Core Curriculum in Nephrology

Biomarkers in Nephrology: Core Curriculum 2013

Gearoid M. McMahon, MB, BCh, and Sushrut S. Waikar, MD, MPH

T he clinical assessment and management of pa-

tients with known or suspected kidney disease
has been aided for decades by biomarkers, a term
CONVENTIONAL BIOMARKERS OF KIDNEY
FUNCTION AND DISEASE
Modern definitions of CKD and AKI emphasize a
defined by a National Institutes of Health working
stage of decreased glomerular filtration rate (GFR)
group as “[a] characteristic that is objectively mea-
and a stage of kidney damage, defined as structural or
sured and evaluated as an indicator of normal biologi- functional abnormalities before a decrease in GFR.
cal processes, pathogenic processes, or pharmacologi- Corresponding biomarkers include filtration markers,
cal responses to a therapeutic intervention.” The such as creatinine and cystatin C, and markers of
characteristics of an ideal biomarker are listed in Box kidney damage, such as urine sediment abnormalities
1. Historically, the first biomarker of kidney disease and albuminuria (Table 1).
was the finding on physical examination of interstitial
edema or ascites, a condition termed dropsy, that was Creatinine
not specific to what eventually became recognized as
The use of creatinine as a marker of GFR dates
kidney failure, but that rather encompassed a number
back to the 1920s. Creatinine is a 113-Da amino acid
of clinical conditions, including congestive heart fail-
derivative that is the product of nonenzymatic break-
ure and cirrhosis. More objective biomarkers in the down of creatine in muscle. It is not protein bound, it
early days of nephrology included examination of is not metabolized in the kidney, and it is freely
urine sediment, followed by measurement of blood filtered in the glomerulus, making it an excellent
urea nitrogen and serum creatinine. marker of glomerular filtration. However, non-GFR
Recently, there has been an explosive growth in the determinants of creatinine concentration limit its util-
search for more sensitive, specific, and prognostically ity in AKI and CKD. Proximal tubular secretion of
accurate biomarkers to assist in the care of patients creatinine accounts for 10%-20% of its excretion,
with or at risk of kidney disease. In this Core Curricu- leading to overestimation of the true GFR, particu-
lum, we aim to discuss both conventional and novel larly in patients with CKD. Gut bacteria also degrade
biomarkers of kidney disease in the settings of acute creatinine and contribute to its clearance, the relative
kidney injury (AKI), chronic kidney disease (CKD), magnitude of which becomes more important as kid-
nephrotoxin exposure, and glomerulonephritis. Ana- ney function decreases. Creatinine can be reabsorbed
tomical localization of selected biomarkers along the after glomerular filtration in patients with very low
nephron is shown in Fig 1. urine and tubular flow rates. Certain medications,
such as cimetidine and trimethoprim, can increase
Additional Reading serum creatinine concentration by inhibiting tubular
» Biomarkers Definitions Working Group. Biomarkers and
secretion. Creatinine is produced at a relatively con-
surrogate endpoints: preferred definitions and conceptual stant rate, which in turn is proportional to muscle
framework. Clin Pharmacol Ther. 2001;69(3):89-95. mass. Between-person variability in creatinine genera-
tion rate—related to age, sex, muscle mass, race, and
perhaps other factors—limits the use of creatinine in
Box 1. Characteristics of an Ideal Biomarker
the estimation of GFR. To account for this variability,
1. Noninvasive, easily measured, inexpensive, and provides a number of creatinine-based equations have been
rapid results
2. From easily available sources (blood or urine)
3. High sensitivity
4. High specificity From the Renal Division, Department of Medicine, Brigham
5. Allows early detection of disease and changes in response and Women’s Hospital, Harvard Medical School, Boston, MA.
to treatment Received August 14, 2012. Accepted in revised form December
6. Predicts prognosis and allows stratification into categories 17, 2012. Originally published online March 1, 2013.
of risk Address correspondence to Sushrut S. Waikar, MD, MPH,
7. Biologically plausible: provides information about the mecha- Brigham and Women’s Hospital, MRB-4, 75 Francis St, Boston,
nisms of disease MA 02115. E-mail: swaikar@partners.org
© 2013 by the National Kidney Foundation, Inc. Published by Elsevier
Adapted from Edelstein CL. Biomarkers in Kidney Disease. Inc. All rights reserved.
1st ed. Amsterdam, the Netherlands: Academic Press/Elsevier; 0272-6386/$36.00
2011. http://dx.doi.org/10.1053/j.ajkd.2012.12.022

Am J Kidney Dis. 2013;62(1):165-178 165

 McMahon and Waikar

Biomarkers of abnormal glomerular

filtra
on or glomerular permeability:
Albumin
β-Trace Protein
Serum Urea Nitrogen
Crea
nine
Cysta
n C
Clusterin
Monocyte Chemoaractant
GST-pi
Protein-1
CTGF (visceral and NGAL
parietal epithelial
cells, inters

um)

Biomarkers of proximal tubular dysfunc
on or injury:

Calbindin D28
Albumin Alkaline Phosphatase
ADMA Alanine Aminopep
dase
β2-microglobulin Clusterin
Cysta
n C GGT NGAL
GST-alpha IL-18 Osteopon
n
KIM-1 LDH Trefoil Factor 3
L-FABP NAG Uromodulin
Netrin NGAL
Trefoil Factor-3 Re
nol Binding Protein-4

Figure 1. Anatomical localization of biomarkers along the nephron. Abbreviations: ADMA, asymmetric dimethyl arginine; CTGF,
connective tissue growth factor; GGT, ␥-glutamyl transpeptidase; GST, glutathione-S-transferase; IL-18, interleukin 18; KIM-1, kidney
injury molecule 1; LDH, lactate dehydrogenase; L-FABP, liver-type fatty acid binding protein; NAG, N-acetyl glucosaminidase; NGAL,
neutrophil gelatinase-associated lipocalin.

developed to estimate GFR, including the Cockroft- » Perrone RD, Madias NE, Levey AS. Serum creatinine as an
Gault, Modification of Diet in Renal Disease (MDRD) index of renal function: new insights into old concepts. Clin
Chem. 1992;38(10):1933-1953.
Study, and CKD-EPI (CKD Epidemiology Collabora- » Rehberg PB. Studies on kidney function: the rate of filtration
tion) equations for adults and the Schwartz equation and reabsorption in the human kidney. Biochem J. 1926;20(3):
for children. Despite improving the estimation of 447-460.
true GFR, all the equations have shortcomings. For
example, at lower creatinine concentrations, the Cystatin C
MDRD Study equation generally underestimates GFR,
Cystatin C is a 13-kDa cysteine protease inhibitor
whereas the Cockroft-Gault and Schwartz equations
that is a marker of GFR. Cystatin C is expressed by all
may overestimate GFR.
nucleated cells and has multiple biological functions,
including controlling extracellular proteolysis and
Additional Readings modulation of the immune system. Its utility in estimat-
» Carrie BJ, Golbetz HV, Michaels AS, Myers BD. Creatinine: ing kidney function derives from the fact that after
an inadequate filtration marker in glomerular diseases. Am J being freely filtered in the glomerulus, it then is
Med. 1980;69(2):177-182.
absorbed in the kidney tubules, where it is fully
» Hankins DA, Babb AL, Uvelli DA, Scribner BH. Creatinine
degradation I: the kinetics of creatinine removal in patients degraded locally.
with chronic kidney disease. Int J Artif Organs. 1981;4(1):35- There is no active tubular secretion or significant
39. extrarenal elimination, making it an excellent marker

