Clinica Chimica Acta 438 (2015) 350357

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Biomarkers of renal function, which and when?

Michael E. Wasung a, Lakhmir S. Chawla b, Magdalena Madero a,
a
Division of Nephrology, Department of Medicine, Instituto Nacional de Cardiologa Ignacio Chvez, Mexico City, Mexico
b
Department of Medicine, Division of Intensive Care Medicine, and the Division of Nephrology, Washington, DC, Veterans Affairs Medical Center, and the Department of Anesthesiology and Critical
Care Medicine, George Washington University

a r t i c l e i n f o a b s t r a c t

Article history: Purpose of the review: Acute kidney injury (AKI) and chronic kidney disease (CKD) are conditions that substantial-
Received 2 April 2014 ly increase morbidity and mortality. Although novel biomarkers are being used in practice, the diagnosis of AKI
Received in revised form 27 August 2014 and CKD is still made with surrogate markers of GFR, such as serum creatinine (SCr), urine output and creatinine
Accepted 29 August 2014 based estimating equations. SCr is limited as a marker of kidney dysfunction in both settings and may be inaccu-
Available online 3 September 2014
rate in several situations, such as in patients with low muscle mass or with uid overload. New biomarkers have
the potential to identify earlier patients with AKI and CKD and in the future potentially intervene to modify out-
Keywords:
Biomarkers
comes.
Acute kidney injury Recent ndings: In particular KIM-1 and NGAL are considered excellent biomarkers in urine and plasma for the
Chronic kidney disease early prediction of AKI; however cycle arrest biomarkers have emerged as novel markers for risk stratication
of AKI. Urine TIMP-2 and IGFBP7 performed better than any other biomarkers reported to date for predicting
the development of moderate or severe AKI. Biomarker combinations are required to increase diagnostic accura-
cy in an acute setting. NGAL, cystatin C, and FGF-23 are promising and accurate biomarkers for CKD detection.
Equations combining cystatin C and SCr perform better than the equations using either cystatin C or SCr alone,
especially in situations where CKD needs to be conrmed. Combining creatinine, cystatin C and urine albumin
to creatinine ratio improves risk stratication for kidney disease progression and mortality.
Summary: Recent advances in molecular biology have resulted in promising biomarkers for AKI and CKD diagnoses;
however more research is necessary to implement them successfully into clinical practice in order to facilitate early
diagnosis, guide interventions and monitor disease progression. The following review describes the most important
biomarkers studied in kidney disease and will discuss the use and the value of these biomarkers in different clinical
settings.
2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
2. Biomarkers in acute kidney injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
2.1. Biomarkers for the early diagnosis of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
2.2. Prognosis of AKI severity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
2.3. Differential diagnosis of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
2.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
3. Biomarkers in chronic kidney disease (CKD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
3.1. Biomarkers for glomerular ltration rate (GFR) measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
3.2. Cardiovascular disease prediction in CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
3.3. CKD progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
3.4. Inammatory and brotic markers associated with CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
3.5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Abbreviations: AKI, acute kidney injury; CKD, chronic kidney disease; SCr, serum creatinine; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; TIMP-2,
tissue inhibitor of metalloproteinases-2; IGFBP7, insulin-like growth factor-binding protein; FGF-23, broblast growth factor-23.
Corresponding author at: Juan Badiano No. 1, Tlalpan 14000 Mxico, D.F., Mexico. Tel.: +52 55 55 73 69 02; fax: +52 55 55 73 77 16.
E-mail address: madero.magdalena@gmail.com (M. Madero).

http://dx.doi.org/10.1016/j.cca.2014.08.039
0009-8981/ 2014 Elsevier B.V. All rights reserved.
 M.E. Wasung et al. / Clinica Chimica Acta 438 (2015) 350357
1. Introduction
The incidence of acute kidney injury (AKI) and chronic kidney dis-
ease (CKD) is increasing worldwide and it is associated with increased
morbidity and mortality. Identifying patients early is of paramount im-
portance in order to offer a prompt intervention and to improve the
prognosis in both settings. A biomarker is dened as a parameter of
structural, biochemical, physiologic or genetic change that indicates
the presence, severity or progress of a disease [1]. The measurement
of kidney function is commonly made using serum creatinine concen-
tration (SCr), blood urea nitrogen (BUN) level and urinalysis; however
accumulating evidence has shown that these biomarkers are subopti-
mal to detect kidney disease in early stages [24].
Kidney injury begins by inducing biological and molecular changes
that ultimately evolve into cellular damage [5] allowing a wide array
of molecules to be measured. Biomarker research has increased sub-
stantially, and each year new potential markers are introduced. Even
though there is better understanding of which biomarkers are useful
in kidney diseases there is still doubt toward when they should be
measured. Before we begin our review, we outline the characteristics
of an ideal biomarker in Table 1. As we will see throughout the review,
the presence of biomarkers in kidney have diagnostic and prognostic
implications in AKI and CKD, however there is currently not enough
data regarding the role of biomarkers on decision making. Clinical judg-
ment is still critical, biomarkers should not be measured indistinctly and
their measurement must be considered in situations that add value to
current available markers.
