphosphatemia
as a potential modifiable risk factor to the field of kidney transplant. Merhi et al.
[18] have reported that higher serum phosphate levels may be associated with
increased risk of transplant failure (HR 1.36; 95% CI 1.15–1.62) and mortality
risk (HR 1.21; 95% CI 1.04–1.40) in a cohort of 3,138 stable kidney transplant
recipients followed up for a median time of 4.0 years.
Trimethylamine N-Oxide
Trimethylamine-N-oxide (TMAO), a circulating organic compound produced
by the metabolism of dietary L-carnitine and choline, has emerged as a novel
cardiovascular risk factor, likely due to its pro-atherogenic effect. Both L-carni-
tine and choline, abundantly present in eggs and red meat, are metabolized by
intestinal bacteria to trimethylamine which, after intestinal absorption, is oxi-
dized by flavin mono-oxygenase enzymes in the liver to form TMAO. Urinary
excretion is the most exclusive route to eliminate TMAO from the body.
It has been reported that serum levels of TMAO are inversely associated with
renal function, being markedly elevated in hemodialysis patients
[19] . TMAO
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22 Barreto · Barreto · Canziani
accumulates during the interdialytic interval, but it is efficiently removed by he-
modialysis
[20] . Beyond renal function impairment, dysbiosis of the gut micro-
biota, diet seems to increase TMAO levels and might explain the great variabil-
ity in its urinary levels
[21] .
T M A O s e e m s n o t t o b e o n l y a s u r r o g a t e m a r k e r f o r r e n a l d y s f u n c t i o n , b u t
likely contributes to the risk of death in CKD patients. TMAO levels in the highest
quartile were independently associated with a 1.9-fold increase in all-cause mor-
tality at 5 years among subjects with either normal or elevated cystatin C levels
[22] . T M A O c o n c e n t r a t i o n s p r e d i c t e d t h e c o r o n a r y a t h e r o s c l e r o s i s b u r d e n a n d
long-term mortality, independent of traditional cardiac risk factors in a cohort of
patients with variable degree of renal impairment
[19] . I n a l a r g e o b s e r v a t i o n a l
study that enrolled 2,529 CKD (stages 3b and 4) patients from the CanPREDDICT
cohort, TMAO was independently associated with cardiovascular events after ad-
justing for potential risk factors. Importantly, even among those with stage 3b
CKD, TMAO levels were capable of identifying those exposed to the highest risk
for cardiovascular events, independent of kidney function
[23] . T M A O l e v e l s a l s o
seem to be associated with higher risk of cardiovascular events and any-cause
death in hemodialysis patients, an effect apparently modulated by race
[24] .
Middle Molecules
Fibroblast Growth Factor 23
FGF23 (MW 32 kDa) is a phosphaturic hormone that regulates mineral homeo-
stasis, mostly secreted by osteocytes and osteoblasts. FGF23 regulates phosphate
levels directly, by inhibiting the renal tubular reabsorption of phosphate, and
indirectly, by reducing calcitriol levels. FGF23 also inhibits the secretion of para-
thyroid hormone (PTH) by the parathyroid glands. These effects depend on the
binding of FGF23 to the complex FGF receptor (FGFR)-klotho, a co-factor that
greatly increases the affinity of FGF23 for FGFR.
Serum levels of FGF23 increase since the early stages of CKD, reaching con-
centrations of up to 1,000-fold above the normal range in patients on hemodi-
alysis. Of note, conventional hemodialysis is not capable of removing FGF23.
This increase in the FGF23 levels results from (i) an increased production to
counteract phosphate accumulation and to overcome FGF23 resistance due to
lower expression of FGFR-klotho; (ii) decreased renal clearance; and (iii) de-
creased degradation of FGF23. The cleavage of the FGF23 molecule is impaired
in uremia leading to the accumulation of the carboxy-terminal fragments that
are not considered biologically active. Interestingly, inflammation and iron de-
ficiency may contribute to the increased FGF23 concentrations noted in CKD.
