Evidence-Based Nephrology

Biomarkers for the Early Detection and Prognosis

of Acute Kidney Injury
Rakesh Malhotra* and Edward D. Siew†‡§

Abstract
AKI is an increasingly common disorder that is strongly linked to short- and long-term morbidity and mortality. Despite a
growing heterogeneity in its causes, providing a timely and certain diagnosis of AKI remains challenging. In this review,
*Division of
we summarize the evolution of AKI biomarker studies over the past few years, focusing on two major areas of Nephrology and
investigation: the early detection and prognosis of AKI. We highlight some of the lessons learned in conducting AKI Hypertension,
biomarker studies, including ongoing attempts to address the limitations of creatinine as a reference standard and the Department of
recent shift toward evaluating the prognostic potential of these markers. Lastly, we suggest current gaps in knowledge Medicine, University
of California San
and barriers that may be hindering their incorporation into care and a full ascertainment of their value. Diego, San Diego,
Clin J Am Soc Nephrol 12: 149–173, 2017. doi: 10.2215/CJN.01300216 California; †Division
of Nephrology and
Hypertension,
Department of
Introduction emerged and are available as a potential aid for AKI Medicine, Vanderbilt
AKI is becoming increasingly common and is linked risk stratification (23). University Medical
to progressive loss of kidney function, cardiovascular center, Nashville,
disease, and death (1–7). Despite often being reduced Tennessee; ‡Tennessee
Early Detection of AKI Valley Healthcare
to its rudimentary manifestations (i.e., a change in Following early studies demonstrating the ability System, Veteran’s
serum creatinine), AKI is comprised of several dis- of several markers to identify established AKI (10,14,16,18, Administration
eases, highlighting a need to improve phenotyping 24–26), large prospective cohorts determined their Medical Center,
beyond the circumstantial. Developing therapies capacity to provide an earlier diagnosis (Appendix Veterans Health
will require a timely description of the onset, location, Administration,
Table 2). These investigations were inspired by the Nashville, Tennessee;
and severity of parenchymal injury, its underlying hypothesis that delays in intervention have hindered clin- and §Vanderbilt
mechanisms, and prognosis (2,8). Next-generation ical trials and small studies demonstrating that markers Center for Kidney
biomarkers of AKI propose to fill these knowledge such as NGAL could provide timelier identification of Disease and
gaps (Figure 1). This review will discuss knowledge Integrated Program for
AKI (27,28). The landmark Translational Research In- Acute Kidney Injury
gained over the past decade and attempts to better vestigating Biomarkers and End Points for Acute Kid- Research, Nashville,
integrate these markers into care. ney Injury (TRIBE-AKI) consortium expanded upon Tennessee
this work by testing whether postoperative levels of
urine NGAL (uNGAL), plasma NGAL (pNGAL), and Correspondence:
urine IL-18 (uIL-18) could detect a doubling of baseline Dr. Edward D. Siew,
The Growing Role of Biomarkers: From Division of
Detection to Prognosis serum creatinine (sCR), acute dialysis, and other end- Nephrology and
Appendix Table 1 lists the several putative bio- points in 1219 adults and 311 children undergoing car- Hypertension,
markers for AKI in humans, including enzymes diac surgery in the United States and Canada (29,30). Vanderbilt Center for
(n-acetyl-b-d-glucosaminidase [NAG], a- and Postoperative biomarker levels were higher in patients Kidney Disease and
Integrated Program for
p-glutathione s-transferase [a-GST/p-GST], alkaline who experienced AKI than those who did not, were
AKI Research,
phosphates [AP], and alanine aminopeptidase associated with AKI after adjusting for clinical risk fac- Vanderbilt University
[AAP]), proinflammatory mediators (IL-18; neutro- tors, and helped reclassify risk in some patients. How- Medical Center, 1161
phil gelatinase–associated lipocalin [NGAL]), and ever, discrimination was modest, yielding the 21st Avenue South,
other structural upregulated proteins (kidney injury following area under the receiver operating curves MCN S3223,
Nashville, TN 37232-
molecule-1 [KIM-1], liver-type fatty acid–binding (AUCs): uNGAL, 0.67 and 0.71, pNGAL, 0.70 and 2372. Email: edward.
protein [L-FABP], sodium–hydrogen (Na+/H+) ex- 0.56, and uIL-18, 0.74 and 0.72 in adults and children, siew@vanderbilt.edu
changer isoform) that are released during tubular respectively (29,30). The observed incidence of AKI was
damage (9–20). Other markers of glomerular filtration 5% in adults and 17% in children, and respective cut-off
reabsorbed by healthy tubular epithelium (cystatin-C values for uNGAL (102 ng/ml and 17 ng/ml), pNGAL
[Cys-C], retinol binding protein [RBP], and a1/b2 mi- (293 ng/ml and 154 ng/ml), and uIL-18 (60 pg/ml and
croglobulin), and hormonal markers (angiotensino- 125 pg/ml) yielded negative predictive values (NPVs)
gen) have also been studied (9,15,21,22). Recently, of 86%–97% and positive predictive values (PPVs) of
markers thought to play a role in cell cycle regula- 11%–30%.
tion, including tissue inhibitor of metalloproteinases-2 Among the critically ill, a study of 632 patients in
(TIMP-2) and IGF-binding protein-7 (IGFBP7), have Europe showed that pNGAL and uNGAL measured

www.cjasn.org Vol 12 January, 2017 Copyright © 2016 by the American Society of Nephrology 149
150 Clinical Journal of the American Society of Nephrology

Figure 1. | How biomarkers may improve the current approach to AKI. The top panel illustrates the current paradigm highlighting how serum
creatinine does permit recognition of AKI, albeit delayed, and an estimate of severity based on loss of function. However, much is left to clinical
impression, including its anatomic location, etiology, appropriateness for a trial, the presence and extent of tissue damage, and risk of progression.
Similarly, tissue damage is presumed retrospectively only after loss of function becomes persistent and prospects for recovery often remain uncertain.
The lower panel shows the complementary roles that AKI biomarkers may play, including aiding in detection of subclinical/early injury, providing
information on its type and location with potential applications including as a tool for trial enrollment, detailing the extent of damage, as a beacon for
ongoing injury during recovery or indicating repair/fibrosis, and risk stratifying for future complications. CV, cardiovascular, HTN, hypertension, sCR,
serum creatinine.