One of the most considerable limitations of biomarkers in kidney
disease is the discriminatory value between AKI and CKD. AKI and
CKD are not distinct syndromes, and actually share common grounds
[6]. Clinical studies have demonstrated that AKI increases the risk for
CKD development [7,8], and CKD has repeatedly shown to be a risk fac-
tor for AKI [911]. Some mechanisms explaining CKD progression after
AKI include nephron loss, glomerular hypertrophy, interstitial inam-
mation and brosis, endothelial injury and cell cycle and maladaptive
repair [6]. AKI and CKD share biomarkers, reecting the integrative
pathophysiology between both entities.
We review the current status of the most important biomarkers in
AKI and CKD and mention the possible settings in which their measure-
ment is useful. This review will rst address biomarkers in AKI and, sec-
ond, biomarkers in CKD. Each section will discuss the use and the value
of biomarkers in different clinical settings. Tables 2 and 3 summarize
some of the most promising biomarkers in AKI and CKD.
2. Biomarkers in acute kidney injury
AKI is characterized by a sudden and sustained decrease of renal
function, resulting in retention of nitrogenous and non-nitrogenous
waste products [12]. The incidence of AKI requiring dialysis ranges
from 4 to 6% in patients hospitalized in the intensive care unit (ICU)
with a mortality rate that varies from 60 to 90% depending on the sever-
ity score of each patient [911]. Even though important advances have
been made in critical care medicine and in renal replacement therapies
(RRT), the outcome of critically ill patients with AKI that require dialysis
Table 1
Characteristics of an ideal biomarker.
1. Noninvasive
2. Highly sensitive and specic
3. Increases rapidly and reliably in response to kidney disease.
4. Correlates with the amount of kidney injury
5. Provides risk stratication and prognostic information
6. Is site specic
7. Applicable across different populations
8. Identies possible mechanisms of injury (prerenal, intrarenal, postrenal)
9. Highly stable over time and across different temperatures and PH
10. Does no interfere with drugs
351
is still poor. In-hospital mortality has not changed signicantly in the
past 50 years [1,13] and AKI is now considered an independent risk fac-
tor for long-term mortality and for the future development of CKD [68,
14]. One possible reason for the poor outcome of severe AKI in the ICU
may be delay in the diagnosis of AKI.
The current practice to diagnose AKI involves measuring the surro-
gate markers of glomerular ltration rate (GFR) such as an increase in
SCr and/or a decrease in urine output with the RIFLE and/or AKIN
criteria. However, the measurement of SCr is limited as a diagnostic
test in the early stages of AKI for several reasons. First, SCr can increase
in cases of prerenal azotemia when there is no tubular injury [15]. Sec-
ond, many non-renal factors can inuence its levels (body weight, mus-
cle metabolism, total body volume and drugs) [12]. Third, SCr in AKI
rises up to 72 h after the initial insult to the kidney [12]; fourth, renal in-
jury can exist with no change in SCr due to renal reserve or tubular se-
cretion of creatinine [16]. Urine chemistry such as the fractional
excretion rate of sodium or urea may differentiate prerenal azotemia
from acute tubular necrosis (ATN) in selected patients; however, results
may be confounded easily with diuretic use and volume status. Urine
microscopy with evaluation of the urine sediment for renal tubular
casts is helpful in differentiating prerenal azotemia from ATN [17]; how-
ever it requires expertise from the examiner.
AKI is characterized by vascular and tubular alterations that express
proteins that can ultimately be measured in urine and in plasma. In AKI,
cells produce biomarkers up to 3 days before the clinical syndrome de-
velops [12] and therefore a biomarker should be measured in this peri-
od. Table 2 outlines the most promising biomarkers. Biomarkers have
been tested in various AKI settings and in diverse populations; in the
following section we will discuss some clinical situations wherein
biomarker measurement has demonstrated to be useful.
2.1. Biomarkers for the early diagnosis of AKI
A considerable amount of evidence demonstrated that plasma and
urine neutrophil gelatinase-associated lipocalin (NGAL), urinary cystatin
C, urinary kidney injury molecule (KIM-1), urinary interleukin-18 (IL-
18), and glutathione-S-transferase are early diagnostic biomarkers of
AKI in different settings. These biomarkers have been studied extensively
in different situations such as in post-cardiac surgery patients, after con-
trast material administration and in kidney transplantation. Evidence
points out that they are present approximately 2 days before the clinical
syndrome of AKI develops [5,12]. Mishra and colleagues demonstrated
the clinical value of NGAL as a marker of kidney injury in 71 children un-
dergoing cardiopulmonary bypass [18]. The 2 hour NGAL levels in urine
and plasma were predictors of AKI, with an AUC of 0.998 for the urine
NGAL and 0.91 for the plasma NGAL. Further research, particularly in a
cohort of adults with a high prevalence of CKD did not have such impres-
sive results as the initial trials [19,20]. Although the follow-up studies
failed to show the outstanding performance of the rst trials, the perfor-
mance of NGAL and its elevation early in AKI remains very good. A meta-
analysis revealed that in cardiac surgical patients, urinary NGAL had an
AUC of 0.775 when measured within 6 h of cardiopulmonary bypass
[21].