166 Am J Kidney Dis. 2013;62(1):165-178

Biomarkers in Nephrology

Table 1. Uses and Limitations of Conventional Biomarkers

Uses Limitations

Creatinine ● Glomerular filtration marker ● Variability in generation rates across individuals

● GFR estimation ● Significant tubular secretion leading to overestimation
● Biomarker of acute and chronic of GFR
decreased kidney function ● Significant extrarenal elimination
● Tubular reabsorption in low urine flow states
● Increases late after AKI

Cystatin C ● GFR estimation (plasma) ● Increases late after AKI

● Biomarker of proximal tubular ● May increase in inflammatory states and when there is
dysfunction (urine) thyroid dysfunction independent of kidney function
● Biomarker of acute and chronic ● Urinary cystatin C is altered in the presence of
decreased kidney function albuminuria

Albuminuria ● Biomarker of glomerular filtration barrier ● 24-hour collections unreliable

dysfunction ● Significant intraindividual variability in
● Biomarker of proximal tubular albumin-creatinine ratio over short periods
dysfunction
● Early biomarker of AKI
● Independent risk factor for
cardiovascular and all-cause mortality
and ESRD

Urine sediment ● Biomarker of AKI ● Poor interobserver agreement

examination ● Biomarker of glomerular disease ● Lack of standardization of reporting of results
● Biomarker of tubulointerstitial disease ● Heavily dependent on experience of the reader
● Uncertain correlation with histopathology
Abbreviations: AKI, acute kidney injury; ESRD, end-stage renal disease; GFR, estimated glomerular filtration rate.

of GFR. Cystatin C also may be superior to creatinine should be considered to determine the source of error
as a better predictor of cardiovascular mortality while (body mass, corticosteroids, etc). A recent study has
providing a more accurate estimation of GFR. suggested that the combination of creatinine and cys-
Serum cystatin C concentration is independent of tatin C levels for estimation of GFR is superior to
muscle mass, nutritional status, and sex, although it creatinine-based equations and may be useful as a
may be altered in patients with derangements in confirmatory test for CKD.
thyroid function, certain cancers, and glucocorticoid Cystatin C is distributed in the extracellular space,
therapy. Although cystatin C production has been well whereas creatinine is distributed in total-body water.
characterized in healthy individuals, less is known As a result, it has a volume of distribution approxi-
about its production in disease states; for example, mately one-third that of creatinine, meaning that it
cystatin C levels are higher at baseline in patients with reaches a steady-state concentration 3 times faster: the
acute leukemia. There are a number of different meth- half-life is 1.5 versus 4 hours. As a result, after kidney
ods for measuring cystatin C and there is significant injury, cystatin C concentration increases earlier than
interassay variation, with some assays performing creatinine concentration. This has been borne out in
better for the diagnosis of AKI and CKD. studies of patients with AKI including contrast-
A major advantage of cystatin C over creatinine induced nephropathy when the increase in cystatin C
level is that it is not as influenced by changes in level precedes that of the creatinine level by 5-24
muscle mass; therefore, estimating equations for esti- hours. However, in a large study of patients undergo-
mated GFR are more accurate across a range of body ing cardiac surgery, cystatin C did not show favorable
types, including infants and the elderly. In patients at kinetic characteristics compared to serum creatinine
extremes of body mass, creatinine-based equations in the diagnosis of AKI.
perform poorly while cystatin C is more accurate. Urinary cystatin C also is a potential marker of
However, the increase in accuracy is moderate at best, acute tubular injury. Because cystatin C usually is
and it is not certain that it is clinically relevant. Some reabsorbed in the proximal tubule, the finding of
have proposed combining creatinine and cystatin C cystatin C in urine suggests some form of proximal
equations for more precise estimations of GFR. In this tubular injury. However, in patients with albuminuria,
setting, agreement between the 2 estimates can be there is competitive inhibition of cystatin C uptake
taken as a more accurate estimation of GFR. How- (both are transported by megalin), and as a result,
ever, if there is disagreement, the clinical picture significant quantities can appear in urine even in the

Am J Kidney Dis. 2013;62(1):165-178 167

 McMahon and Waikar

absence of tubular injury. This effect has been shown Table 2. Scoring System for Urine Sediment Based on
in rats, and similar effects have been noted in patients Numbers of Granular Casts and Kidney Tubular Epithelial Cells
with diabetes and children with nephrotic syndrome. Granular Casts per HPF
It should be noted that cystatin C is not the only
urinary biomarker that is affected by increasing levels Kidney tubular epithelial 0 1-5 >6
of albuminuria: all the low-molecular-weight proteins cells per HPF (0 points) (1 point) (2 points)

under investigation as potential urinary biomarkers

0 0 1 2
potentially could be affected by this process, includ-
(0 points)
ing neutrophil gelatinase-associated lipocalin (NGAL),
1-5 1 2 3
liver-type fatty-acid binding protein (L-FABP), and (1 point)
␤2-microglobulin. ⬎6 2 3 4
(2 points)
Additional Readings
Abbreviation: HPF, high-power field.
» Briguori C, Visconti G, Rivera NV, et al. Cystatin C and Adapted from Perazella MA, et al. Urine microscopy is associ-
contrast-induced acute kidney injury. Circulation. 2010; ated with severity and worsening of acute kidney injury in hospi-
121(19):2117-2122. talized patients. Clin J Am Soc Nephrol. 2010;5(3):402-408.
» Demirtas S, Akan O, Can M, Elmali E, Akan H. Cystatin C
can be affected by nonrenal factors: a preliminary study on
leukemia. Clin Biochem. 2006;39(2):115-118.
more invasive testing. Examination of urine sediment
» Edelstein CL. Biomarkers in Kidney Disease. 1st ed. Amster-
dam, the Netherlands: Academic Press/Elsevier; 2011. can help discriminate proliferative glomerular disease
» Grubb A. Diagnostic value of analysis of cystatin C and from nonproliferative diseases. Hematuria and red
protein HC in biological fluids. Clin Nephrol. 1992;38(suppl blood cell casts are a sensitive early sign of relapse in
1):S20-S27. patients with lupus nephritis. White blood cell casts
» Herget-Rosenthal S, Marggraf G, Husing J, et al. Early
may be seen in tubulointerstitial nephritis, but also
detection of acute renal failure by serum cystatin C. Kidney
Int. 2004;66(3):1115-1122. may been seen in glomerulonephritis and pyelonephri-
» Inker LA, Schmid CH, Tighiouart H, et al. Estimating tis. Muddy brown casts, granular casts, and kidney
glomerular filtration rate from serum creatinine and cystatin tubular epithelial cells and casts can indicate acute
C. N Engl J Med. 2012;367(1):20-29. tubular injury, but can be seen in a variety of other
» Manzano-Fernandez S, Januzzi JL Jr, Boronat-Garcia M, et
acute and chronic kidney diseases.
al. Beta-trace protein and cystatin C as predictors of long-
term outcomes in patients with acute heart failure. J Am Coll In the setting of AKI, recent carefully performed
Cardiol. 2011;57(7):849-858. studies have suggested that a semiquantitative scoring
» Nejat M, Hill JV, Pickering JW, Edelstein CL, Devarajan P, system based on the number of kidney tubular epithe-
Endre ZH. Albuminuria increases cystatin C excretion: impli- lial cells and granular casts may provide useful infor-
cations for urinary biomarkers. Nephrol Dial Transplant.
mation on the differential diagnosis and prognosis of
2011;27 Suppl 3:iii96-103.
» Nejat M, Pickering JW, Walker RJ, Endre ZH. Rapid detec- AKI (Table 2). The combination of this score with
tion of acute kidney injury by plasma cystatin C in the novel biomarkers, including kidney injury molecule 1
intensive care unit. Nephrol Dial Transplant. 2010;25(10): (KIM-1), NGAL, and interleukin 18 (IL-18), has been
3283-3289. shown to be more sensitive than creatinine or urine
» Priem F, Althaus H, Jung K, Sinha P. Beta-trace protein is not
electrolyte levels alone in predicting the need for
better than cystatin C as an indicator of reduced glomerular
filtration rate. Clin Chem. 2001;47(12):2181. dialysis in patients with AKI.
» Shlipak MG, Sarnak MJ, Katz R, et al. Cystatin C and the risk However, the diagnostic utility of urine sediment
of death and cardiovascular events among elderly persons. analysis results in clinical practice is uncertain due to
N Engl J Med. 2005;352(20):2049-2060. a lack of standardization in reporting of results and the
» Spahillari A, Parikh CR, Sint K, et al. Serum cystatin C-
potential for significant inter- and intraobserver vari-
versus creatinine-based definitions of acute kidney injury
following cardiac surgery: a prospective cohort study. Am J ability. Most studies of the performance of urine
Kidney Dis. 2012;60(6):922-929. sediment involve clinicians with many years of expe-
rience and an interest in examining urine sediment.
Urine Sediment Analysis This may contrast with the experience of the general
Examination of urine sediment is a time-honored population of practicing nephrologists, internists, and
test relied on by generations of nephrologists to aid in trainees. There is a need for a single standard for
the diagnosis of kidney diseases. The actual diagnos- reporting urine sediment analysis results that corre-
tic performance characteristics of urine sediment ex- lates with results of kidney biopsies and clinical
amination and its inter- and intraobserver variability outcomes. Future studies are needed to quantify the
have not been well characterized. The presence of diagnostic characteristics of urine sediment examina-
specific findings in urine sediment can aid in the tion in a variety of kidney diseases and assess the
diagnosis of acute and chronic kidney disease prior to inter- and intraobserver reliability.