at intensive care unit admission discriminated between
patients who developed AKI within 7 days and those
who did not (pNGAL: AUCs of 0.7760.05 and 0.8060.06
and RIFLE risk [R] and injury [I]. uNGAL: AUCs of
0.8060.04 and 0.8560.04 for RIFLE Risk and Injury cate-
gories, respectively). Notably, 58% of patients fulfilled
creatinine-based criteria for AKI on enrollment day (31).
In another study of 451 critically ill adults in the US,
uNGAL provided moderate discrimination for AKI de-
veloping within 24 and 48 hours of measurement (AUC,
0.71; 95% confidence interval [95% CI], 0.63 to 0.78; and
AUC, 0.64; 95% CI, 0.57 to 0.71, respectively) (32). A sub-
group analysis performed among patients with an
eGFR$75 ml/min per 1.73m2 to enrich for incident AKI
improved discrimination to 0.77 (95% CI, 0.64 to 0.90)
(32). However, a subsequent case-control study by the
same authors did not confirm improvement in the perfor-
mance of urine NGAL, Cys-C, and L-FABP for detecting
AKI within 48 hours in patients with preserved kidney
function (33). Several other studies evaluating NGAL,
Cys-C, KIM-1, and IL-18 in the critically ill have also
been performed, showing varying levels of performance
(32–37).
Recently, a large multicenter study (SAPPHIRE) was con-
ducted to identify novel biomarkers for AKI in critically ill
adults (23). In a discovery cohort, the ability of 340 protein
biomarker candidates to detect Kidney Disease Improving
Global Outcomes (KDIGO) stage 2 or 3 injury within 12 hours
was examined in 522 patients. Of the markers examined,
the combined AUC of TIMP-2 and IGFBP7 was 0.77 (95%
CI, 0.72 to 0.82), a finding subsequently replicated in a sep-
arate validation cohort of 728 patients (AUC, 0.80; 95% CI,
0.75 to 0.84). After adjusting for nine clinical covariates,
TIMP-2*IGFBP7 was associated with a 4.1-fold increase in
the risk of developing KDIGO stage 2 or 3 AKI. As one of
the shared functions of these molecules is to induce G1 cell
cycle arrest, the investigators hypothesized TIMP-
2*IGFBP7 may serve a protective effect by minimizing the
proliferation of damaged cells. In a separate study of 408
patients, a value of 0.3 ng/ml2/1000 for AKI demonstrated
good discrimination (AUC, 0.82; 95% CI, 0.76 to 0.88) (38).
The primary endpoint of stage 2 or 3 AKI occurred in 17%
Clin J Am Soc Nephrol 12: 149–173, January, 2017
of patients yielding PPVs ranging from 27%–62% for cutoff
values of 0.3 and 2.0 ng/ml2/1000. These observations re-
cently led the Food and Drug Administration to approve the
marketing of TIMP-2*IGFBP7 as a potential aid in the risk
assessment for moderate or severe AKI in critically ill pa-
tients within 12 hours, when used in conjunction with clin-
ical evaluation, including confirmation by alternative
methods (38). Notably, NPVs for TIMP-2*IGFBP7 ranged
from 88%–96%, suggesting that a strength might lie in the
ability to identify patients at low risk for severe AKI. The
12-hour window in the SAPPHIRE study was also shorter
than in other biomarker studies. Whether early knowledge
of increased risk for severe AKI within this window can
translate into improved outcomes remains to be determined
(see Limitations and Future Directions, below).
Early detection has also been examined in other acute
care settings (18,26,39–41). In one study of 121 hospitalized
cirrhotic patients, uNGAL accurately discriminated pa-
tients who developed AKI from those who did not, with
an AUC of 0.83 (95% CI, 0.76 to 0.91), a PPV of 54%, and
an NPV of 89% (41). Similarly, in a single-center study of
91 patients with acute decompensated heart failure, ele-
vated sNGAL levels ($140 ng/ml) were associated
with a 7.4-fold increase in the risk of developing AKI (39).
Challenges of Early Detection Studies
While showing some ability to provide a timelier di-
agnosis, interpretive and practical issues have emerged.
One of these includes a need to expand the framework
for assessing biomarker performance beyond sCR. This has
been suggested by studies demonstrating that some injury
markers increase in concert after percutaneous coronary
intervention in patients with preserved kidney function
without an increase in sCR (42), and in critically ill patients
with prerenal injury (43). Whether this reflects nonspecific
expression or unrecognized tubular damage due to renal
functional reserve or other confounders is unknown. Con-
versely, low biomarker levels in the presence of elevated
creatinine may not necessarily constitute a false-negative.
These limitations were illustrated in a simulation study
demonstrating that a biomarker with a 100% sensitivity
in a 10% prevalent population may be seen as being only
47% sensitive when compared with sCR, assuming creati-
nine itself is 80% sensitive and 90% specific (44). Notably,
the Predictive Safety Testing Consortium found that mark-
ers such as uKIM-1 and NAG outperformed sCR in
preclinical studies when a tissue standard was used (45).
However, tissue standards in human AKI have been lim-
ited to a few small studies (46–48). These results have sug-
gested that AKI biomarkers might be more valuable when
viewed as complementary rather than in competition with
sCR, leading the Acute Dialysis Quality Initiative to
propose a framework to characterize AKI using both func-
tional and damage markers in 2014 (49). This delineation
allows for subclinical AKI where damage markers are
elevated without an accompanying change in function
(biomarker1/sCR2), functional changes in the absence of
damage (biomarker2/sCR1), and both functional and
damage (biomarker1/sCR1). Subsequent proof of concept
studies have illustrated that patients with elevated NGAL
without elevated sCR were at higher risk for death, and
that patients with elevations of both NGAL and sCR
Biomarkers of AKI, Malhotra and Siew 151
were at higher risk for death and dialysis than patients
with elevated sCR alone (50,51).
The effect of differences in case mix on performance also
remain poorly understood. Although the timing of tubular
damage and their contributions to increases in sCR likely
vary with the cause of AKI, clinical phenotyping remains
limited in most studies. Some studies have shown improve-
ment in biomarker discrimination when requiring more
persistent increases in sCR (32,37), whereas others have
shown some ability to distinguish prerenal versus intrinsic
AKI (26,52–54). In one study of 1635 adults presenting to an
emergency room, uNGAL, uL-FABP, and uKIM-1 showed
moderate-to-good utility for discriminating between intrinsic
and prerenal AKI, CKD, and normal kidney function (54).
However, there were multiple contributors in the intrinsic
AKI group, including, but not limited to, hypotension, obstruc-
tion, sepsis, nephrotoxicity, and GN/vasculitis. It remains un-
clear how biomarker expression patterns differ across these
subtypes.
Clinical uncertainty as to when injury actually occurs
and the kinetics of biomarker expression outside of cardiac
surgery or planned nephrotoxin exposure may also affect
performance. As AKI can occur at different times before
creatinine rises, a biomarker measurement before injury
or one that only transiently increases can reduce its
observed sensitivity (55). The former would reflect more of a
prediction rather than early detection scenario and not be
expected to perform well. In 528 patients from the Early In-
tervention with Erythropoietin Does Not Affect the Outcome
of Acute Kidney Injury (EARLY-ARF) trial, GGT, NGAL,
Cys-C, KIM-1, and IL-18 were used to triage patients for
enrollment but failed to adequately distinguish patients
who developed AKI from those who did not (AUCs, 0.59–
0.67) (56). However, performance of some biomarkers im-
proved after stratifying by the timing of likely kidney insult
determined by clinical adjudication (AUCs, 0.85–0.94) (56).
Much also remains to be learned about the biologic variabil-
ity of biomarker expression. For example, preclinical and
clinical studies indicate that genetic background and comor-
bidities, such as CKD, can have a substantial effect on chronic
and acute biomarker expression (57,58). An improved under-
standing and ability to quantify this variability is needed to
better interpret values in the diagnosis and prognosis of AKI
(59). Standardization of assays also remains an important
goal. Although reported intra- and interassay coefficients of
variation appear reasonable, studies comparing performance
of commercially available platforms has been limited (60) and
further collaboration is required to integrate and standardize
different methodologies.
The Move toward More Meaningful
Clinical Endpoints
Predicting AKI Severity
Given the debated significance and specificity of smaller
or transient changes in sCR, studies have investigated
whether biomarkers measured during early stages of AKI can
predict progression to a higher stage (21,22,61,62). The TRIBE-
AKI investigators tested if tubular injury markers could pre-
dict worsening of AKI stage in 380 cardiac surgery patients
with stage 1 AKI. Although individual discrimination was
moderate (AUCs, 0.63 for uIL-18 to 0.74 for pNGAL), the
152 Clinical Journal of the American Society of Nephrology
highest quintiles of uIL-18 and pNGAL associated with pro-
gression to a higher stage (adjusted odds ratios of 3.0 (95%
CI, 1.3 to 7.3) and 7.7 (95% CI, 2.6 to 22.5), respectively) (61).
All biomarkers improved risk classification, although uIL-18
was more effective in reclassifying nonevents to a lower level
of risk whereas uNGAL and pNGAL appeared to help re-
classify events to a higher level of risk (61).
Another study tested the ability of uL-FABP, uNGAL,
uIL-18, and uKIM-1 to predict injury progression, dialysis,
or death within 7 days in 152 critically ill patients with
stage 1 AKI (62). Discrimination of uL-FABP was highest
(AUC, 0.79; 95% CI, 0.70 to 0.86) with uNGAL, uIL-18, and
uKIM-1 showing more modest performance (AUC range,
0.62–0.65). Adding uL-FABP raised the AUC of the clinical
model from 0.74 (95% CI, 0.68 to 0.84) to 0.82 (95% CI, 0.75
to 0.90), an improvement not observed with the other bio-
markers. uL-FABP correctly reclassified a higher proportion
of nonevents and uNGAL reclassified a higher proportion of
events than predicted by the clinical model. Comparable
studies in patients with cirrhosis and hospitalized patients
have shown similar results (24,63).
In children, the ability of biomarkers to enhance clini-
cal risk prediction tools has been investigated. The renal
angina index (RAI) is a clinical tool developed to identify
critically ill children with evidence of early injury who are
at risk for developing severe AKI (64). While effectively
ruling out patients at low risk for severe AKI, the PPV of
renal angina for predicting severe AKI was only 40% in the
population studied, highlighting the need for additional
tools beyond established clinical predictors. The authors
hypothesized that RAI might also help guide biomarker
measurement and improve performance by enriching the
population for the outcome of interest and reduce false
positives, a problem observed by less selective measure-
ment of biomarkers, such as troponin (65–67). Subse-
quently, the investigators found that elevated matrix