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Uremic Toxins and Outcomes 23
Several observational studies have reported that elevated serum levels of
FGF23 are independently associated with adverse clinical outcomes. A large
prospective study that included more than 10,000 incident hemodialysis pa-
tients followed up for 12 months showed that elevated FGF23 level was asso-
ciated with almost 6-fold increase in the risk of one-year mortality
[25] . The
Homocysteine in Kidney and End-Stage Renal Disease (HOST) study ( n =
1,099; CKD stages 2–4) found that elevated FGF23 levels are independently
associated with greater risk of ESRD
[26] . High serum levels of FGF23 have
also been associated with left ventricular hypertrophy and vascular altera-
tions, such as atherosclerosis and vascular calcifications
[27, 28] . Interestingly,
the cardiac hypertrophic action of FGF23 depends on the FGF23 binding to
the FGFR4 present in the myocardium (not to the complex FGFR-klotho),
and the signaling pathway occurs through the PLCγ-calcineurin-NFAT path-
way rather than via the MAPK cascade
[29] . Higher FGF23 levels have been
associated with a greater risk of heart failure
[30] . Giving further support to
the relevance of FGF23 as a predictor of adverse outcomes, a recent meta-
analysis that evaluated 19 prospective studies showed that increased FGF23
serum levels were independently associated with a higher risk of all-cause (RR
1.68; 95% CI 1.48–1.92) and cardiovascular mortalities (RR 1.68; 95% CI 1.38–
2.04)
[31] .
Parathyroid Hormone
PTH (MW 9.4 kDa) is secreted by the parathyroid glands in response to a de-
crease in the concentration of extracellular calcium. PTH is essential for the
bone and mineral homeostasis due to its action in the kidneys, where it promotes
calcium reabsorption, phosphate excretion, and stimulates the activity of
1-α-hydroxylase, the enzyme responsible for the production of calcitriol; and
bone, where it regulates bone remodeling and FGF23 production.
Increased levels of PTH are generally found since the early stages of CKD,
namely sHPT. Besides and beyond the myriad of clinical manifestations related
to sHPT that include fatigue, pruritus, psychological symptoms, and bone de-
formities, elevated serum levels of PTH have been associated with bone fracture,
cardiovascular complications, and mortality. In a large observational study that
enrolled 40,538 hemodialysis patients, PTH concentrations ≥ 600 pg/mL were
associated with an increase in the relative risk of death, all-cause, cardiovascular,
and fracture-related hospitalization
[32] . Moreover, it has been reported that
high PTH levels are associated with vascular calcification and left ventricular
hypertrophy
[33, 34] . PTH may lead to deleterious effects on the cardiovascular
system due to the ubiquitous expression of its main receptor, PTH1R. The hy-
pertrophic effects of PTH on cardiomyocytes, and the bidirectional and
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24 Barreto · Barreto · Canziani
stimulatory relationship between aldosterone and PTH are some of the potential
mechanisms that might, at least partly, explain why PTH may damage the car-
diovascular system
[35] .
Beta2-Microglobulin
Beta 2-microglobulin (β2M) is a polypeptide (MW 11,729 Da) found ubiqui-
tously on the surface of nucleated cells as part of the major histocompatibility
complex I. β2M is freely filtered by the glomerulus and metabolized by the tu-
bules. Hence, its levels inversely correlate with the renal function. Other condi-
tions that may increase β2M serum levels are hematologic, inflammatory, and
infectious diseases. Factors beyond β2M accumulation, such as molecular struc-
tural changes by AGES and limited proteolysis, may corroborate to its toxicity.
The classical complication related to β2M accumulation in ESRD patients is
its deposition on bone and joints leading to pain, carpal tunnel syndrome, hem-
arthrosis, and cystic bone lesions, the so-called dialysis-related amyloidosis
[36] .
More recently, there is a growing interest on the effects of β2M on the cardio-
vascular system. Higher levels of β2M have been associated with arterial stiff-
ness, vascular calcification, and higher mortality
[37] . In the HEMO study, high
serum levels of β2M was associated with an increased risk of mortality, mainly
due to infectious disease
[38] . Interestingly, in transplant recipients, the serum
levels of β2M at the moment of hospital discharge was a potent predictor of mor-
tality and loss of the renal allograft, suggesting that β2M may be a more valuable
surrogate marker for these outcomes than creatinine
[39] .