The predictive value of NGAL has shown to be inuenced by several
factors, including baseline renal function, severity of AKI and age [22].
NGAL is an acute phase reactant that is released from immune cells
[23] and it is found to be elevated in inammatory conditions. In a
study performed by Wagener et al. in 426 adult patients submitted to
cardiac surgery, urinary NGAL levels were found to be elevated in all pa-
tients after the procedure. Urinary NGAL levels correlated signicantly
with cardiopulmonary bypass and aortic cross-clamp times; raising
the possibility that CPB induced inammation led to increased levels
of NGAL [24]. NGAL levels are also inuenced by other medical condi-
tions such as preeclampsia and cancer [2527].
Urinary NGAL, KIM-1, L-FABP, IL-18 and cystatin C seem to have less
specicity and sensitivity in patients with comorbid conditions [28]. In a
352 M.E. Wasung et al. / Clinica Chimica Acta 438 (2015) 350357

Table 2
Examples of biomarkers used for the detection of AKI.

Legend Source Physiological action

Tubular enzymes released in the urine

Alpha glutathione S transferase GST Expressed in almost all tissues Released from lysosomes, brush-border and cytoplasm
Kidney: proximal tubular cells of proximal tubular epithelial cells
Pi glutathione S transferase GST Expressed in almost all tissues.
Kidney: distal tubular cells
N-acetyl beta glucosaminidase NAG Several tissues (liver, brain, spleen etc.).
Kidney: proximal tubular cells
Inammatory urinary markers
Interleukin 18 IL-18 Monocytes, dendritic cells, macrophages Filtered freely by the glomeruli, reabsorbed, non-secreted
Interleukin 1, 6, 8 IL-1, -6, -8 and epithelial cells
Low molecular proteins
Urinary cystatin C Cystatin C All nucleated cells Filtered freely by the glomeruli, reabsorbed, non-secreted
Hepatocyte Growth Factor HGF Mesenchymal cells
Alpha 1 microglobulin 1MG Liver
Beta 2 microglobulin 2MG All nucleated cells
Proximal tubule proteins
Kidney injury molecule 1 KIM-1 Kidney: proximal tubular cells Up-regulated in proximal tubular epithelial cells
Neutrophil gelatinase-associated lipocalin NGAL Leucocytes, loop of Henle and collecting ducts
Urinary cystatin C Cystatin C All nucleated cells
Liver-type fatty acid-binding protein L-FABP Hepatocytes, kidney: proximal tubular cells
Cell cycle arrest proteins
Insulin-like growth factor-binding protein 7 IGFBP7 Expressed in almost all epithelial cells Renal tubular cells enter a short period of G1 cell-cycle
Tissue inhibitor of metalloproteinases-2 TIMP-2 Kidney: tubular epithelial cells arrest following AKI.

prospective cohort of 529 patients conducted by Endre and colleagues,
none of the biomarkers evaluated had an AUC above 0.7 [29]. The per-
formance of these biomarkers improved when adjustment was made
with the baseline renal function and with the duration of renal injury.
Cycle arrest biomarkers have emerged as novel markers for risk
stratication of AKI. Kashani and colleagues studied two new bio-
markers, insulin-like growth factor-binding protein 7 (IGFBP7) and tis-
sue inhibitor of metalloproteinases-2 (TIMP-2), in the urine of patients
at high risk of AKI from multiple causes [30]. Both biomarkers are in-
ducers of G1 cell-cycle arrest, considered as a key mechanism of AKI
and performed better than any other biomarkers reported to date. The
study consisted of a discovery and validation phase. In the discovery
phase, blood and urine samples were obtained from three distinct co-
horts to identify novel biomarkers for AKI among 340 proteins. The
best biomarkers were then compared in the validation phase to a num-
ber of previously described biomarkers such as NGAL, KIM-1, and IL-18,
among others. Urinary IGFBP7 and TIMP-2 performed the best in both
phases. Measuring both biomarkers exhibited an AUC of 0.80 for the de-
velopment of AKI within 12 h and when measured alone, IGFBP7 and
TIMP-2 exhibited an AUC of 0.76 and 0.79 respectively. Importantly,
neither TIMP-2 nor IGFBP-7 was elevated in patients without AKI and
important co-morbidities such as CKD, diabetes and sepsis. In a
follow-up trial, which utilized the same enrolment criteria, TIMP-2
and IGFBP-7 were found to have similar performance. In this trial, AKI
experts in a blinded fashion clinically adjudicated the AKI endpoint.