metalloproteinase-8 and neutrophil elastase increased the
probability of a patient experiencing severe AKI compared
with patients with RAI alone by 31% (68).
Informing Long-Term Prognosis of AKI.
Renal Recovery after AKI. The transition of care is an
opportunity to improve longitudinal outcomes after AKI
(69,70). As recovery is a key prognosticator (71), identify-
ing poor recovery may help facilitate nephrology referral,
inform clinical decision making, guide enrollment in trials,
and, most importantly, provide key information to AKI
survivors (72–75). AKI biomarkers may also be better
suited to characterizing recovery compared with more pe-
ripherally associated outcomes, including mortality.
The Biologic Markers of Recovery for the Kidney study
examined the ability of blood and urine biomarkers to
predict recovery among patients with dialysis-requiring
AKI (72–74). In 76 patients with urine available 1, 7, and 14
days after dialysis initiation, changes in urinary hepatocyte
growth factor and uNGAL individually predicted
dialysis-free survival by day 60 with AUCs of 0.74 (95%
CI, 0.60 to 0.89) and 0.70 (95% CI, 0.55 to 0.85), respectively,
and 0.84 (95% CI, 0.73 to 0.94) when combined (72). Sim-
ilarly, the addition of biomarkers raised the c-statistic of
the clinical model from 0.74 to 0.93. Among 817 patients
with plasma available, each natural log increase in plasma
IL-8 and TNF receptor-1 levels were associated with a
lower likelihood of dialysis-free survival at 60 days (hazard
ratios [HRs], 0.80 [95% CI, 0.70 to 0.91] and 0.63 [95% CI,
0.50 to 0.71], respectively) (73,74). Notably, these studies
have been limited by use of highly select populations and
variability in the timing of dialysis in relation to injury rec-
ognition. In addition, as these markers reflect mainly tubular
injury, their ability to provide information on repair itself
may be limited, highlighting the need to identify additional
markers of recovery and prognosis (76,77).
Long-Term Outcomes. In cardiology, major adverse car-
diac events (all-cause mortality, myocardial infarction or
unplanned repeat revascularization, stroke, and heart failure)
have been approved as a primary end point by the Food and
Drug Administration for cardiovascular-outcomes studies
(78,79). Similarly, others have advocated using long-term out-
comes (e.g., major adverse kidney events—sustained loss of
kidney function, dialysis, or death) for both intervention and
biomarker studies in patients with established AKI (80,81).
The TRIBE-AKI study observed a graded risk for mor-
tality between the highest and lowest tertiles of biomarker
levels (uNGAL, uKIM-1, uIL-18, uL-FABP, albumin) and
3-year mortality in 1199 adults in patients with sCR1AKI
(adjusted HR, 2.0 to 3.2) and in patients without sCR1AKI
(adjusted HR, 0.9 to 1.8) (82). Notably, the risk of mortality
in patients within the highest biomarker tertile in patients
without sCR1AKI was similar to patients with sCR1AKI
with the lowest tertiles of biomarker expression (except for
uL-FABP). These results are consistent with the hypothesis
that AKI biomarkers can provide additional prognostic in-
formation, although the extent to which elevated markers
are specifically reflecting mortality risk attributable to kid-
ney injury is uncertain.
Studies have also begun to examine the association be-
tween AKI biomarkers and long-term loss of kidney function.
In a secondary analysis of 692 patients from the SAPPHIRE
study, uTIMP-2*IGFBP7 levels above 2.0 (n544) at inten-
sive care unit admission were associated with death or di-
alysis at 9 months (HR, 2.16; 95% CI, 1.32 to 3.53) (83).
Among patients receiving kidney transplant, median post-
operative levels of uNGAL (.422 ng/ml) and uIL-18(.137
pg/ml) have been associated with a 6.0- and 5.5-fold ad-
justed risk for poor graft function (dialysis or eGFR,30) at
1 year (84).
Limitations and Future Directions
Despite progress, important challenges remain in de-
termining whether biomarkers can improve outcomes in
AKI. One key question is how to apply biomarkers in current
practice. For example, while timelier evaluation may benefit
some patients, recent studies of electronic alerting systems
have failed to demonstrate that earlier knowledge of in-
creased risk translates to better outcomes on a population
level, possibly due to the lack of specific or effective in-
terventions (85,86). A future trial that randomizes patients to
biomarker measurement plus protocol-driven guidelines for
AKI evaluation versus no biomarker measurement may be
informative. Another potential application is their use in clin-
ical trials. In the recent ELAIN study randomizing critically
ill patients with stage II AKI to early versus delayed initia-
tion of RRT, investigators excluded patients with plasma
NGAL levels ,150 ng/dl. Most patients had values of
Clin J Am Soc Nephrol 12: 149–173, January, 2017
.400 ng/dl and .90% of patients in the delayed arm even-
tually received RRT, suggesting that high levels might help
enrich populations for patients likely to progress or experi-
ence complications of severe AKI (87). In a similar study, the
Standard versus Accelerated Initiation of RRT in Acute Kid-
ney Injury STARRT-AKI trial researchers also measured
NGAL in whole blood; however, they found that whole-
blood NGAL was able to confirm the presence of severe
AKI but had difficulty distinguishing between patients in
the standard arm who did and did not go on to require
RRT (88). The latter is consistent with observational studies,
indicating that some of the stronger aspects of performance
observed for several biomarkers may be in their NPV
(29,33,68,89). Whether biomarkers have value as surrogate
outcomes in interventional studies has not been studied.
To some extent, the use of markers such as TIMP2*IGFBP7
may be instructive. A strong association with more spe-
cific and persistent losses of kidney function (e.g., major
adverse kidney events outcomes) may alone be sufficient
to inform decision making; however, full realization of
their potential will depend on broadening the scope of
investigation. Preclinical and epidemiologic data now
support the contention that the sequelae of AKI extend
beyond eGFR and include potential mediators (e.g., hy-
pertension) or alternative outcomes (e.g., cardiovascular
disease). Yet studies that examine the association between
injury biomarkers and related elements of tubular func-
tion, including sodium, water handling, solute transport,
and novel imaging, remain lacking (90,91).
An important opportunity for biomarkers to help im-
prove outcomes in AKI may lie in refining the current
approach to clinical phenotyping. Determining whether
biomarkers can help fill an unmet need to noninvasively
align prospective therapies with target AKI subpopulations
may benefit from tissue standards for comparison. In
support of this idea, the National Institute of Diabetes
and Digestive and Kidney Disease NIDDK recently high-
lighted in their Kidney Research National Dialogue the
need for ‘more intensive efforts to procure human biologic
samples’ (92). Specifically highlighted was the paucity of
kidney biopsies for diseases unrelated to the glomerulus
and a call for ‘reappraisal of the standard indications for
biopsy.’ While the goals of such studies need to be delin-
eated and the risk to patients carefully weighed, outcomes
in this disease will depend on improving confidence in our
ability to identify the types of underlying lesions seen in
modern-day AKI. It is worth recalling that pilot biopsy
studies in the African American Study of Kidney Disease
and Hypertension (AASK) and Irbesartan Diabetic
Nephropathy Trial (IDNT) helped to confirm that the clin-
ical diagnoses of hypertensive nephrosclerosis and dia-
betic nephropathy were reliable enough to ensure
enrollment of the target populations of interest (93,94).
The extent to which heterogeneity in the diagnosis of
AKI may be currently impacting the appropriate enroll-
ment in clinical trials is unknown, although we know
that considerable variability exists between clinicians in
simply distinguishing between prerenal AKI versus acute
tubular necrosis (95). The effect of even small degrees of
clinical uncertainty can be illustrated by a hypothetical trial
of an intervention with a 90% power (a of 0.05) to reduce
dialysis dependence in sepsis-associated acute tubular
Biomarkers of AKI, Malhotra and Siew 153
necrosis by 10% (50%–40% reduction, n5518/arm). If the
same study population contains even a 20% subpopulation
of drug-induced AIN (assuming no response to interven-
tion), the power of the study would be reduced to 73%
(PS software, v3.0; Dupont and Plummer).
Similarly, developing future therapies will also depend
on gaining improved mechanistic understanding of the
pathogenesis of AKI. Whether current AKI markers can
adequately capture these complexities remains to be ex-
amined. For example, the hypothesis that the expression
of TIMP-2*IGFBP7 may be protective is provocative but
has yet to be confirmed. Another recent study examining
patients with septic shock who underwent rapid postmor-
tem kidney biopsy found marked increases in KIM-1 im-
munohistochemical staining in corticomedullary junction
and cortical labyrinth tubules that was more widespread
than the amount of frank necrosis observed (48). Whether
these results provide insight into a specific distribution of
injury in sepsis or its ‘reversibility’ and the ability of urine
KIM-1 to capture some of this information are worth ex-
ploring. Future use of rapidly evolving “omic” techniques
and emerging systems biology approaches may facilitate
the identification of novel markers that can provide greater
biologic and mechanistic insights into the pathogenesis of
clinical AKI (96–104) (Appendix Tables 2 and 3).
In conclusion, the investigation of next-generation AKI
biomarkers over the past decade has prompted a needed
reevaluation of long-standing knowledge gaps in the tradi-
tional approach to evaluating and treating AKI. Early
experiences viewed within this traditional framework sug-
gest potential value for prediction and prognosis that remains
to be confirmed by ongoing use in practice and in clinical
trials. However, advancing understanding of AKI and de-
termining the full value of biomarkers will require creatively
expanding the lens of investigation, including addressing
fundamental questions of the etiology and underlying
mechanisms that current methods of investigation alone
may not elucidate. The latter may hold the key to eventually
translating long-overdue care treatment strategies into rou-
tine practice.
Acknowledgments
R.M. and E.D.S. are supported by the Vanderbilt Center for
Kidney Disease. E.D.S. is also supported by grants from the National
Institutes of Health NIDDK DK92192-07 and the Veterans Health
Affairs HSRD & Merit Award IIR 13-073-3. At the time of prepa-
ration of this manuscript, R.M. was pursuing a nephrology fellow-
ship at Vanderbilt University.
Disclosures
None.
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See related commentary, “Commentary on Biomarkers for Early
Detection and Prognosis of AKI” on pages 174–175.
Appendix
158