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Biomarkers in Nephrology

Table 3. Examples of Clinical Settings in Which Novel Biomarkers of Kidney Disease Have Been Studied in Humans

Scenario Selected Biomarkers

Cardiac surgery B2M, CyC, GST, IL-18, KIM-1, L-FABP, NAG, netrin, NGAL
Intensive care unit B2M, CyC, GST, IL-18, KIM-1, L-FABP, NAG, NGAL
Contrast-induced nephropathy CyC, IL-18, KIM-1, L-FABP, NAG, NGAL
Drug-induced nephrotoxicity AAP, AP, B2M, CyC, calbindin D, clusterin, GGT, GST, KIM-1, LDH, L-FABP, NAG, NGAL,
osteopontin, RBP4
Chronic kidney disease ADMA, BTP, CTGF, FGF-23, KIM-1, L-FABP, NGAL, RBP4, TFF3, uromodulin
Glomerular disease IL-18, IP-10, NGAL, MCP-1, NAG, osteopontin, TGF␤
Abbreviations: AAP, alanine aminopeptidase; ADMA, asymmetric dimethyl arginine; AP, alkaline phosphatase; BTP, beta-trace
protein; B2M, ␤2-microglobulin; CyC, cystatin C; CTGF, connective tissue growth factor; FGF-23, fibroblast growth factor 23; GGT,
␥-glutamyl transpeptidase; GST, glutathione-S-transferase; IL-18, interleukin 18; IP-10, interferon ␥–induced protein 10; KIM-1, kidney
injury molecule 1; L-FABP, Liver-type fatty acid binding protein; LDH, lactate dehydrogenase; MCP-1, monocyte chemoattractant
protein 1; NAG, N-acetyl glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; RBP4, retinol binding protein 4; TFF3,
trefoil factor 3; TGF␤, transforming growth factor ␤.

Additional Readings Additional Readings

» Fogazzi GB, Ponticelli C, Ritz E. The Urine Sediment: An » Chronic Kidney Disease Prognosis Consortium, Matsushita
Integrated View, 3rd edition. Milan, Italy: Elsevier Srl; 2010. K, van der Velde M, Astor BC, et al. Association of estimated
» Hall IE, Coca SG, Perazella MA, et al. Risk of poor outcomes glomerular filtration rate and albuminuria with all-cause and
with novel and traditional biomarkers at clinical AKI diagno- cardiovascular mortality in general population cohorts: a
sis. Clin J Am Soc Nephrol. 2011;6(12):2740-2749. collaborative meta-analysis. Lancet. 2010;375(9731):2073-
» Perazella MA, Coca SG. Traditional urinary biomarkers in 2081.
the assessment of hospital-acquired AKI. Clin J Am Soc » Levey AS, de Jong PE, Coresh J, et al. The definition,
Nephrol. 2011;7(1):167-174. classification, and prognosis of chronic kidney disease: a
» Perazella MA, Coca SG, Hall IE, Iyanam U, Koraishy M, KDIGO Controversies Conference report. Kidney Int. 2011;
80(1):17-28.
Parikh CR. Urine microscopy is associated with severity and
worsening of acute kidney injury in hospitalized patients.
Clin J Am Soc Nephrol. 2010;5(3):402-408. NOVEL BIOMARKERS
The primary rationale for the development of novel
Albuminuria biomarkers of kidney diseases is that early and more
Proteinuria has long been recognized as a conse- accurate diagnosis may allow for interventions that
quence of kidney damage. Measurement of urinary prevent progression. Because levels of current bio-
protein and urinary albumin is a central component of markers of kidney disease become elevated relatively
screening for and monitoring CKD. The glomerulus late in the injury process, they do not allow for early
acts as a barrier to filtration due to pore size and interventions that could be successful at preventing
charge selectivity, while most filtered albumin is reab- propagation of the injury. Early identification could
sorbed in the proximal tubule. Thus, the presence of allow for injury-specific interventions that currently
are not possible. This could be particularly useful in
albuminuria usually indicates damage to the filtration
AKI when a defined insult, for example, after contrast
barrier, but also may be the result of proximal tubular
administration or cardiac surgery, could allow for
dysfunction.
more appropriate risk stratification and treatment
Multiple studies have demonstrated that albumin-
(Table 3). Thus, novel biomarkers have a number of
uria is an independent risk factor for mortality in the potential roles in nephrology: (1) to more accurately
general population. Albuminuria also is an indepen- diagnose AKI early in the course of the disease, (2) to
dent risk factor for the progression of diabetic and correlate better with GFR in patients with CKD, (3) to
nondiabetic CKD. As a result, it has been proposed determine the anatomical location of injury in patients
that stage 3 CKD be stratified further by the presence with AKI (ie, glomerular, tubular, interstitial, or vascu-
or absence of albuminuria. Reduction in albuminuria lar), (4) to identify the cause of AKI and CKD, (5) to
is a commonly accepted goal of treatment in patients monitor the effectiveness of interventions, and (6) to
with CKD, although it is not as yet an acceptable provide information regarding the prognosis of AKI
surrogate end point in clinical trials. However, the US and CKD.
Food and Drug Administration (FDA) recently quali- One significant issue surrounding the development
fied its use as a biomarker of proximal tubular injury of biomarkers in nephrology is the frequent absence
from nephrotoxin exposure in animal studies. of a reliable gold standard for the diagnosis of kidney