Both of these markers performed better than existing markers and the
operating characteristics are well suited for clinical use [31].
2.2. Prognosis of AKI severity
Several of the biomarkers mentioned previously performed well for
the risk stratication and prognosis of AKI. In children undergoing car-
diac surgery, plasma NGAL was a predictive biomarker of AKI and AKI
severity. Dent and associates measured plasma NGAL at baseline and
at frequent intervals for 24 h after cardiopulmonary bypass (CPB) in a
pediatric cohort [32]. Using multivariate analysis it was determined
that the plasma NGAL at 2 h after CPB was the most powerful predictor
of AKI, and correlated with the duration of AKI and the length of hospital
stay. The 12 hour plasma level of NGAL correlated with mortality.
In the study performed by Liangos and colleagues, urinary KIM-1
and N-acetyl--(D)-glucosaminidase (NAG) levels were able to predict
adverse clinical outcomes such as dialysis and mortality in a cohort of
201 patients with AKI of multiple causes [33]. The role of serum cystatin
C to predict adverse clinical outcomes in AKI is limited; in the study

Table 3
Examples of biomarkers for detection of CKD.

Legend Source Physiological action

Amino acids Impaired clearance or increased production in CKD

Plasma asymmetric dimethylarginine ADMA Endothelial cells
Proteins FGF-23 Osteoblasts
Fibroblast growth factor 23
Chemokines MCP-1 All nucleated cells
Monocyte chemoattractant protein-1 Kidney cells
Tubular injury cell markers
Neutrophil gelatinase-associated lipocalin NGAL Leucocytes, loop of Henle and collecting ducts Released from lysosomes, brush-border and cytoplasm
Urinary cystatin C Cystatin C All nucleated cells of proximal tubular epithelial cells
Liver-type fatty acid-binding protein L-FABP Hepatocytes, kidney: proximal tubular cells
Urinary brosis markers
Connective tissue growth factor CTGF All tissues Excessive production of probrotic growth factors and
Transforming growth factor-1 TGF-1 All tissues extracellular matrix
Collagen IV COLIV Kidney, eye, skins
Glomerular cell injury markers
Plasma cystatin C Cystatin C All nucleated cells Impaired GFR
Podocalyxin PCX Podocytes Podocyte structure defect
Nephrin Nep Podocytes
 M.E. Wasung et al. / Clinica Chimica Acta 438 (2015) 350357
done by Perianayagum et al. [34], serum cystatin C had an AUC of 0.65
for prediction of death and dialysis in 200 patients with AKI, performing
similarly to the SCr level.
In the study performed by Siew et al. [35] in 451 critically ill adult
patients, urinary IL-18 concentrations did not predict the development
of AKI, but did predict death and the need for short term dialysis. In the
prospective study by Endre et al. [29], urinary NGAL, cystatin C and IL-
18 were the strongest predictors of dialysis (AUC N 0.7). All biomarkers
except KIM-1 were moderately predictive of death within 7 days (AUC
0.610.69). Doi and colleagues found that the urinary levels L-FABP,
NGAL and IL-18 were able to predict 14 day mortality (L-FABP AUC
0.9, NGAL AUC 0.83, IL-18 AUC 0.83) and that the prediction improved
when urinary L-FABP and NGAL levels were combined (AUC 0.93)
[36]. In the study performed by Kashani and colleagues [31], risk for
major adverse kidney events (death, dialysis or persistent renal impair-
ment) within 30 days elevated sharply for TIMP-2 and IGFBP7 above 0.3
and doubled when values were N2.0.
Biomarkers are a useful aid for the prediction of renal recovery after
AKI. Srisawat and colleagues conducted a multicenter, prospective ob-
servational cohort in 76 patients that had AKI and required RRT in the
ICU [37]. Renal recovery was dened as being alive and free from dialy-
sis at 60 days from the start of RRT. Patients who recovered had a higher
urinary cystatin C level on day 1, and lower uHGF on days 7 and 14.
Levels of uNGAL and uHGF that decreased in the rst 14 days were asso-
ciated with greater odds of renal recovery.
2.3. Differential diagnosis of AKI
The performance of NGAL and other biomarkers has been evaluated
in different populations with AKI. A single measurement of urinary
NGAL in the emergency department was found to be highly specic
(99%) and sensitive (90%) for AKI diagnosis [38]. Furthermore, urinary
NGAL was able to distinguish patients with AKI from patients with
CKD and prerenal conditions that had an elevated SCr. NGAL levels
have also been found to be useful in identifying other forms of AKI
such as delayed graft function [39] and contrast induced nephropathy
[40].