Appendix Table 1. Properties of Biomarkers of AKI

Biomarker Kinetics in Factors Affecting

Biomarker Types Biomarkers Sources Tested Site of Expression Known Functions Assay Platform
Cardiac Surgery Setting Biomarker Levels

Functional Cys-C (9) Urine, serum Nucleated cell 13-kDa cysteine protease Detected 12–24 h Albuminuria increase Nephelometry,
inhibitor, freely filtered postinjury, and peak urinary excretion of turbidimetry, ELISA
and further reabsorbed levels at 24–48 h Cys-C (competitive (assay analysis is
and metabolized in reabsorption through affected by diabetes,
the PT megalin-facilitated large dose steroids,
endocytosis); blood hyperthyroidism,
Cys-C levels are inflammation,
affected by GFR hyperbilirubinemia,
rheumatoid factor, and
hypertriglyceridemia)
Tubular injury IL-18 (12) Urine, serum PT, mononuclear cells, 24-kDa molecule cleaved Elevation within first 6 h IL-18 levels are also high ELISA
macrophages, by caspase-1 to generate after injury, and peak in inflammation,
fibroblasts, biologically active levels at 12–18 h sepsis, heart failure
dendritic cells, 18-kD molecule;
Clinical Journal of the American Society of Nephrology

intestinal epithelia, upregulation in

adrenal cortex ischemic kidney injury
and is proinflammatory
KIM-1 (10) Urine PT (KIM-1a: renal; 38.7-kDa type-1 Detected 12–24 h KIM-1 is also expressed at ELISA; Luminex
KIM-1b: liver) membrane postinjury and peak high levels in patients
glycoprotein; levels at 48–72 h with clear cell–type
phosphatidylserine renal cell carcinoma,
receptor; upregulated chronic proteinuria,
in tubular injury and CKD, and sickle-cell
activates immune cells nephropathy
TH-1, TH-2, and TH-17;
promotes apoptotic and
necrotic cell clearance
and remodeling of
injured epithelia
L-FABP (20) Urine PT, liver, small 15-kDa cytoplasmic Detected within 1 h after L-FABP levels are also ELISA
intestine, pancreas, protein which injury, and peak elevated in liver
lung, stomach transports free fatty levels within 6 h disease, sepsis,
acids to mitochondria polycystic kidney
and peroxisomes for disease, CKD
metabolism; protects
against damage caused
by reactive oxygen
species; upregulated
during ischemia–
reperfusion injury
Appendix Table 1. (Continued)
Biomarker Types Biomarkers Sources Tested Site of Expression Known Functions
NGAL (13) Urine, serum Kidney (PT.DT), 25-kDa lipocalin protein
neutrophils, liver, associated with
spleen gelatinase from
neutrophils; NGAL
chelates labile Fe
released from damaged
tubules and prevents
formation of hydroxy
radicals; also
upregulates the reno-
protective enzyme
heme-oxygenase-1
NAG (19) Urine PT 140-kDa proximal tubular
lysosomal enzyme, not
filtered
a-/p-GST (11,17) Urine a-GST: PT; p-GST: DT 47–51 kDa cytoplasmic
enzymes; increased
after tubular injury
Cell cycle arrest TIMP-2 (23) Urine All epithelial cells; TIMP-2 stimulates p27
IGFBP7 (23) kidney tubular expression and IGFBP7
epithelial cells increases the expression of
p53 and p21, which block
cyclin-dependent protein
kinase complexes on cell
cycle promotion, thereby
resulting in transient cell
cycle arrest
PT, proximal tubule; KIM-1, kidney injury molecule-1; TH, T-helper cells; L-FABP, liver-type fatty acid–binding protein; NGAL, neutrophil gelatinase–associated lipocalin; DT, distal tubule; Fe, iron; NAG,
n-acetyl-b-d-glucosaminidase; a-GST, a-glutathione s-transferase; p-GST, p-glutathione s-transferase; TIMP-2, tissue inhibitor of metalloproteinases-2; IGFBP7, IGF-binding protein 7.
Biomarker Kinetics in Factors Affecting
Assay Platform
Cardiac Surgery Setting Biomarker Levels
Detected as early as 3 h Plasma NGAL influenced ELISA
after injury, peak at 6 by CKD (really?),
h, and can have chronic hypertension
sustained elevation (really?), systemic
as long as 5 d infections,
inflammatory
conditions, anemia,
hypoxia, and
malignancies
Detected 12 h after renal Urinary NAG is inhibited Colorimetric (false positive
injury by urea, heavy metals, in rheumatoid arthritis,
Clin J Am Soc Nephrol 12: 149–173, January, 2017
industrial solvents hyperthyroidism, and
impaired glucose
tolerance; degraded
over time even when
stored at -80°C)
Detected 12 h after renal — ELISA
injury
Detected within 12 h — ELISA; Luminex
after renal injury
Biomarkers of AKI, Malhotra and Siew
159
 160

Appendix Table 2. Biomarkers Studies for Early Diagnosis of AKI

Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

ICU Bell et al. (105), uTIMP-2*IGFBP7, 94 adults Prospective, single- No KDIGO stage 1, 2, and AUCs for prediction of AKI
2015 uNGAL, uCys-C center 3 within 48 h by uTIMP-2*IGFBP7,
uNGAL, and uCys-C
were 0.40, 0.51, and 0.43,
respectively.
Zwiers et al. uNGAL, uKIM-1 100 children (age Prospective, single- Yes (0–6 h, 6–12 h, RIFLE criteria within AUCs for predicting AKI:
(106), 2015 1 d to 1 yr) center 12–24 h, 24–36 h, 48 h after ICU uNGAL: 0–6 h, 0.81; 6–
36–48 h, and admission 12 h, 0.78; 12–24 h, 0.71;
48–72 h) 24 h peak level, 0.81;
uKIM-1: at 12–24 h, 0.74.
Wong et al. ELA2, FGF13, MMP-8, 241 children Prospective, No Modified KDIGO Multibiomarker-based
(107), 2015 OLFM4, PRTN3 (derivation); multicenter stage 2 model to predict SAKI at
200 children classification (at day 3 had an AUC of 0.95
(validation) least 2-fold (95% CI, 0.91 to 0.99) in
increase of sCR derivation and 0.83 (95%
from baseline CI, 0.72 to 0.95) in test
Clinical Journal of the American Society of Nephrology

serum creatinine), cohort. AUC of SAKI

mortality risk model to predict
mortality was 0.90 (95%
CI, 0.85 to 0.96).
Bihorac et al. uTIMP-2*IGFBP7 408 adults Prospective, No KDIGO Stage 2 and 3 AUCs for predicting AKI
(38), 2014 multicenter criteria within 12 h for uTIMP-2*IGFBP7,
after the 0.82; sCR, 0.63. Clinical
enrollment model had an AUC of
0.70; clinical model 1
uTIMP-2*IGFBP7
improved AUC to 0.86.
Patients with uTIMP-
2*IGFBP7 values
between 0.3 and 2.0 had
5-fold risk of AKI
whereas it was 17-fold
for value above 2.0.
Frank AJ et al. GWAS 1264 adults (887 Retrospective, No 0.3 mg/dl or $50% 2 SNPs in the B-cell CLL/
(100), 2012 with genotype single-center increase in sCR Lymphoma 2 (BCL2):
data) from baseline in rs8094315 (OR 0.68 per
the first 72 h after additional copy of minor
ICU admission G allele; P,0.01) and
rs12457893 gene (OR 0.68
per additional copy of
minor C allele; P,0.01)
and a SNP in the serpin
peptidase inhibitor, clade
A (a-1 antiproteinase,
antitrypsin), member 4
(SERPINA4) gene:
rs2093266 (OR 0.53 per
additional copy of minor
A allele; P,0.01) were
associated with a
decreased risk of
developing AKI.
Appendix Table 2. (Continued)
Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