Am J Kidney Dis. 2013;62(1):165-178 169

 McMahon and Waikar

disease. The limitations of creatinine as a biomarker biomarkers in acute kidney injury (AKI). Am J Physiol Renal
were discussed, but in brief, it is insensitive, not very Physiol. 2011;301(4):F697-F707.
» Haase M, Devarajan P, Haase-Fielitz A, et al. The outcome of
specific, and the level tends to increase relatively late neutrophil gelatinase-associated lipocalin-positive subclini-
in the course of kidney injury, when the injury may cal acute kidney injury: a multicenter pooled analysis of
not be reversible. Currently, the commonly used defi- prospective studies. J Am Coll Cardiol. 2011;57(17):1752-
nitions of AKI (RIFLE, AKIN [AKI Network], and 1761.
KDIGO [Kidney Disease: Improving Global Out- » Nejat M, Pickering JW, Devarajan P, et al. Some biomarkers
of acute kidney injury are increased in pre-renal acute injury.
comes]) incorporate creatinine concentration into the Kidney Int. 2012;81(12):1254-1262.
diagnostic framework, whereas equations for estimat- » Ozer JS, Dieterle F, Troth S, et al. A panel of urinary
ing GFR in patients with CKD (MDRD Study and biomarkers to monitor reversibility of renal injury and a
CKD-EPI equations) also generally require creatinine serum marker with improved potential to assess renal func-
level. tion. Nat Biotechnol. 2010;28(5):486-494.
» Waikar SS, Betensky RA, Emerson SC, Bonventre JV.
It is notable that recent studies have demonstrated Imperfect gold standards for kidney injury biomarker evalua-
the usefulness of novel biomarkers in mild AKI. tion. J Am Soc Nephrol. 2012;23(1):13-21.
Urinary levels of certain biomarkers have been found
to be elevated in patients with prerenal AKI (KIM-1, Plasma/Serum Versus Urinary Biomarkers
cystatin C, and IL-18), whereas others (NGAL and Urine and plasma/serum are potential sources of
␥-glutamyl transpeptidase [GGT]) were not. A recent biomarkers in nephrology. Both are easily collected
meta-analysis found that elevated urinary or plasma and minimally invasive. Urine has a number of advan-
NGAL level predicted the future need for renal replace- tages over serum or plasma for the evaluation of
ment therapy and in-hospital mortality in critically ill potential biomarkers. Urine is a proximal fluid, mean-
patients in the intensive care unit, even in the absence ing that it is in close contact to the site of injury (the
of a clinically significant increase in creatinine level. kidney), and as such, is a site where biomarkers can
This suggests that there is a spectrum of subclinical accumulate after being shed by damaged kidney tis-
but significant AKI that is not being detected by the sue. In patients with AKI, urinary biomarker levels
traditional gold-standard methods. increase relatively early in the course of the disease.
In animal studies, increasing levels of kidney injury In contrast, for serum markers of GFR, such as creati-
biomarkers correlate with the gold standard of increas- nine and cystatin C, there is a significant lag between
ing structural damage on kidney biopsy in models of the time of injury and the time that their concentra-
AKI. However, there have been no large studies of tions will exceed the threshold required to make a
humans correlating structural appearance with bio- diagnosis of AKI. Urinary biomarkers can be sepa-
marker levels. As a result, we continue to use creati- rated into those that are present in the kidney and
nine as the gold standard in biomarker trials. How- released in the event of damage (eg, proximal tubular
ever, biomarker levels may relate to the severity of enzymes) and those induced by tubular injury (eg,
kidney injury and be associated with the occurrence of KIM-1, NGAL, and IL-18). Preformed biomarkers
clinical outcomes, including need for dialysis and appear in urine shortly after injury, whereas there is a
death. Recently, the FDA qualified a set of urinary time lag for the appearance of the induced biomark-
biomarkers of nephrotoxicity for regulatory use in ers.
certain preclinical settings (ie, animal studies). These Urine biomarkers have a number of important po-
included urinary KIM-1, albumin, total protein, albu- tential limitations. It is important that urine is handled
min, ␤2-microglobulin, cystatin C, clusterin, trefoil correctly. Urinary biomarkers may not be stable for
factor 3, and renal papillary antigen 1. The recent long periods and urine should be frozen promptly if it
FDA qualification of novel injury biomarkers demon- is not analyzed immediately. There is a potential for
strates a move away from creatinine as the gold error if urine is not collected appropriately. Midstream
standard for studies in nephrology toward a more urine samples are more informative in women, presum-
nuanced approach. ably because initial voids are more prone to bacterial
contamination. In contrast, for males, prostate cancer
Additional Readings markers are detectable more readily in first-void urine
» Dieterle F, Sistare F, Goodsaid F, et al. Renal biomarker samples. Baseline kidney function is an important
qualification submission: a dialog between the FDA-EMEA modifying influence. As detailed next, a major limita-
and predictive safety testing consortium. Nat Biotechnol. tion of urinary biomarkers is that their concentration
2010;28(5):455-462.
changes with hydration status, and multiple methods
» Edelstein CL. Biomarkers in Kidney Disease. 1st ed. Amster-
dam, the Netherlands: Academic Press/Elsevier; 2011. of dealing with this problem have been suggested,
» Endre ZH, Pickering JW, Walker RJ. Clearance and beyond: including normalizing to urinary creatinine concentra-
the complementary roles of GFR measurement and injury tion.