Parikh and colleagues measured urinary IL-18 in 72 patients in a
mixed population of renal conditions, including healthy subjects with
no evidence of renal disease. The authors reported urinary IL-18 levels
to be signicantly higher in patients diagnosed with ATN than in pa-
tients with prerenal azotemia or in healthy control subjects [41]. In
the study conducted by Siew and colleagues [32], urinary elevations of
IL-18 did not predict AKI, but did predict poor outcomes such as death
and the need for short term dialysis. Elevations in urine IL-18 is specic
for ATN and is associated with a higher risk of adverse clinical outcomes.
KIM-1 excretion in the urine is highly specic for kidney injury be-
cause no other organs have shown to express KIM-1 that could change
its urinary concentration. KIM-1 is a transmembrane glycoprotein that
is upregulated in animal models and in humans in the proximal tubule
cells after ischemic or toxic injury [42,43]. KIM-1 is expressed in kidney
tissue obtained from biopsies from patients with ATN [44], and is effec-
tive at distinguishing ATN from prerenal azotemia and CKD. In adults
submitted to cardiac surgery, urinary KIM-1 achieved the highest AUC
in comparison to other biomarkers (NAG, NGAL, IL-18, cystatin C and
1 microglobulin). The AUC for KIM-1 to predict AKI 2 h postoperative-
ly was 0.74 (95% CI = 0.640.91) followed by IL-18 and NAG [45].
The Topaz study [31] had the objective of validating the ability of uri-
nary levels of TIMP-2 and IGFBP7 in identifying patients at high risk for
development of moderate to severe AKI within 12 h, using a cutoff value
of more than 0.3 (ng/ml)2/1000. Critically ill patients with a single mea-
surement of urinary TIMP-2 and IGFBP7 above this level carried a higher
risk of developing AKI of multiple causes. Measuring these markers was
helpful for distinguishing true AKI in a diverse group of patients in the
intensive care unit.
353
2.4. Discussion
Current evidence supports the concept that the measurement of cer-
tain biomarkers for AKI facilitates early detection, differential diagnosis
and prognosis of AKI. The diagnosis of AKI can be made 48 h earlier
using biomarkers such as NGAL than with conventional assessments.
However their usefulness is still limited in patients with preexisting
renal disease and with comorbidities [22,23] in which the performance
for predicting AKI in this setting is still poor.
Encouraging results have been obtained with cyclic arrest markers
(IGFBP7 and TIMP-2) for early diagnosis and prognosis of AKI in com-
parison to other markers. The AUCs of both biomarkers for severe AKI
prediction within 12 h of sample collection were signicantly higher
than those of other biomarkers [30]. AKI research will be facilitated
with their use and cases of subclinical AKI may be uncovered. Future
studies will be necessary to guide interventions upon their positivity.
There is still a barrier for clinical translation regarding biomarkers in
AKI since there are not enough studies that have evaluated the impact of
biomarker information on decision making. What shall we do when a
biomarker results positive? Should we initiate dialysis? What does an
intervention include? Do early interventions decrease hospital and
long term mortality in AKI? Future studies should answer these ques-
tions before biomarker panels are routinely measured. However, the
use of these biomarkers to identify patients at risk will give clinicians
the opportunity to deploy the KDIGO consensus guidelines [46]. The im-
pact of the deployment of these guidelines in patients at high-risk for
AKI is unknown, but this represents the next frontier in the treatment
of AKI research. Simply put, do our consensus guidelines improve out-
comes? Future studies should address this issue with the aid of specic
biomarkers.
It is still not clear whether biomarkers perform better than the clin-
ical expertise of physicians; however they should be regarded as a com-
plement to a routine assessment and part of a decision tree. None of the
biomarkers in AKI reviewed here can replace careful clinical reasoning.
3. Biomarkers in chronic kidney disease (CKD)
CKD is characterized by a sustained reduction in GFR along with ev-
idence of structural or functional abnormalities of the kidneys on urinal-
ysis, biopsy and imaging [47]. CKD is associated with increased
cardiovascular events, leading to a higher morbidity and mortality
rate. Other important outcomes include infection, AKI, cognitive and
physical impairment, and progression of kidney disease [48]. Premature
death can occur as a result of complications involved with CKD, espe-
cially in the presence of coexistent proteinuria [49]. Table 3 outlines
the most promising biomarkers for CKD. In the following section we
shall discuss which biomarkers are useful in CKD and when to consider
measuring them. Monitoring renal function in CKD requires biomarkers
that provide rapid, non-invasive and specic measurements that corre-
late well with kidney tissue pathology. Early identication of patients
with CKD is important in order to perform early interventions and re-
duce progression to kidney failure or cardiovascular events.