Free Light Chains
Light chains (LCs) are important components of the immunoglobulin mole-
cules synthesized by plasma cells. Both LC isotypes, the monomeric κ (MW 22.5
kDa) and the dimeric λ (MW 45 kDa), are produced more on a small scale than
the heavy chains of the immunoglobulin. This excess is normally excreted and/
or metabolized by the kidneys. Thus, free light chains (FLCs) may accumulate
as renal function declines. The development of more accurate assays capable of
detecting FLC levels below the so-called normal range have allowed the assess-
ment of the relationship between polyclonal FLC serum levels and clinical out-
comes.
Clinical studies in CKD patients have demonstrated that high FLC levels are
independently associated with overall mortality risk across different stages of
CKD
[40, 41] . Not all studies were able to demonstrate this association though
[42] . Differences on the clinical and demographic characteristics of the study
population, number of patients included, and follow-up duration may account
for these conflicting findings. FLC κ and λ levels have also been associated with
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Uremic Toxins and Outcomes 25
inflammation, vascular calcification, and levels of other uremic toxins, such as
β2M, indoxyl sulphate, and para-cresyl sulfate, and with progression to ESRD
[40, 42] .
In conclusion, polyclonal FLC has been considered a valuable marker of in-
flammation that reflects more closely the activity of the adaptive immune system
and chronic inflammation. In addition, its independent association with mortal-
ity suggests that FLC measurement may provide additional information over
traditional markers of inflammation, such as hsRCP, and of renal function. Oth-
erwise, whether free LC may provide prognostic information over-and-above
other middle molecules, such as β2M, and its pathogenic role in uremia remains
to been demonstrated.
Protein-Bound Uremic Toxins
Indoxyl Sulfate
Indoxyl sulfate (IS; MW 212.21 Da) is the main compound of the indole group.
IS is generated in the liver from the metabolism of indole, which is produced by
intestinal microbiota from the degradation of dietary tryptophan. The kidneys
are the main route of excretion of IS by tubular secretion via organic anion trans-
porters (OAT)-1 and OAT-3 located on the basolateral membrane of epithelial
cells of the proximal tubule. As other protein-bound uremic toxins, the major
fraction of IS circulates in the body tightly bound to albumin. As expected con-
sidering its physicochemical characteristics, IS has an inverse relationship with
kidney function and is poorly removed by conventional dialysis therapies.
IS is one of the most vastly studied uremic toxins. Most clinical and experi-
mental evidences suggest that IS acts as a nephro-vascular toxin. Barreto et al.
[43] have reported that IS levels are directly associated with arterial calcification
and stiffness and with overall and cardiovascular mortality, even after adjust-
ment for age, gender, diabetes, albumin, hemoglobin, phosphate, and aortic cal-
cification. A recent meta-analysis has found an association between elevated free
IS levels and increased risk of all-cause mortality (OR 1.10, 95% CI 1.03–1.17),
but not with cardiovascular events
[44] . Furthermore, IS has been independent-
ly associated with first heart failure event in hemodialysis patients
[45] . High
levels of IS were reported to be associated with dialysis graft thrombosis after
endovascular interventions, given further support to the hypothesis that IS acts
as a cardiovascular toxin
[46] .
It has been reported that serum levels of IS may also influence CKD progres-
sion and other uremic manifestations. Higher IS serum levels have been associ-
ated with renal progression (reduction in estimated GFR >50% from baseline or
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26 Barreto · Barreto · Canziani
progression to ESRD); its predictive power, however, was reduced when para-
cresyl sulfate (pCS) was added to the model
[47] . Even though some studies have
suggested that IS may play a role in the uremia-related musculoskeletal disor-
ders, such as renal osteodystrophy
[48] , no study so far has examined the asso-
ciation between IS levels and bone-specific outcomes, such as fracture.