170 Am J Kidney Dis. 2013;62(1):165-178

Biomarkers in Nephrology

Serum or plasma also is a good potential source of The use of short timed urine collections also has been
biomarkers. It is obtainable in anuric patients and is proposed, although this is not always practical in the
less prone to bacterial contamination. However, ambulatory setting. Similarly, normalizing to the ac-
changes in serum biomarker concentrations are not tual urine flow rate at the time of collection would
necessarily related to decreased kidney function alone lead to more unbiased results, although measuring this
and can be the product of a systemic response. The accurately is not without difficulty. Interestingly, one
large number of proteins present in serum makes the recent study suggested that absolute biomarker values
discovery of specific kidney markers more compli- were better at diagnosing AKI, whereas normalizing
cated. to creatinine level was a better predictor of overall
adverse outcomes. This finding may reflect the prog-
Additional Readings nostic significance of creatinine generation rate, which
» Edelstein CL. Biomarkers in Kidney Disease. 1st ed. Amster- influences urinary creatinine concentration and there-
dam, the Netherlands: Academic Press/Elsevier; 2011. fore creatinine-normalized biomarker levels. For the
» Hewitt SM, Dear J, Star RA. Discovery of protein biomarkers moment, most studies continue to normalize to creati-
for renal diseases. J Am Soc Nephrol. 2004;15(7):1677-1689. nine level, understanding that this is an imperfect
» Wu J, Chen YD, Gu W. Urinary proteomics as a novel tool for
biomarker discovery in kidney diseases. J Zhejiang Univ Sci
technique. In a clinical setting, it should be recognized
B. 2010;11(4):227-237. that results should be interpreted with reference to the
factors that may influence urinary creatinine concen-
Normalization of Urinary Biomarkers tration.
Because the kidney has the ability to reabsorb water Additional Readings
over a wide range, the concentration of a biomarker in
» Conti M, Moutereau S, Esmilaire L, et al. Should kidney
urine depends not only on its rate of production, but tubular markers be adjusted for urine creatinine? The example
also on urine flow rate. Because of this, absolute of urinary cystatin C. Clin Chem Lab Med. 2009;47(12):1553-
values of urine biomarkers will change significantly 1556.
depending on urine flow rate, thus making them » Jantos-Siwy J, Schiffer E, Brand K, et al. Quantitative urinary
difficult to interpret. Ideally, urinary biomarkers should proteome analysis for biomarker evaluation in chronic kidney
disease. J Proteome Res. 2009;8(1):268-281.
be normalized by expressing them as a ratio to a » Ralib AM, Pickering JW, Shaw GM, et al. Test characteristics
substance that is excreted at a constant rate by the of urinary biomarkers depend on quantitation method in acute
kidneys. In practice, urinary biomarkers commonly kidney injury. J Am Soc Nephrol. 2012;23(2):322-333.
are expressed as a ratio to urinary creatinine concen- » Waikar SS, Sabbisetti VS, Bonventre JV. Normalization of
tration. This assumes that creatinine production is urinary biomarkers to creatinine during changes in glomeru-
lar filtration rate. Kidney Int. 2010;78(5):486-494.
relatively constant across individuals and relative to
the urinary biomarker concentrations in healthy and NOVEL BIOMARKERS IN CLINICAL CONDITIONS
diseased states. However, urine creatinine concentra-
tion is affected by a number of factors, including Novel Biomarkers of AKI
creatinine generation rate, which varies significantly AKI is a syndrome characterized by a rapid de-
between individuals. As a result, normalizing bio- crease in GFR associated with the accumulation of
marker concentrations to creatinine concentration po- waste products usually excreted by the kidney. It is
tentially will introduce bias. There are 4 settings in relatively common, occurring in up to 9% of all
particular in which normalizing to creatinine concen- hospitalized patients, and is particularly prevalent in
tration could generate misleading results: (1) when patients admitted to the intensive care unit. The mor-
there are significantly higher or lower creatinine gen- tality of patients requiring dialysis in the intensive
eration rates (ie, increased or reduced muscle mass) or care unit is 40%-60%, and this has remained high
more extrarenal degradation of creatinine (which is during the past 20 years. Current definitions of AKI
substantially higher in patients with advanced CKD), include a change in serum creatinine level over a
(2) in the setting of an acute change in GFR in which defined period as one of the major criteria. As men-
excretion of creatinine has not reached a steady state, tioned, this leads to a delay in diagnosis because of the
(3) in the setting of markedly increased or decreased lag time between the occurrence of the injury and an
tubular secretion of creatinine, and (4) in low urine increase in serum creatinine level (Fig 2). As a result,
flow states in which there may be reabsorption of much effort is being focused on biomarkers of AKI
creatinine in the proximal tubules. that can make the diagnosis earlier and more accu-
As a result, alternative methods of urine normaliza- rately and possibly enable the use of strategies to
tion have been proposed. There is some interest in the prevent progression of this syndrome.
use of alternative urinary peptides, although the devel- One of the best-studied new biomarkers of AKI is
opment of these techniques is in the very early stages. NGAL, a 21-kDa protein. It is involved in innate

Am J Kidney Dis. 2013;62(1):165-178 171

 McMahon and Waikar

700 0.9

0.8
600
0.7
500
0.6
ng/ml or pg/ml

400 Urine NGAL

0.5

mg/dl
(ng/ml) Figure 2. Pattern of change in bio-
0.4 Urine IL-18 (pg/ml) markers over time in patients with acute
300
kidney injury following cardiac surgery.
0.3 Serum Creatinine
200 (mg/dl) Abbreviation: NGAL, neutrophil gelati-
0.2 nase-associated lipocalin. Conversion
100 factor for units: serum creatinine in mg/dL
0.1 to ␮mol/L, ⫻88.4. Data from Parikh CR,
0 0 et al. Postoperative biomarkers predict
Pre-op 6 12 18 24 48 acute kidney injury and poor outcomes
after pediatric cardiac surgery. J Am Soc
Hours Post Surgery Nephrol. 2011;22:1737-1747.

immunity and is expressed primarily by immune cells, rently subclinical kidney disease. This was borne out
but also by hepatocytes and kidney tubular cells. It by a meta-analysis that found that elevations in NGAL
originally was identified through transcriptome analy- level even in the absence of an elevated creatinine
sis in a mouse model of ischemia-reperfusion injury in level predicted worse outcomes. This finding suggests
which its production in kidney tubules was noted to that there may need to be further changes in the way
increase rapidly after kidney injury. It is readily detect- that we define AKI to incorporate these biomarkers.
able in urine and resistant to degradation by proteases, IL-18 is an 18-kDa proinflammatory cytokine that
making it a potentially ideal biomarker of AKI. NGAL is expressed primarily by macrophages, but also by
appears in urine early after injury, and studies of monocytes, dendritic cells, and kidney epithelial cells.
cultured human tubular cells confirmed that it is It has a role in the innate and adaptive immune
produced in response to hypoxic injury. Urinary and response and is upregulated in inflammatory states.
plasma NGAL levels have been shown to correlate IL-18 is thought to be one of the mediators of injury in
with degree of kidney injury, and levels return to ischemic AKI. Inhibition of IL-18 has been shown to
baseline on resolution of AKI. Interestingly, the source be effective in treating inflammatory disorders in
of plasma and urinary NGAL appears to be different. mice, and interstitial IL-18 expression is increased in
Most urinary NGAL is produced in tubules in re- mouse models of AKI. Mice deficient in caspase, an
sponse to injury, whereas most plasma NGAL origi- IL-18–activating enzyme, develop less acute tubular
nates in distant organs, where its production is upregu- necrosis than wild-type mice in models of ischemia.
lated in the setting of AKI. Commercial assays are Urinary IL-18 originates in the kidney tubular epithe-
available for NGAL measurement. lium and thus was proposed as a potential marker of
Urinary NGAL has been shown to be a possible AKI.
early biomarker of AKI in adults and children after IL-18 level has been shown to increase early in
cardiac surgery and after administration of radiocon- patients with sepsis in the intensive care unit and was
trast. Similarly, elevations of NGAL levels predict a good predictor of AKI, particularly when combined
AKI in patients presenting to the emergency depart- with NGAL level. IL-18 levels increased in patients
ment. A large meta-analysis of studies of NGAL in with acute tubular necrosis, but not those with prer-
AKI found that there was no advantage in using enal AKI or CKD and healthy controls. Similarly,
urinary NGAL as opposed to serum NGAL. NGAL increasing IL-18 levels predicted AKI in pediatric
performed better in children, although it remained patients undergoing cardiac surgery. Higher postopera-
useful in adults, suggesting that there may be other tive urinary IL-18 excretion also has been shown to
factors that influence NGAL levels in this population. identify AKI and predict progression of AKI after
A wide range of cutoff values for urinary NGAL were cardiac surgery in adults. Potential limitations of
used in the various studies, but a level ⬎150 ng/mL IL-18 derive from the fact that it may be a more
appeared to be the most appropriate, particularly when generalized marker of inflammation rather than a
commercial assays were used. Interestingly, normal- specific marker of AKI, particularly in older age
ization of results to urinary creatinine level did not groups, for whom there may be underlying baseline
appear to affect the test accuracy. Elevated NGAL decreased kidney function.
level in the absence of elevated serum creatinine level Another biomarker that has attracted considerable
may be prognostically significant and suggests that interest is KIM-1. KIM-1 initially was found to be
creatinine level may misclassify individuals with cur- highly upregulated in a rat model of ischemic kidney