3.1. Biomarkers for glomerular ltration rate (GFR) measurement
Evidence suggests that GFR is the best index of renal function in CKD
[50] and it is measured as the clearance rate of a ltration marker from
plasma by the kidneys. Normal values are approximately 130 ml/min/
1.73 m2 in young men and 120 ml/min/1.73 m2 in young women and
values decline as people age [49]. Urine isotope collections (inulin,
iothalamate, EDTA and iohexol) are considered as gold standards for
measuring GFR, however they are impractical, expensive, time consum-
ing and invasive in the clinical setting. For this reason, GFR is estimated
with equations that take into account endogenous ltration markers
such as SCr and demographic factors. Creatinine generation is affected
by age, sex, race and body weight independently from GFR [51]. GFR
354 M.E. Wasung et al. / Clinica Chimica Acta 438 (2015) 350357
estimating equations appear to be a more accurate estimate of GFR than
SCr alone [5255]; however they cannot account for individual differ-
ences in muscle mass for a given age, sex or race. Table 4 outlines the
various validated equations for GFR estimation. Muscle wasting is com-
mon in CKD and it is associated with lower creatinine generation there-
fore, providing bias toward a higher eGFR.
Cystatin C is a 13-kDa protein synthesized at a constant rate in all nu-
cleated cells [56]. It is freely ltered in the glomerulus and is reabsorbed
and catabolized completely in the proximal tubule with a lack of tubular
secretion [56]. Cystatin C is less affected by muscle mass than SCr, and is
considered to be a better marker of early kidney dysfunction [5761]
and a more reliable marker of renal function. In a study of 164 patients
with stage 2 or 3 CKD (GFR of 30 to 89 ml/min/1.73 m2), serum cystatin
C had a higher accuracy in detecting a GFR of less than 60 ml/min/
1.73 m2 than SCr, but only in women [61]. Investigators from CKD-EPI
developed 3 GFR estimating equations for cystatin C: using cystatin C
alone, cystatin C with demographic factors, and cystatin C with creati-
nine and demographic factors. The equation that included cystatin C,
creatinine and demographic coefcients provided the most accurate
GFR estimates [62].
Inker and colleagues [63] established an international reference
standard for cystatin C to develop a GFR estimating equation based on
5352 subjects from 13 cohorts. The equations were than validated in
1119 participants from 5 different studies. Two new equations that in-
volved cystatin C alone and in combination with creatinine were devel-
oped. The 2012 CKD-EPI cystatin C and 2009 CKD-EPI creatinine-based
equations had similar performances in this setting for the outcome of
measured GFR. The best GFR estimation resulted from the combined
creatininecystatin C equation. Near the CKD threshold (GFR of 45
75 ml/min/1.73/m2), the combined equation meaningfully improved
staging of CKD (GFR b 60 ml/min/1.73/m2) in comparison to the 2009
creatinine equation. The combined creatininecystatin C equation ap-
pears to be the optimal GFR estimate.
3.2. Cardiovascular disease prediction in CKD
Cystatin C levels have been associated with adverse clinical events.
In prior studies in the general population and in the elderly, cystatin C
has been shown to be a better predictor of mortality and adverse cardio-
vascular events than SCr [6467]. Cystatin C levels are also associated
with a higher rate of non-cardiovascular disease outcomes such as
pulmonary disease, cancer and infection [68]. Endothelial nitric oxide
synthase converts the amino acid L-arginine into L-citrulline and nitric
oxide [69]. Nitric oxide (NO) is considered an important vasodilator
that also inhibits several processes involved in vascular diseases, such
as leucocyte adhesion, platelet aggregation and vascular smooth muscle
cell proliferation [70]. An alteration in vascular NO synthesis is known to
Table 4
Estimating equations for glomerular ltration rate. MDRD and CKD-EPI equations incorpo-
rating creatinine, cystatin C or both.
MDRD
GFR (ml/min/1.73 m2) = 175 (SCr)1.154 (age)0.203 (0.742
if female) (1.212 if African American)
2009 CKD-EPI creatinine
GFR = 141 min (SCr / , 1) max(SCr / , 1)1.209 0.993age 1.018
[if female] 1.159 [if Black]
2012 CKD-EPI cystatin C
eGFR = 133 min(Scys / 0.8, 1)0.499 max(Scys / 0.8, 1)1.328 0.996age
[0.932 if female]
2012 CKD-EPI creatinine and cystatin C
eGFR = 135 min(SCr / k, 1)a max(SCr / k, 1)0.601 min(Scys / 0.8,
1)0.375 max(Scys / 0.8, 1)0.711 0.995age [0.969 if female] [1.08 if
Black]
SCr is serum creatinine and Scys is serum cystatin C; k is 0.7 for females and 0.9 for males; a
is 0.248 for females and 0.207 for males.
MDRD, modication of diet in renal disease equation.