Para-Cresyl Sulfate
pCS (MW 188.2 Da) is a phenol compound produced from the metabolism of
tyrosine and phenylalanine by intestinal bacteria. In the distal portion of the co-
lon, these amino acids are converted into phenolic compounds, such as p-cresol,
which suffer conjugation in the liver to generate pCS, the main metabolite, and
p-cresylglucoronide (pCG). In the blood, these compounds circulate mostly
bound to albumin. pCS and pCG are excreted by the kidneys mainly via the tu-
bular secretion by OAT-1 and OAT-3.
Serum levels of pCS increase as renal function declines, particularly in the late
stages of CKD. Moreover, due to its binding to albumin, pCS is hardly removed
by traditional dialysis therapies. High serum levels of pCS have been significant-
ly associated with overall and cardiovascular mortalities independent of well-
known predictors of survival, and with renal progression
[47, 49] . The clinical
importance of pCS has been further supported by 2 recent meta-analyses that
demonstrated its association with increased risk of mortality and of cardiovas-
cular events in CKD patients
[44] . More recently, Liabeuf et al. [50] were the first
to report that pCG, the minor metabolite of p-cresol, is independently associ-
ated with overall and cardiovascular mortalities, suggesting that it may have
much the same predictive power for mortality as pCS.
The relationship between pCS with infection-related hospitalizations and
septicemia has been recently investigated in 2 cohorts of hemodialysis patients,
Choices for Healthy Outcomes in Caring for ESRD (CHOICE) study and HEMO
study. In CHOICE, free pCS was associated with 50% higher risk of infection-
related hospitalizations in patients with no gastrointestinal disease. A significant
trend was noted between greater levels of free pCS and septicemia in both co-
horts in patients with no gastrointestinal disease
[51] .
Conclusions
The field of uremic toxicity has gained a lot of interest during the last years. A
number of observational studies have suggested that uremic toxins contribute
to the high cardiovascular burden and mortality observed in CKD. The obser-
vational design of these studies precludes establishing a causal relationship be-
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Uremic Toxins and Outcomes 27
tween uremic retention solutes and clinical outcomes, particularly when analy-
ses are not adjusted for GFR. Experimental studies, both in vitro and those based
on animal models of CKD, have complemented the clinical studies providing
consistent evidences that support the role of uremic toxins in the pathogenesis
of uremic syndrome, including cardiovascular manifestations and CKD pro-
gression ( Fig.1 ). Importantly, the acquired knowledge may shift treatment par-
adigms in Nephrology. New therapeutic targets, such as the gut, have been pro-
posed and the concept of dialysis adequacy relied only on urea removal has been
questioned. The development of novel technologies on dialysis therapy aimed
to remove middle molecules and protein-bound uremic toxins seems to be an
interesting approach as an attempt to change the high cardiovascular mortality
observed in CKD.
Chronic kidney disease
Renal clearance Gut generation
(dysbiosis)
Uremia retention
molecules
Oxidative stress
Inflammation
Apoptosis
Fibrosis
Endothelial dysfunction
Cardiovascular
disease
Cerebrovascular
disease
Progression of
renal impairment
Protein-rich
diet
Cardiovascular events (e.g., ischemic coronariopathy)
Cerebrovascular events (e.g., stroke)
Bone fractures
End-stage renal disease
Overall and cardiovascular mortalities
Catabolism
Musculoskeletal
alterations
Fig. 1. Schematic view of uremia retention molecules accumulation and toxicity.
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28 Barreto · Barreto · Canziani
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Maria Eugênia Fernandes Canziani
Service of Nephrology, Department of Internal Medicine
Federal University of São Paulo
Rua Pedro de Toledo 282
São Paulo, SP, CEP 04039-000 (Brazil)
E-Mail dialisefor@uol.com.br
Ronco C (ed): Expanded Hemodialysis – Innovative Clinical Approach in Dialysis.
Contrib Nephrol. Basel, Karger, 2017, vol 191, pp 18–31 ( DOI: 10.1159 /000479253 )
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