172 Am J Kidney Dis. 2013;62(1):165-178

Biomarkers in Nephrology

injury. It is a transmembrane protein that is expressed factors, for example, although NAG has been shown
at very low levels in normal kidney, but its production to be a sensitive marker of AKI in certain situations,
increases in dedifferentiated proximal tubular cells in the presence of heavy metals and other nephrotoxins
the presence of ischemic or nephrotoxic AKI. The in urine inhibits its activity, reducing the sensitivity of
extracellular domain of KIM-1 appears in urine shortly the assay. There is a suggestion that panels of en-
after ischemic injury and can be detected readily by a zymes may be more useful than individual enzymes in
new KIM-1 urinary dipstick, potentially making it a diagnosing kidney disorders, although their primary
convenient and readily measured marker of AKI. use may lie in the diagnosis of drug nephrotoxicity, in
Early studies of humans found that, in common which levels of these enzymes increase prior to any
with the rat, KIM-1 expression was increased in increase in creatinine level and may help guide drug
kidney biopsy specimens from patients with acute dosing.
tubular necrosis. Elevated urinary KIM-1 level pre- Apart from cystatin, there are a number of other
dicted increased risk of mortality or need for dialysis proteins that normally are filtered at the glomerulus,
in hospitalized patients with AKI and was a sensitive but do not appear in urine because of uptake and
predictor of AKI in children undergoing cardiac sur- metabolism in the proximal tubule. The presence of
gery. Its performance as a biomarker of AKI may these biomarkers in urine suggests tubular dysfunc-
prove to be better in patients with normal baseline tion (although, as mentioned previously, in the pres-
kidney function, as is the case for many novel biomark- ence of sufficient albuminuria, the normal uptake
ers. This may be because although expression is very mechanism can be overwhelmed and they can become
limited in normal kidney tissue, there is upregulation present even with normal tubular function). The most
of expression in various CKDs, with the degree of important of these are ␤2-microglobulin and retinol
expression correlating with tubulointerstitial fibrosis. binding protein 4 (RBP-4). ␤2-Microglobulin is an
Its role in CKD currently is under investigation. 11.8-kDa protein component of the major histocompat-
The kidneys contain large amounts of enzymes that ibility complex class I molecule that is filtered freely
perform specialized functions in tubular cells. These in the glomerulus and reabsorbed and metabolized by
enzymes can be released into urine in the event of the tubules, with ⬍1% appearing in urine. Urinary
kidney injury both as a result of leakage from cells ␤2-microglobulin excretion increases in the setting of
(preformed enzymes) or by upregulation of their pro- nonselective proteinuria and also as a result of tubular
duction in response to injury. Because they are local- damage, particularly as a result of exposure to tubular
ized to specific cells along the nephron, the presence toxins. Urinary ␤2-microglobulin has been studied in
of certain enzymes in urine can give a clue to the a wide range of clinical contexts and appears to be
specific location of the kidney injury. N-Acetyl- useful in distinguishing prerenal azotemia from acute
glucosaminidase (NAG) is a lysosomal enzyme pri- tubular necrosis, but is of uncertain utility in patients
marily localized to the proximal tubule. Glutathione- with sepsis, in whom its levels can increase in the
S-transferase (GST) is a family of enzymes with 8 absence of AKI. RBP-4 is synthesized primarily by
different classes found throughout the nephron. How- hepatocytes and is involved in the transport of retinol
ever, the ␣ isoform is present in proximal tubular cells in blood. The C-terminally processed form is excreted
alone, whereas the ␲ isoform is found in only distal in urine and accumulates in patients with decreased
tubular cells. Alkaline phosphatase, GGT, alanine GFR. RBP-4 is reabsorbed in the proximal tubule and
aminopeptidase, and lactate dehydrogenase are brush- appears in large quantities when there is significant
border enzymes originating in the proximal tubule proximal tubular damage. It is a particularly sensitive
that normally are present in urine in small quantities marker of nephrotoxicity and levels are elevated in
and increase significantly in the setting of AKI. Tubu- decreased kidney function caused by heavy metals
lar enzymuria has been noted in patients with a broad and in patients with diabetic nephropathy. One advan-
range of kidney diagnoses, including acute tubular tage that it carries over ␤2-microglobulin is that it is
necrosis, interstitial nephritis, nephrotoxicity, and acute more stable at low pH.
transplant rejection. No individual enzyme has been There are a large number of other biomarkers that
shown to be a consistent predictor of AKI or need for are under investigation to determine their utility in the
dialysis across all studies, and although some have early diagnosis of kidney diseases, including AKI.
shown promise in certain homogenous populations, Most are proteins expressed in tubules for which
these results have not been replicated across heteroge- urinary concentrations change in the event of tubular
neous groups. This may be due in part to the fact that injury. Trefoil factor 3 is a protein involved in mainte-
enzymuria is increased in many settings and is not nance of the integrity of mucosal surfaces. It normally
specific for AKI. There also may be settings in which is found in urine, and decreasing levels are a sensitive
activity of the enzymes is decreased by exogenous marker of proximal tubular/collecting duct dysfunc-