CKD-EPI, chronic kidney disease epidemiology collaboration equation.
inuence the progression of atherosclerosis and carotid stenosis [71,72].
Asymmetric dimethylarginine (ADMA) is a nitric oxide synthase (NOS)
inhibitor [73,74] that plays an important role in the vascular tone. The
levels of ADMA are particularly high in patients with endothelial dys-
function, hypertension and proteinuria. ADMA levels increase as kidney
function declines because of a reduced clearance [74] and is found to be
associated with several cardiovascular events. ADMA is considered as
the missing link between cardiovascular disease and CKD [73] by
some authors, and is a strong biomarker that predicts a higher mortality
in CKD [7577] as well as a faster progression of kidney injury [78].
Counteracting the effects of ADMA in CKD should be explored as a strat-
egy to prevent cardio-renal complications.
3.3. CKD progression
NGAL is expressed by tubular epithelial cells in response to injury
and tubulointerstitial damage that is a common pathway in the progres-
sion of most forms of kidney disease [79]. NGAL has performed well as a
biomarker in CKD in several small studies. In one of them performed in
45 children with CKD stages 24, plasma NGAL levels were inversely as-
sociated with GFR [80]. As kidney function declined under 30 ml/min,
NGAL outperformed cystatin C as a biomarker of kidney function.
Smith and colleagues [81] conducted a prospective observational cohort
in 158 adult patients with stage 3 or 4 CKD. Urinary NGAL to creatinine
ratio (uNCR) was associated with a higher risk of death and initiation of
renal replacement therapy independent of renal and cardiovascular risk
factors. The authors concluded that measuring urinary uNCR improves
the prediction of renal progression in this population. Plasma NGAL
levels also predict progression of CKD in adults. The study performed
by Bolignano and colleagues [82] demonstrated that plasma and urinary
NGAL had a predictive power for CKD progression even after adjust-
ment for eGFR after an 18.5 month follow-up in a cohort of 96 patients.
NGAL was an independent predictor of CKD progression and reected
the severity of renal disease.
Warnock and associates evaluated whether combining creatinine,
cystatin C, and urine albumin to creatinine ratio would improve identi-
cation of risks associated with CKD compared with creatinine alone, in
a prospective cohort study involving 26,643 adults enrolled in the Rea-
sons for Geographic and Racial Differences in Stroke (REGARDS) study
[83]. In this study, the adjusted risk of mortality was threefold higher
in patients with CKD dened by both GFR cystatin and GFR creatinine
than in those with CKD dened by GFR creatinine alone and approxi-
mately sixfold higher in patients with CKD identied by all three
markers. The authors concluded that adding cystatin C to the measures
with SCr and albumincreatinine ratio improved the accuracy for all-
cause mortality and end-stage renal disease prediction.
Disturbances of mineral metabolism are common in CKD. As kidney
function declines phosphate is retained by the kidney, renal calcitriol
production declines and as a result calcium tends to decrease. FGF-23
is a 32-kDa protein secreted by osteoblasts primarily that plays an im-
portant role in controlling phosphate concentration. It acts as an endo-
crine hormone by inhibiting parathyroid hormone secretion, decreasing
levels of calcitriol, and by inducing phosphaturia [84,85]. Circulating
levels increase progressively as kidney function decreases below
60 ml/min/1.73 m2 [86,87]. A considerable amount of evidence has
shown that an elevated level of FGF-23 is an independent risk factor
for adverse outcomes in CKD such as a faster progression, higher inci-
dence of cardiovascular disease and increased mortality [88,89].
3.4. Inammatory and brotic markers associated with CKD
Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic pro-
tein that is expressed by a variety of cells, including endothelial cells, -
broblasts, mononuclear cells and kidney cells upon a proinammatory
stimulus [9092]. Expression of MCP-1 is up-regulated in kidney dis-
eases that have a sustained inammatory response, such as in diabetic
 M.E. Wasung et al. / Clinica Chimica Acta 438 (2015) 350357
nephropathy and lupus [93,94]. Studies have demonstrated that
podocytes and tubular cells produce MCP-1 in response to high glucose
levels and advanced glycosylation end products and that urinary levels
of MCP-1 are signicantly higher in patients with diabetic nephropathy
[95,96]. Urinary levels of MCP-1 are also elevated in patients with lupus
nephritis reecting intrarenal expression [94,97]. Serum concentrations
of MCP-1 are also high in patients with diabetic nephropathy and lupus
nephritis; however, the levels of serum MCP-1 do not correlate with the
degree of renal activity and damage in both entities [94,98].
Connective tissue growth factor (CTGF) is a member of the CCN fam-
ily of matricellular proteins, which is implicated in many cellular func-
tions, which include cell proliferation, adhesion and wound healing
[99102]. CTGF is considered an important mediator of tissue brosis
and several brotic conditions such as liver brosis and scleroderma
have been found to overexpress it [103105]. Renal diseases that ex-
press higher levels of CTGF levels include diabetic nephropathy, focal
and segmental glomerulosclerosis and IgA nephropathy [106,107].