Kashani et al. uTIMP-2*IGFBP7, 522 adults Prospective, No Three KDIGO stage 2 AUCs for predicting AKI:
(23), 2013 uKIM-1, IL-18, (discovery) multicenter and 3 criteria uTIMP-2*IGFBP7, 0.80;
uL-FABP, uNGAL, 728 adults within 12 h of IGFBP7, 0.76; uTIMP-2,
pNGAL, pCys-C, (validation) sample collection 0.79; sCR, 0.75. Clinical-
a-/p-GST model AUC for AKI
stage 2/3 was 0.81.
Clinical model 1 uTIMP-
2*IGFBP7 improved
AUC to 0.87, IDI to 0.08,
and NRI to 68%.
Nejat et al. (35), pCys-C, sCR 444 adults Prospective, Yes (0 h, 12 h, 24 h, AKIN criteria (50% or AUCs for predicting AKI:
2010 multicenter and daily for the 0.3 mg/dl increase pCys-C, 0.78; sCR ,0.87.
next 6 d) in sCR), sustained pCys-C predicted
Clin J Am Soc Nephrol 12: 149–173, January, 2017

AKI, dialysis, and sustained AKI (AUC,

mortality within 0.80) and need of
30 d dialysis or death
(AUC, 0.68).
Siew et al. (37), uIL-18 451 adults Prospective, single- No AKIN criteria (50% or AUCs for predicting AKI
2010 center 0.3 mg/dl increase (uIL-18: at 24 h 0.62, 48 h
in sCR), AKI 0.60, AKIN stage 1 0.59,
severity, dialysis AKIN stage 2/3 0.62).
within 28 d, uIL-18 independently
mortality predicts need of
dialysis or death
(OR 1.86). Addition
of uIL-18 did not
improve ability of
uNGAL to predict AKI
as assessed by change
in AUC curve.
Cruz et al. (34), pNGAL 301 adults Prospective, single- Yes (daily for 4 d) RIFLE criteria, need AUCs for predicting AKI
2010 center of dialysis during within 48 h (pNGAL
ICU stay 0.78, sensitivity 73%,
specificity 81%, cut-off
150 ng/ml). pNGAL
was good predictor for
RRT (AUC 0.82);
whereas moderate
predictor for ICU
mortality (AUC 0.67).
Siew et al. (32), uNGAL 451 adults Prospective, single- No AKIN criteria (50% or AUCs for predicting AKI:
2009 center 0.3 mg/dl increase clinical model, 0.81;
in sCR), dialysis uNGAL at 24 h, 0.71;
within 28 d, 48 h, 0.64; clinical
mortality model 1 uNGAL, 0.82.
uNGAL independently
predicts severe AKI
during hospitalization
(HR, 2.6).
Biomarkers of AKI, Malhotra and Siew
161
 162

Appendix Table 2. (Continued)

Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

Cardiac surgery Elmariah S et al. Mass spectrometry 44 adults Prospective, single- No Increase in plasma Baseline levels of 5‐
(99), 2016 (metabolite center creatinine to adenosyl-
profiling) 150%–199% of homocysteine predicted
baseline or AKI after adjustment for
increase of $0.3 eGFR (OR per 1 SD
mg/dl within increase, 5.97; 95% CI,
7 d of TAVR 1.62 to 22.0; P,0.001).
Zacharias et al. NMR spectra 85 adults Prospective single- No AKIN staging 1,2, A random forests classifier
(104), 2015 center and 3 prognosticated AKI
across all stages with an
average accuracy of 806
0.9% and an AUC of
0.87.
Torregrosa uNGAL, uKIM-1, 193 adults Prospective, single- No RIFLE criteria In coronary angiography
et al. (108), uL-FABP center group, AUCs for
2015 predicting AKI:
uNGAL, 0.96; uKIM-1,
Clinical Journal of the American Society of Nephrology

0.71, whereas L-FABP

had poor prediction. In
CABG group, AUCs for
predicting AKI:
uNGAL, 0.92; uKIM-1,
0.72; and uL-FABP, 0.74.
Zhang et al. IL-6, IL-10 960 adults Prospective, Yes (preoperative AKIN criteria (50% or Elevated first postoperative
(109), 2015 multicenter and daily up to 0.3 mg/dl increase IL-16 and IL-10 were
day 5) in sCR from significantly associated
baseline), severe with AKI (OR, 2.13 and
AKI, all-cause 1.57, respectively; 3rd
mortality tertile versus 1st tertile).
Elevated IL-10 was
associated with lower
risk of mortality
(adjusted HR, 0.72).
Meersch et al. uTIMP-2*IGFBP7, 51 children Prospective, single- Yes (preoperative, 4 RIFLE criteria within AUCs for predicting AKI:
(110), 2014 uNGAL, uKIM-1 center h, and 24 h after 72 h after surgery uTIMP-2*IGFBP7, 0.85;
initiation of uNGAL, 0.87; and
CPB) uKIM-1, 0.64. Adding
uTIMP-2*IGFBP7 to
clinical model improved
AKI risk prediction
significantly (P,0.001).
Pilarczyk et al. uTIMP-2*IGFBP7 60 adults Prospective, single- Yes (4 h after KDIGO stage 2 or 3 AUC for predicting AKI
(111), 2015 center surgery and within 48 h after stage 2/3: uTIMP-
then every 12 h surgery 2*IGFBP7, 0.86, whereas
up to 4 d) sCR had poor prediction
(AUC, 0.36).
Ho et al. (112), NGAL, KIM-1, NAG, 28 studies Meta-analysis — RIFLE (eight studies), Composite AUCs for AKI:
2015 Cys-C, IL-18, L- AKIN(16studies),or NGAL, KIM-1, and L-
FABP, Hepcidin- KDIGOcriteriathree FABP, 0.72; and Cys-C,
25, uTIMP-2, studies); 1 study IL-18, u-a-GST,
uIGFBP7, u-a-GST, used combined u-p-GST, ,0.70).
u-p-GST AKIN/RIFLE
Appendix Table 2. (Continued)
Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

Sabbisetti et al. Blood KIM-1, uKIM-1 92 adults Prospective, single- No KDIGO criteria AUCs for predicting AKI:
(113), 2014 center within 48 h blood KIM-1, 0.96;
normalized uKIM-1,
0.98; uKIM-1, 0.91.
Koyner et al. uCys-C 1203 adults, 299 Prospective, Yes (preoperative, AKIN criteria (50% or uCys-C was not associated
(114), 2013 children multicenter 0–6 h, and 6–12 0.3 mg/dl increase with the development of
h postoperative) in sCR), severe AKI after cardiac surgery
AKI (doubling of in both adults and
sCR or need of children. Addition of
dialysis) uCys-C to clinical model
did not significantly
increase AUC (adults:
mild AKI: clinical model,
Clin J Am Soc Nephrol 12: 149–173, January, 2017

0.67; uCys-C, 0.68; clinical

model 1 uCys-C, 0.68;
severe AKI clinical
model, 0.72; uCys-C,
0.71; clinical model 1
uCys-C, 0.72).
Parikh et al. uKIM-1, uL-FABP, 1219 adults, 311 Prospective, Yes (preoperative AKIN stage 2/ Clinical model for AKI had
(115), 2013 uNGAL, uIL-18 children multicenter and every 6 h for RIFLE-I or an AUC of 0.69 in adults
the first 24 h after dialysis, and 0.78 in children.
surgery) progression to Addition of uKIM-1
higher stage, to clinical model
mortality marginally improved
discrimination and
classification in adults
(AUC, 0.73; NRI, 19%).
uKIM-1 and uL-FABP
were not significantly
associated with AKI in
adults or children after
adjusting for other kidney
injury biomarkers
(uNGAL and uIL-18).
Parikh et al. uNGAL, pNGAL, 1219 adults Prospective, Yes (preoperative AKIN stage 2/ AUCs for predicting AKI:
(29), 2011 uIL-18 multicenter and every 6 h for RIFLE-I or dialysis clinical model, 0.69;
the first 24 h after uNGAL, 0.67; pNGAL,
surgery) 0.70; and uIL-18, 0.74.
Adding uIL-18 and
pNGAL separately to
clinical model improved
AUCs (0.76 and 0.75,
respectively),NRI,andIDI.
Devarajan P SELDI-TOF MS 30 children Prospective, single- $50% increase in sCR AUCs for urine a1-
et al. (98), (proteomics) (discovery), 365 center from baseline microglobulin, a1-acid
2010 children within 72 h of CPB glycoprotein, and albumin
(validation) measured 6 h post-CPB
were 0.84 (95% CI, 0.79 to
0.89), 0.87 (95% CI, 0.83 to
Biomarkers of AKI, Malhotra and Siew