Am J Kidney Dis. 2013;62(1):165-178 173

 McMahon and Waikar

tion, particularly due to nephrotoxicity. L-FABP is a cal acute kidney injury: a multicenter pooled analysis of
cytoplasmic protein expressed in proximal tubules prospective studies. J Am Coll Cardiol. 2011;57(17):1752-
1761.
that binds free fatty acids and transports them to the
» Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre
mitochondria for metabolism. Increased urinary L- JV. Kidney injury molecule-1 (KIM-1): a novel biomarker for
FABP excretion has been noted in patients with AKI human renal proximal tubule injury. Kidney Int. 2002;62(1):
(particularly ischemic), nephrotoxicity, and severe sep- 237-244.
sis in the absence of AKI. » Liangos O, Perianayagam MC, Vaidya VS, et al. Urinary
Osteopontin is a 44-kDa protein synthesized primar- N-acetyl-beta-(D)-glucosaminidase activity and kidney injury
molecule-1 level are associated with adverse outcomes in
ily in bone, but also in macrophages, activated T cells, acute renal failure. J Am Soc Nephrol. 2007;18(3):904-912.
and endothelial cells. In the kidney, it is expressed in » Lisowska-Myjak B. Serum and urinary biomarkers of acute
the loop of Henle and distal tubules and is involved in kidney injury. Blood Purif. 2010;9(4):357-365.
kidney protection against oxidative stress and isch- » Mishra J, Ma Q, Prada A, et al. Identification of neutrophil
emia. Increased urinary osteopontin levels have been gelatinase-associated lipocalin as a novel early urinary bio-
marker for ischemic renal injury. J Am Soc Nephrol. 2003;
noted in some chronic kidney diseases, including 14(10):2534-2543.
immunoglobulin A (IgA) nephropathy, membra- » Parikh CR, Coca SG, Thiessen-Philbrook H, et al. Postopera-
nous nephropathy, and glomerulonephritis, as well tive biomarkers predict acute kidney injury and poor out-
as in patients with AKI. Netrin-1 is an axonal- comes after adult cardiac surgery. J Am Soc Nephrol. 2011;
guidance molecule that is not expressed in normal 22(9):1748-1757.
» Parikh CR, Jani A, Melnikov VY, Faubel S, Edelstein CL.
tubular cells but is highly expressed after kidney Urinary interleukin-18 is a marker of human acute tubular
injury, with levels increasing within 2 hours of an necrosis. Am J Kidney Dis. 2004;43(3):405-414.
acute insult. Levels correlate with degree of injury » Parikh CR, Mishra J, Thiessen-Philbrook H, et al. Urinary
and return to normal as the injury resolves. Clus- IL-18 is an early predictive biomarker of acute kidney injury
terin is an 80-kDa protein that is expressed constitu- after cardiac surgery. Kidney Int. 2006;70(1):199-203.
» Urbschat A, Obermuller N, Haferkamp A. Biomarkers of
tively during normal kidney development. Its pro-
kidney injury. Biomarkers. 2011;16(suppl 1):S22-S30.
duction is upregulated in response to kidney injury » Waikar SS, Betensky RA, Bonventre JV. Creatinine as the
and it is expressed throughout the nephron. It has gold standard for kidney injury biomarker studies? Nephrol
shown promise as a biomarker of tubular injury and Dial Transplant. 2009;24(11):3263-3265.
regeneration in animal models, but has not yet been » Waring WS, Moonie A. Earlier recognition of nephrotoxicity
investigated in large studies in humans. using novel biomarkers of acute kidney injury. Clin Toxicol
(Phila). 2011;49(8):720-728.
» Yu Y, Jin H, Holder D, et al. Urinary biomarkers trefoil factor
Additional Readings 3 and albumin enable early detection of kidney tubular injury.
» Amin AP, Salisbury AC, McCullough PA, et al. Trends in the Nat Biotechnol. 2010;28(5):470-477.
incidence of acute kidney injury in patients hospitalized with
acute myocardial infarction. Arch Intern Med. 2012;172(3): Novel Biomarkers of CKD
246-253.
» Bagshaw SM, Langenberg C, Haase M, Wan L, May CN, CKD affects ⬃11% of the US population and is a
Bellomo R. Urinary biomarkers in septic acute kidney injury. major contributor to morbidity and mortality. Early
Intensive Care Med. 2007;33(7):1285-1296. identification of those at risk of CKD is important and
» Bellomo R, Kellum JA, Ronco C. Acute kidney injury.
Lancet. 2012;380(9843):756-766.
has been facilitated by the routine use of estimating
» Bonventre JV. Kidney injury molecule-1 (KIM-1): a urinary equations for GFR (MDRD Study and CKD-EPI
biomarker and much more. Nephrol Dial Transplant. 2009; equations). Classification of CKD into stages and
24(11):3265-3268. stratification by degree of proteinuria has helped iden-
» Edelstein CL. Biomarkers in Kidney Disease. 1st ed. Amster- tify patients at risk of progression to end-stage renal
dam, the Netherlands: Academic Press/Elsevier; 2011.
» Koyner JL, Garg AX, Coca SG, et al. Biomarkers predict
disease. However, current techniques for estimating
progression of acute kidney injury after cardiac surgery. J Am GFR rely on serum creatinine level, with all its
Soc Nephrol. 2012;23(5):905-914. attendant deficiencies, and newer biomarkers are be-
» Haase M, Bellomo R, Devarajan P, Schlattmann P, Haase- ing sought that may better stratify patients by risk
Fielitz A; NGAL Meta-analysis Investigator Group. Accuracy category. In common with AKI, no single biomarker
of neutrophil gelatinase-associated lipocalin (NGAL) in diag-
nosis and prognosis in acute kidney injury: a systematic
has been identified that can reliably predict progres-
review and meta-analysis. Am J Kidney Dis. 2009;54(6):1012- sion of CKD.
1024. As discussed, creatinine and cystatin C are the
» Haase M, Bellomo R, Story D, Davenport P, Haase-Fielitz A. biomarkers used most often to determine GFR. An-
Urinary interleukin-18 does not predict acute kidney injury other promising marker of GFR is beta-trace protein
after adult cardiac surgery: a prospective observational cohort
study. Crit Care. 2008;12(4):R96.
(BTP). BTP is a low-molecular-mass protein belong-
» Haase M, Devarajan P, Haase-Fielitz A, et al. The outcome of ing to the lipocalin protein family that is produced at a
neutrophil gelatinase-associated lipocalin-positive subclini- constant rate by glial cells in the central nervous

174 Am J Kidney Dis. 2013;62(1):165-178

Biomarkers in Nephrology

system. It is filtered freely at the glomerulus and Fibroblast growth factor 23 (FGF-23) is a phospha-
reabsorbed and metabolized in the proximal tubule. turic hormone that is elevated in patients with CKD.
There is almost no nonrenal elimination. It has been The degree of elevation correlates with stage of CKD,
shown to be as good as cystatin C and creatinine and elevations in FGF-23 levels are noted prior to
levels at detecting mild abnormalities in kidney func- elevation in phosphate levels.
tion and also may be better than creatinine level at In order to be useful for clinical practice, new
predicting adverse outcomes in patients with acute biomarkers of CKD progression will have to prove
heart failure. Estimating equations are being derived superior to the impressive prognostic ability of esti-
that include serum BTP but they have not been vali- mated GFR and albuminuria. New biomarkers of
dated in large studies. BTP can aid in the prediction of GFR estimation will have to improve precision, bias,
progression of CKD, but similar to cystatin C, cortico- and the prognostic ability of creatinine and cystatin C
steroids can influence levels independent of GFR. levels.
As well as its potential role in the diagnosis of AKI, Studies from the National Institutes of Health CKD
NGAL may be a useful biomarker in patients with Biomarkers Consortium and other investigators are
CKD, particularly for identifying patients at risk of a underway to identify and validate novel biomarkers of
significant decrease in GFR, because of its ability to CKD.
detect subtle changes in tubular function. Urinary and
serum NGAL levels are elevated in a wide range of Additional Readings
kidney diseases, including IgA nephropathy, auto- » Bolignano D, Lacquaniti A, Coppolino G, et al. Neutrophil
somal dominant polycystic kidney disease, and dia- gelatinase-associated lipocalin (NGAL) and progression of
chronic kidney disease. Clin J Am Soc Nephrol. 2009;4(2):337-
betic nephropathy. Urinary NGAL level has been
344.
shown to differentiate HIV (human immunodefi- » Fassett RG, Venuthurupalli SK, Gobe GC, Coombes JS,
ciency virus) nephropathy from other forms of kidney Cooper MA, Hoy WE. Biomarkers in chronic kidney disease:
disease, whereas higher levels were associated with a review. Kidney Int. 2011;80(8):806-821.
increased risk of progression in a diverse group of » Kamijo-Ikemori A, Sugaya T, Yasuda T, et al. Clinical
significance of urinary liver-type fatty acid-binding protein in
patients with CKD. diabetic nephropathy of type 2 diabetic patients. Diabetes
AKI itself is a risk factor for future CKD, but there is Care. 2011;34(3):691-696.
no reliable means of determining who will recover » Lu TM, Chung MY, Lin CC, Hsu CP, Lin SJ. Asymmetric
entirely and who will be left with some kidney impair- dimethylarginine and clinical outcomes in chronic kidney
ment after an episode of AKI. NGAL is a potential disease. Clin J Am Soc Nephrol. 2011;6(7):1566-1572.
» Manzano-Fernandez S, Januzzi JL, Boronat-Garcia M, et al.
marker of future progression. Similarly, KIM-1 may Impact of kidney dysfunction on plasma and urinary N-
have a role in predicting the transition from AKI to terminal pro-B-type natriuretic peptide in patients with acute
CKD. Urinary KIM-1 levels have been shown to corre- heart failure. Congest Heart Fail. 2010;16(5):214-220.
late with proteinuria, decreasing in response to treatment » Priem F, Althaus H, Jung K, Sinha P. Beta-trace protein is not
with angiotensin-converting enzyme inhibitors or a low- better than cystatin C as an indicator of reduced glomerular
filtration rate. Clin Chem. 2001;47(12):2181.
sodium diet, suggesting a potential role as a measure of » Ravani P, Tripepi G, Malberti F, Testa S, Mallamaci F, Zoccali
therapeutic efficacy. C. Asymmetrical dimethylarginine predicts progression to
Urinary L-FABP level correlates with degree of dialysis and death in patients with chronic kidney disease: a
proteinuria in patients with CKD. In patients with competing risks modeling approach. J Am Soc Nephrol.
diabetes, it has been shown to correlate with GFR and 2005;16(8):2449-2455.
» Spanaus KS, Kollerits B, Ritz E, et al. Serum creatinine,
predict progression to end-stage renal disease. Base- cystatin C, and beta-trace protein in diagnostic staging and
line levels predicted the future development of albu- predicting progression of primary nondiabetic chronic kidney
minuria, suggesting a potential role in stratifying disease. Clin Chem. 2010;56(5):740-749.
patients who might benefit from early preventative » Waanders F, Vaidya VS, van Goor H, et al. Effect of
renin-angiotensin-aldosterone system inhibition, dietary so-
therapies. Other potential urinary biomarkers of CKD
dium restriction, and/or diuretics on urinary kidney injury
include connective tissue growth factor, L-FABP, and molecule 1 excretion in nondiabetic proteinuric kidney dis-
trefoil factor 3. ease: a post hoc analysis of a randomized controlled trial.
Asymmetric dimethylarginine (ADMA) is an inhib- Am J Kidney Dis. 2009;53(1):16-25.
itor of nitric oxide synthase and a marker of endothe-
lial function. Increasing plasma levels predict progres- Biomarkers of Nephrotoxicity
sion of CKD and mortality in patients with chronic Nephrotoxicity is an unfortunate side effect of
kidney failure in diabetic and nondiabetic kidney many drugs and a relatively common cause of acute
disease. Increased plasma levels also are predictive of and chronic kidney injury. Currently, clinical nephro-
future cardiovascular mortality in patients with known toxicity is diagnosed when there is an increase in
CKD and cardiovascular disease. serum creatinine concentration or a decrease in urine