CTGF has been found to be an independent predictor of ESRD and corre-
lates well with the rate of decline of GFR in diabetic nephropathy [107,
108].
3.5. Discussion
Biomarkers in CKD help us estimate GFR, assess cardiovascular dis-
ease, determine metabolic abnormalities associated with CKD and dif-
ferentiate inammatory and brotic conditions of the kidney. The
pathophysiology of CKD is complex and involves multiple processes in
its progression and in some cases an underlying primary renal disease
is involved. Comorbidity is present in practically all patients with CKD,
therefore a single biomarker is insufcient to meet all of these
expectations.
Cystatin C is a very useful biomarker in CKD and used for GFR estima-
tion and for cardiovascular disease assessment. The value of estimating
GFR with cystatin C over conventional assessments is to improve the
specicity of CKD diagnosis. KDIGO guidelines [108] recommend
cystatin C measurement in patients in which CKD would be missed
when using eGFR with SCr alone. Cystatin C based GFR equations are
more accurate than the CKDEPI equation in certain subgroups of pa-
tients in which the SCr level may be insensitive to capture reduced kid-
ney function such as hospitalized patients, patients with chronic liver
disease and cirrhosis, frail elders, and patients with HIV infection and
malignancy. Cystatin C is less inuenced by muscle mass than creatinine
and may reect a better marker of GFR in these populations. Cystatin C
levels however are affected by inammatory conditions that present
high levels of C reactive protein [57], thyroid disease, glucocorticoid
use and obesity, and this should be implemented with caution in
these conditions. Combining creatinine, cystatin C and urine albumin
to creatinine ratio improves risk stratication and helps predict progres-
sion to end stage kidney disease and mortality.
FGF-23 is an accurate marker of CKD metabolic bone disease and
may serve as a complement of CKD assessment. FGF-23 as well as
other biomarkers such as NGAL, ADMA, MCP-1 and CTGF are emerging
biomarkers that may help predict progression and stratify risk in CKD,
however more research is needed in the eld before these can be imple-
mented in clinical practice.
4. Conclusion
Biomarker research has increased signicantly in the past ten years
with the purpose of allowing nephrologists to identify kidney injury ear-
lier. SCr, eGFR and proteinuria are not sensitive enough, and identify kid-
ney diseases late when valuable interventions can be applied. Biomarkers
may revolutionize the diagnosis of AKI and CKD, however they must be
validated in larger populations with comorbidities and with hard clinical
end points. Of those reviewed, cycle arrest markers appear as the most
promising for AKI, performing better than other existing biomarkers for
355
early diagnosis and prognosis of AKI. NGAL is a very useful marker in
AKI diagnosis; however it does not perform well in patients with pre-
existing renal failure. Renal recovery is an interesting topic in AKI
research, and decreasing levels of urinary NGAL and urinary HGF demon-
strated to be useful in predicting recovery in different populations.
During the last century, SCr has been the most used biomarker to
screen and diagnose kidney disease. Creatinine however has several
limitations and should be utilized only in estimating equations
(Table 3). The CKD-EPI seems to be more generalizable and accurate
than the MDRD estimating equation. More recently the GFR estimating
equation combining SCr and cystatin C performed better than equations
that used either SCr or cystatin C alone and is recommended in specic
conditions, such as when conrmation of CKD is required [108]. Cystatin
C is a valuable biomarker of kidney function in CKD, kidney disease pro-
gression and cardiovascular disease prediction. Emerging biomarkers
for CKD (NGAL, ADMA, MCP-1, CTGF) can serve as a complement in rou-
tine assessments in CKD patients.
Biomarkers in AKI and CKD share similarities; both entities have a
reduction in nephron number, vascular insufciency and cell cycle dis-
ruption. For this reason biomarkers such as NGAL, KIM-1 and cystatin-
C are present in both. Kidney diseases are complex, have multiple
causes, and may accompany systemic diseases and CKD and AKI may co-
exist; therefore a single biomarker is unlikely to meet all the expecta-
tions and biomarker panels will be necessary. Biomarkers should not
replace clinical assessments of patients with CKD and AKI and should
be considered a complement to clinical reasoning. Indiscriminate use
of biomarker panels can distract physicians from taking adequate deci-
sions affecting outcomes.
In conclusion, relevant biomarkers have been developed to diag-
nose, predict outcome and stratify risk patients with AKI and CKD. It is
important to know which biomarkers are useful and when can they
be measured. Currently, the use of biomarkers for decision making is
not dened; and still measured bench-work and not bed-side. Al-
though some of the biomarkers are highly sensitive and specic, larger,
multicenter and long term trials are still necessary to make translation
and eventually make decisions upon their positivity.
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