0.91), and 0.76 (95% CI,

0.71 to 0.81), respectively.
163
 164

Appendix Table 2. (Continued)

Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

Liangos et al. uKIM-1, uNAG, 103 adults Prospective, Yes (preoperative, $50% increase in sCR AUCs for predicting AKI:
(116), 2009 uNGAL, uCys-C, multicenter 2 h, 24 h, and in the first 72 h clinical model, 0.83;
uIL-18, urinary a-1 48 h after after termination uKIM-1, 0.78; uNAG,
microglobulin discontinuation of CPB 0.62; uIL-18, 0.66.
of CPB) Adding uKIM-1 to
clinical model improved
AUC to 0.88.
Bennett et al. uNGAL 196 children Prospective, single- Yes (preoperative, 50% increase in sCR, AUCs for predicting AKI:
(27), 2008 center 2 h, 4 h, 6 h, 8 h, dialysis, mortality uNGAL, 0.93 sensitivity,
12 h, up to 72 h) 82%, specificity, 90%,
cut-off 100 ng/ml. Need
of dialysis: AUC, 0.86;
and mortality: AUC,
0.91.
Stafford-Smith GWAS 1671 adults Prospective, single- No Delta sCR Two alleles (IL-6 2572C
et al. (103), center and angiotensinogen
2005 842C) showed a strong
Clinical Journal of the American Society of Nephrology

association with renal

injury in white patients
(P,0.001). Adding
genetic to clinical factors
provided 2–4-fold
improvement in
prediction of renal
injury post–cardiac
surgery.
Cardiac catheterization Hirsch et al. uNGAL, pNGAL 91 children Prospective, single- Yes (precontrast, 50% increase in sCR AUCs for predicting CIN at
(117), 2007 center 2 h, and 6 h) above baseline 2 h: uNGAL, 0.92; and
pNGAL, 0.91; PPV 100%
and 80%; at 6 h: uNGAL,
0.97; and pNGAL, 0.95;
PPV 90%; and 100%; for
cut-off 100 ng/ml.
Hospitalized Vaidya et al. uKIM-1, uIL-18, 204 adults Cross-sectional No a $50% increase in AUCs for predicting AKI:
(18), 2008 uNGAL, uNAG, sCR from uKIM-1, 0.93; uNAG,
uVEGF, uHGF, admission value 0.83; uNGAL, 0.89; uIL-
uCys-C, uCXCL10 or known 18, 0.83; uCys-C, 0.85).
baseline, dialysis,
mortality
Ferguson et al. uL-FABP 154 adults Cross-sectional No AKI was defined as a AUC for predicting AKI:
(25), 2010 $50% increase in uL-FABP, 0.93. uL-FABP
sCR from was also significant
admission value predictor of RRT
or known (P50.02) and composite
baseline, dialysis, end point of death/RRT
mortality (P50.03). Higher levels
of uL-FABP were seen in
ATN and sepsis AKI,
whereas lower levels in
contrast nephropathy.
Appendix Table 2. (Continued)
Authors and
End Point (Urine/
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers Summary of Findings
Serum)
Publications

Cirrhosis Treeprasertsuk uNGAL 121 adults Prospective, single- No AKIN criteria (50% or AUCs for predicting AKI:
et al. (41), center 0.3 mg/dl increase uNGAL, 0.83;
2015 in sCR from sensitivity, 77.1%;
baseline), 30- specificity 73.3%; cut-
d mortality off, 56 ng/ml; baseline
sCR, 0.58. In
multiadjusted model,
uNGAL could not
predict 30-d mortality.
ICU, NICU, cardiac Shao et al. (118), uKIM-1 2979 patients Meta-analysis (five —- AKIN/RIFLE/ Pooled analysis of studies
surgery, cardiac 2014 prospective, two KDIGO showed an AUC of 0.86
catheterization, cross-sectional, for prediction of AKI;
hospitalized,ER and four case- sensitivity, 74%; and
Clin J Am Soc Nephrol 12: 149–173, January, 2017

control studies) specificity, 86%.

uTIMP-2, urinary tissue inhibitor of metalloproteinases-2; IGFBP7, IGF-binding protein 7; uNGAL, urinary neutrophil gelatinase-associated lipocalin; uCys-C, urinary cystatin-C; KDIGO,
Kidney Disease Improving Global Outcomes; uKIM-1, urinary kidney injury molecule-1; RIFLE, risk, injury, failure, loss of kidney function, and ESRD; ICU, intensive care unit; AUC, area under
curve; ELA2,elastase 2; FGF13, fibroblast growth factor 13; MMP-8, matrix metalloproteinases 8; OLFM4, olfactomedin 4; PRTN3, proteinase; sCR, serum creatinine; 95% CI, 95% confidence
interval; SAKI, septic acute kidney injury; GWAS, genome-wide association studies; SNP, single-nucleotide polymorphism; OR, odds ratio; uL-FABP, urinary liver-type fatty acid–binding
protein; pNGAL, plasma neutrophil gelatinase-associated lipocalin; pCys-C, plasma cystatin-C; a-GST, a-glutathione s-transferase; p-GST, p-glutathione s-transferase; IDI, integrated dis-
crimination improvement; NRI, net-reclassification index; uIL-18, urinary IL-18; AKIN, Acute Kidney Injury Network; HR, hazard ratio; TAVR, transcatheter aortic valve replacement; NMR,
nuclear magnetic resonance spectroscopy; CABG, coronary artery bypass graft; NGAL, neutrophil gelatinase-associated lipocalin; NAG, n-acetyl-b-d-glucosaminidase; L-FABP, liver-type fatty
acid–binding protein; uIGFBP7, urinary IGF-binding protein 7; u-a-GST, urinary a-glutathione s-transferase; u-p-GST, urinary p-glutathione s-transferase; KIM-1, kidney injury molecule-1;
SELDI-TOF MS. surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; CPB, cardio-pulmonary bypass; CIN, contrast nephropathy; PPV, positive predictive value;
uVEGF-A, urinary vascular endothelial growth factor-A; uHGF, urinary hepatocyte growth factor; uCXCL10, urinary chemokine (c-x-c motif) ligand 10; ATN, acute tubular necrosis; NICU,
neonatal intensive care unit; ER, emergency room.
Biomarkers of AKI, Malhotra and Siew
165
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Appendix Table 3. Biomarker AKI Prognostic Studies

Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

ICU Parr et al. (62), uL-FABP, uIL-18, 152 adults Prospective, Stage 1 AKI No Composite outcome AUCs for predicting
2015 uKIM-1, uNGAL single-center was comprised of composite outcome
persistent doubling (uL-FABP 0.79,
of sCR ($2 d), uIL-18 0.64, uKIM-1
dialysis, and 0.62, uNGAL 0.65,
mortality and combination
of biomarkers 0.
81). Clinical-
model AUCs for
composite outcome
was 0.74; adding
uL-FABP to clinical
model improved
AUC (0.82), NRI
(31%) and IDI
Clinical Journal of the American Society of Nephrology

(0.09).
Pike et al. (73), IL-6, IL-8, IL-10, IL- 817 adults Prospective, AKI with RRT No Renal recovery was AUCs for renal
2015 18, MMIF, TNFR- nested defined as being recovery (IL-6 0.61,
I, TNFR-II, DR-5 observational alive and IL-8 0.63, IL-10
cohort, independent from 0.57, IL-18 0.58,
multicenter RRT by day 60 after MIF 0.57).
hospital discharge; Clinical-model
60-d mortality AUCs for renal
recovery (0.73) and
mortality (0.74);
Adding IL-8 to
clinical model
improved the
prediction of renal
recovery and
mortality (AUCs
0.76 and 0.78,
respectively). IL-8
improved IDI and
NRI for renal
recovery and
mortality.
Koyner et al. uTIMP-2*IGFBP7 692 adults Prospective, No AKI/stage No 9-mo composite UTIMP-2*IGFBP7.2.0
(83), 2015 multicenter 1 AKI endpoint of all-cause and sCR showed
mortality and/or (adjusted HRs
RRT of 2.16 and
1.40, respectively)
for death or
RRT within 9 mo;
Clinical model 1
uTIMP-2*IGFBP7
did not improved
AUC (0.70), but
did improve NRI
by 23% and IDI
(0.01).
Appendix Table 3. (Continued)
Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Koyner et al. uNGAL, pNGAL, 77 adults Prospective, Stage 1/stage 2 No Progression to AKIN AUCs for prediction
(119), 2015 uKIM-1, uIL-18, multicenter AKI stage 3, need of RRT, of progression to
uTIMP-2*IGFBP7, mortality AKI stage 3
FST (uNGAL 0.65,
pNGAL 0.75,
uKIM-1 0.63,
uIL-18 0.65,
uTIMP-2*IGFBP7
0.69, and FST 0.87),
Combining FST
with uTIMP-
2*IGFBP7 resulted
Clin J Am Soc Nephrol 12: 149–173, January, 2017

in nonsignificant
improvement in
AUC 0.90. FST (2 h
urine output) had
AUCs of 0.86 and
0.70 for RRT and
mortality.
Dewitte et al. uTIMP-2*IGFBP7, 57 adults Prospective, Stage 1/stage Yes (at inclusion Recovery defined uTIMP-2*IGFBP7 and
(75), 2015 pNGAL single-center 2/stage 3 and 24 h) as return to pNGAL AUCs for
sCR,1.53baseline early renal
or 0.35 mg/dl above recovery (0.70 and
the baseline with 0.78, respectively);
reversal of oliguria uTIMP-2*IGFBP7
within 48 h; major showed good
adverse kidney ability to predict
events was defined major adverse
as death, RRT, or kidney events,
persistence of renal with AUC-ROC
dysfunction values close to 0.8;
(sCR$200% above where as pNGAL
baseline) at hospital had AUCs
discharge 0.68–0.76. Clinical
Model AUC for
renal recovery was
0.87; adding
uTIMP-2*IGFBP71
pNGAL to clinical
model improved
AUC (0.89), NRI,
and IDI.
Murugan et al. IL-1b, IL-6, IL-8, IL- 817 adults Prospective, AKI with RRT No Renal recovery was Increased
(74), 2014 10, IL-18, MIF, nested defined as being concentrations of
TNF, TNFR-I, observational alive and plasma IL-8, IL-18,
TNFR-II, DR-5, cohort, independent from MIF, and TNFR-I
GM-CSF multicenter RRT by day 60 after were associated
hospital discharge; with slower renal
60-d mortality recovery and
Biomarkers of AKI, Malhotra and Siew

increased
mortality.
167
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Appendix Table 3. (Continued)

Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Aregger et al. uIGFBP7, pNGAL 64 adults Prospective, Stage 1/stage No Predicting early AUCs of uIFGBP-7 for
(120), 2014 (includes 12 multicenter 2/stage 3 recovery (defined as early recovery 0.74,
adults as not classifying for need of RRT 0.65,30-d
control) any RIFLE class mortality 0.65 and
during 7-d follow- in-hospital mortality
up), RRT, mortality 0.68. Addition of
uIGFBP-7 to clinical
model improved
prediction of renal
recovery from 76.6%
–82.1% concordant
values (IDI: 0.0816
0.04). Clinical Model 1
uIGFBP-71pNGAL
did not improved
AUCs.
Clinical Journal of the American Society of Nephrology

Yamashita et al. uTIMP-2, uNAG, 98 adults Prospective, No AKI/stage No AKI was defined on the AUCs for prediction of
(121), 2014 pNGAL, pIL-6 single-center 1/stage 2 basis of KDIGO severe AKI: uTIMP-2,
criteria; progression 0.80; pNGAL, 0.87;
of AKI stage (non- pIL-6, 0.70; uNAG,
AKI to AKI any 0.83; and progression
stage, stage 1–2, of AKI: uTIMP-2, 0.73;
stage 2–3) pNGAL, 0.76; pIL-6,
0.74; uNAG, 0.77.
uTIMP-2 showed the
highest AUCs for 7-
d (0.83) and in-
hospital mortality
(0.74), whereas sCR
had AUCs of 0.67 and
0.61, respectively.
AUC of the clinical
model for severe AKI
was 0.87; adding
uTIMP-2 to the clinical
model improved
AUC (0.89), NRI
(41%), and IDI (0.04).
Kashani et al. uTIMP-2*IGFBP7, 522 adults Prospective, No AKI/stage No MAKE30 includes death, MAKE30 elevated
(23), 2013 uKIM-1, IL-18, (discovery) multicenter 1 AKI dialysis and sharply for uTIMP-
uL-FABP, 728 adults persistent renal 2*IGFBP7 above
uNGAL, pNGAL, (validation) dysfunction (sCR $ 0.30 and doubled for
pCys-C, p-GST 200% above baseline values above 2.0.
at hospital discharge)
Zhang et al. sCys-C 232 adults Retrospective, AKI with RRT No Renal recovery was sCys-C showed better
(122), 2012 single-center defined as dialysis performance in
independence and predicting renal
final sCR,50% function recovery
above baseline than sCR (AUC, 0.87
value versus 0.63; P,0.01).
Appendix Table 3. (Continued)
Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Srisawat et al. uNGAL, uCys-C, 76 adults Prospective, AKI with RRT Yes (days 1, 7, Renal recovery was AUC for renal recovery:
(72), 2011 uIL-18, uHGF, multicenter and 14) defined as alive and uNGAL, 0.70; uCys-
uNGAL/MMP-9, free of dialysis at C, 0.61; uIL-18, 0.42;
and urinary 60 d from the start of uHGF, 0.74;
creatinine RRT uNGAL/MMP-9,
0.53; urinary
creatinine, 0.66.
Clinical model had
an AUC of 0.74;
clinical model 1
relative change of
uHGF 1 uNGAL 1
Clin J Am Soc Nephrol 12: 149–173, January, 2017

uCys-C 1 uNGAL/
MMP-91uIL-18
improved AUC
(0.94) and NRI
(63.3%).
Lorenzen JM mRNAs 77 adults, Prospective, AKI with RRT No Survival 4 wk after MIR-210 was identified
et al. (102), (transcriptome) 30 age- single-center initiation of AKI as an independent
2011 matched prognostic factor for
controls 28-d survival with
AUC of 0.7 (95% CI,
0.53 to 0.78; P50.03),
PPV 0.41, and NPV
1.0.
Kumpers et al. pNGAL 109 adults Prospective, AKI with RRT Yes Renal recovery was AUCs for predicting
(123), 2010 single-center defined as no need 14-d mortality:
for RRT at day 28 pNGAL, 0.74; cut-
after the study off, 360 ng/ml); no
enrollment, association was
mortality found between renal
recovery and
pNGAL.
Cardiac surgery Moledina et al. pNGAL 1191 adults Prospective, No AKI Yes (preoperative, All-cause mortality (3-y Elevated first
(124), 2015 multicenter 0–6 h, peak 1–3 follow-up) preoperative and
d postsurgery) postoperative
pNGAL levels were
significantly
associated with
mortality (adjusted
HR, 1.48 and 1.31,
respectively; 3rd
tertile versus first
tertile). No
association persists
between
postoperative NGAL
and mortality after
adjusting for
Biomarkers of AKI, Malhotra and Siew

perioperative sCR
changes.
169
 170

Appendix Table 3. (Continued)

Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Arthur et al. uL-FABP, uIL-18, 95 adults Prospective, Stage 1 AKI No AKI progression was AUCs for primary and
(22), 2014 uKIM-1, uNGAL, multicenter defined as secondary
32 biomarkers worsening of renal outcomes: clinical
dysfunction (AKIN model, 0.63 and 0.68;
stage 1 to a higher uIL-18, 0.74 and
AKIN stage 2/3) 0.89; uL-FABP, 0.67
within 10 d or and 0.85; uKIM-1,
mortality within 0.73 and 0.81;
30 d; secondary uNGAL, 0.72 and
outcome was AKIN 0.83. Combination of
stage 3 or death uIL-18 and uKIM-1
improves prediction
of AKIN-3 or death
with an AUC of 0.93.
Coca et al. (82), uL-FABP, uIL-18, 1199 adults Prospective, No AKI Yes (preoperative, 3-y mortality Among patients with
2014 uKIM-1, uNGAL, multicenter 0–6 h AKI, adjusted HRs
Clinical Journal of the American Society of Nephrology

albumin postoperative, for 3-year mortality

daily for up to for individual
5 d) urinary biomarkers
were: uNGAL, 2.52;
uIL-18, 3.16; uKIM-
1, 2.01; uL-FABP,
2.35 and urine
albumin 2.85
(highest tertile
versus lowest
tertile). Addition of
uIL-18, uL-FABP,
and uKIM-1 to
clinical model did
not change AUC
(0.69–0.71), but
improved NRI (44%,
44%, and 18%,
respectively).
Meersch et al. uTIMP-2*IGFBP7, 50 adults Prospective, No AKI Yes (preoperative, Renal recovery was AUCs for prediction of
(125), 2014 pNGAL single-center 4 h, 12 h, and defined as sCR value renal recovery for
24 h after CPB) at hospital discharge uTIMP-2*IGFBP7
equal to or lower was 0.79.
than the Combination of
preoperative NGAL and uTIMP-
creatinine value 2*IGFBP7 did not
improve AUC.
Appendix Table 3. (Continued)
Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Alge et al. (21), Urinary 97 adults Prospective, Stage 1 AKI No Progression to a higher uAnCR AUCs for
2013 angiotensinogen single-center AKIN stage, AKIN worsening of AKI
stage 3, RRT, (0.70), AKIN stage 3
mortality (0.71), RRT (0.71),
AKIN 3 or mortality
(0.75) and RRT or
mortality (0.71).
Prognostic
predictive power of
uAnCR was
improved when
only patients with
Clin J Am Soc Nephrol 12: 149–173, January, 2017

AKIN stage 1 were

analyzed. Adding
uAnCR to clinical
model improved
prediction of
worsening of AKI
(NRI, 45.7%).
Kidney transplant Hall et al. (126), uNGAL, IL-18, 91 adults Prospective, Transplant Yes (0 h, 6 h, 12 h, Graft recovery was AUCs for predicting
2010 KIM-1 single-center 18 h, 1st, and defined in three dialysis: uNGAL
2nd categories: DGF, 0.82, IL-18 0.82;
postoperative SGF, and IGF; RRT whereas KIM-1 had
day) within 1 wk of poor prediction
kidney transplant (AUC 0.50); Clinical
model 1 uNGAL1
IL-18 did improve
reclassification by
110% (P,0.001).
Reese et al. uNGAL, uKIM-1, 2441 adults Prospective, Transplant No DGF was defined as a High donor uNGAL
(127), 2015 uIL-18, uL-FABP, multicenter requirement for RRT levels were
urinary within 7 d after significantly
microalbumin transplant, 6-mo associated with
eGFR recipient DGF with
RR 1.21 (highest
versus lowest
tertiles). Addition of
urinary biomarkers
does not improve
AUC, IDI, and NRI.
At 6-mo, donor
urinary biomarkers
added minimal
value in predicting
recipient allograft
function.
Biomarkers of AKI, Malhotra and Siew
171
 172

Appendix Table 3. (Continued)

Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Pianta et al. uTIMP-2*IGFBP7, 56 adults Prospective, Transplant Yes (4 h, 8 h, and DGF was defined as AUCs for predicting
(128), 2015 VEGF-A, MMIF, single-center 12 h) requirement for RRT DGF: uTIMP-
MCP-1, TFF3, within 7 d 2*IGFBP7, 0.76;
CXCL16, sCR VEGF-A, 0.81;
uTIMP-2, 0.73; and
uIGFBP7, 0.71;
whereas sCR
showed poor
prediction for DGF
(AUC 0.56). Clinical
model AUC for DGF
was 0.70; adding
TIMP-2 (0.81 and
0.11) and VEGF-A
(0.85 and 0.19)
separately to clinical
Clinical Journal of the American Society of Nephrology

model improved
AUC and IDI.
Hospitalized Wang et al. uL-FABP 114 adults Prospective, Stage 3 AKI No Recovery was defined AUCs for renal
(129), 2015 single-center as alive and neither recovery: uL-FABP,
requiring RRT nor 0.91; sensitivity,
having persistent 85.5; and specificity,
AKIN 3 with a 86.4% for cut-off
minimum crcl of 20 102.1 ng/mg). uL-
mL/min at hospital FABP modestly
discharge predicted hospital
death (AUC, 0.83)
and RRT (AUC,
0.71). u-FABP
improved renal
recovery
classification
compared with
clinical model (NRI,
35%; P50.02)
Westhoff et al. uTIMP-2*IGFBP7 46 children Prospective, Stage 1/stage No Predicting mortality AUCs for 30-d and 90-d
(130), 2015 single-center 2/stage 3 and need of RRT mortality: uTIMP-
2*IGFBP7, 0.79 and
0.84, respectively).
AUC for RRT was
0.67.
Singer et al. uNGAL 145 adults Prospective, Stage 1/stage No Progression to a higher uNGAL had an AUC of
(63), 2011 single-center 2/stage 3 AKIN stage, 0.71 (95% CI, 0.62 to
dialysis, mortality 0.8), as compared
within 7 d of ICU with sCR level,
admission; type of which had an AUC
AKI of 0.61 (95% CI, 0.51
to 0.71).
Appendix Table 3. (Continued)
Patient Type at
Authors and
Time of
Clinical Settings Year of Biomarkers Sample Size Study Design Serial Biomarkers End Point Summary of Findings
Biomarker
Publication
Measurement

Srisawat et al. pNGAL 189 adults Prospective, Stage 3 No Recovery was defined pNGAL AUCs for renal
(89), 2011 multicenter as alive and not recovery was 0.74.
requiring RRT Clinical model 1
during pNGAL did not
hospitalization and improve AUC, but
nor having a did improve NRI by
persistent RIFLE-F 17%.
classification at
hospital discharge
Cirrhosis Belcher et al. uL-FABP, uIL-18, 188 adults Prospective, Stage 1/stage Yes (daily for 3 d) Progression to a higher AUCs for AKI
(24), 2014 uKIM-1, uNGAL multicenter 2/stage 3 AKIN stage, progression and
dialysis, mortality death: uL-FABP,
Clin J Am Soc Nephrol 12: 149–173, January, 2017

0.76; uIL-18, 0.71;

uKIM-1, 0.66;
uNGAL, 0.77. IL-18
independently
improved NRI
(51%).
Heart Failure Verbrugge et al. uIL-18, uKIM-1, 83 adults Prospective, No AKI No Persistent renal uIL-18 was a predictor
(131), 2013 uNGAL single-center impairment was of persistent renal
defined as persistently impairment with an
elevated sCR levels AUC of 0.67. uIL-18
($0.3 mg/dl) after 6 was also associated
mo compared with with all-cause
baseline; all-cause mortality (HR 1.48).
mortality

uL-FABP, urinary liver-type fatty acid–binding protein; uIL-18, urinary IL-8; uKIM-1, urinary kidney injury molecule-1; uNGAL, urinary neutrophil gelatinase–associated lipocalin; sCR, serum
creatinine; AUC, area under curve; NRI, net-reclassification index; IDI, integrated discrimination improvement; MMIF, macrophage migration inhibitory factor; TNFR-I, TNF receptor 1; TNFR-II, TNF
receptor II; DR-5, death receptor-5; MIF, migration inhibitory factor; uTIMP-2, urinary tissue inhibitor of metalloproteinases-2; IGFBP7, IGF-binding protein 7; pNGAL, plasma neutrophil gelatinase–
associated lipocalin; uIL-18, urinary IL-18; FST, furosemide stress test; AKIN, Acute Kidney Injury Network; AUC-ROC, area under the receiver operating characteristic curve; GM-CSF, granulocyte
macrophage stimulating factor; RIFLE, risk, injury, failure, loss of kidney function, and ESRD; uIGFBP7, urinary IGF-binding protein 7; uNAG, urinary n-acetyl-b-d-glucosaminidase; pIL-6, plasma IL-
6; KDIGO, Kidney Disease Improving Global Outcomes; pCys-C, plasma cystatin-C; p-GST, p-glutathione s-transferase; MAKE, major adverse kidney events; sCys-C, serum cystatin-C; uHGF,
urinary hepatocyte growth factor; MMP-9, matrix metalloproteinase-9; NPV, negative predictive value; CPB, cardio-pulmonary bypass; HR, hazard ratio; NGAL, neutrophil gelatinase–associated
lipocalin; uAnCR, urinary-angiotensinogen-creatinine ratio; DGF, delayed graft function; SGF, slow graft function; IGF, immediate graft function; KIM-1, kidney injury molecule-1; RR, relative risk;
VEGF-A, vascular endothelial growth factor-A; MCP-1, monocyte chemotactic protein 1; TFF3, trefoil factor 3; CXCL16, chemokine (c-x-c motif) ligand 16; TIMP-2, tissue inhibitor of metal-
loproteinases-2; crcl, creatinine clearance; ICU, intensive care unit.
Biomarkers of AKI, Malhotra and Siew
173