Am J Kidney Dis. 2013;62(1):165-178 175

 McMahon and Waikar

output. These are relatively late complications of As noted earlier, the FDA has qualified the follow-
drug-induced kidney injury and also are nonspecific. ing biomarkers—urinary KIM-1, albumin, total pro-
Often, nephrotoxicity is a diagnosis of exclusion when tein, ␤2-microglobulin, cystatin C, clusterin, trefoil
other causes are ruled out. Nephrotoxicity can be factor-3, and renal papillary antigen 1—along with
localized to a specific part of the nephron, which traditional clinical chemistry markers and histopathol-
serum creatinine cannot distinguish. Proteinuria (eg, ogy for the detection of acute drug-induced nephrotox-
due to vascular endothelial growth factor inhibitors) icity in rat toxicology studies.
and subtle signs of defects in proximal tubular func-
tion (eg, related to the use of antiretroviral medica- Additional Readings
tions) can suggest specific forms of nephrotoxicity, » Ali BH, Al-Salam S, Al-Husseini I, Nemmar A. Comparative
but these generally arise late in the process. Specific protective effect of N-acetyl cysteine and tetramethylpyrazine
biomarkers that have increased levels earlier in the in rats with gentamicin nephrotoxicity. J Appl Toxicol.
2009;29(4):302-307.
course of nephrotoxicity and more accurately localize » Hoffmann D, Fuchs TC, Henzler T, et al. Evaluation of a
the site of the lesion would be of great benefit in both urinary kidney biomarker panel in rat models of acute and
the clinical management of nephrotoxicity (by identi- subchronic nephrotoxicity. Toxicology. 2010;277(1-3):49-58.
fying patients at risk and allowing the withdrawal of » Waring WS, Moonie A. Earlier recognition of nephrotoxicity
using novel biomarkers of acute kidney injury. Clin Toxicol
an offending agent) and drug development. Because it (Phila). 2011;49(8):720-728.
is a defined insult at a specific time, drug nephrotoxic- » Zhou Y, Vaidya VS, Brown RP, et al. Comparison of kidney
ity is a good model for the study of AKI and there is injury molecule-1 and other nephrotoxicity biomarkers in
much ongoing research in this field. urine and kidney following acute exposure to gentamicin,
mercury, and chromium. Toxicol Sci. 2008;101(1):159-170.
NAG is produced by proximal tubular cells in
response to ischemic and oxidative stress. In animal
Biomarkers of Glomerular Disease
studies, increased urinary NAG excretion is a sensi-
tive marker of gentamicin and cisplatin toxicity, with The clinical course of glomerular diseases is vari-
decreasing levels after antioxidant therapy suggesting able and difficult to predict without serial kidney
a potential role for this class of drugs in some cases. biopsies. Treatments generally are toxic, and repeated
Other tubular enzymes also are potential markers of courses of immunosuppression sometimes are indi-
nephrotoxicity. Increased GST-␣ levels suggest proxi- cated in relapsing cases. An ideal biomarker in pa-
mal tubular damage in models of methotrexate- tients with glomerular diseases would be one that
induced kidney damage, and GST-␲ is a marker of gives information about disease activity, prognosis,
distal tubular dysfunction. Levels of GGT, alanine and likelihood of clinical relapse.
aminopeptidase, and lactate dehydrogenase all in- This is particularly relevant in patients with lupus
nephritis. Lupus nephritis is a highly variable disease
crease in the setting of gentamicin and vancomycin
with multiple classes requiring different treatments.
use, in the absence of a decrease in GFR, suggesting a
Patients can move from one class to another, and rates
potential role in monitoring for potential toxicity.
of relapse are high. Biomarkers that could noninva-
Urinary calbindin-D may be a marker of distal renal
sively identify the class of lupus, predict response to
tubular injury caused by cisplatin-based chemo-
therapy, and alert to relapse could revolutionize treat-
therapy. ment. Urinary NGAL has been investigated as a
One study evaluated the performance of a panel of potential biomarker of disease activity in patients with
biomarkers in a model of acute and subacute gentami- lupus. It has been shown to predict kidney flares better
cin toxicity. The panel included urinary cystatin C, than traditional methods without any correlation with
NGAL, KIM-1, ␤2-microglobulin, clusterin, GST-␣, extrarenal disease. Urinary NGAL excretion also was
and osteopontin. Urinary cystatin C and NGAL were higher in patients with lupus nephritis than in those
the most sensitive markers of gentamicin toxicity, with clinical lupus without kidney involvement. A
with level changes appearing within 1 day, whereas recent study examined a panel of biomarkers in a
KIM-1 levels best correlated with histologic appear- diverse group of patients with lupus nephritis and
ance in the subacute model. In another model of found that the combination of urinary NGAL and
gentamicin nephrotoxicity, urinary KIM-1 levels in- monocyte chemotactic peptide 1 (MCP-1) was an
creased within 24 hours of administration, while uri- excellent test of lupus nephritis chronicity, whereas
nary NAG and serum urea nitrogen and creatinine the combination of MCP-1, ␣1-acid glycoprotein, and
levels remained in the normal range. This was despite ceruloplasmin predicted disease activity. MCP-1 is a
histologic evidence of necrosis in half the kidney chemotactic chemokine that is specific for monocytes.
tubules, thus elegantly illustrating the need for more Increased urinary levels have been noted in patients at
sensitive biomarkers than the traditional panel. the time of lupus flares, and the increase can be seen

176 Am J Kidney Dis. 2013;62(1):165-178

Biomarkers in Nephrology

up to 4 months before the clinical flare. A recent pilot serum creatinine level at baseline and fractional
study has suggested that a combination of urinary excretion of IgG was able to classify patients as
proteins and clinical variables could be used to derive responders or nonresponders to therapy in all cases.
a potentially useful composite biomarker panel that One limitation of this study of course was the
reflects specific pathologic lesions, such as tubuloin- requirement for a kidney biopsy. More recently,
terstitial inflammation in lupus nephritis. Currently, urinary IL-18 has been shown to discriminate pa-
no single biomarker can definitively predict a lupus tients who experience progression from those who
flare. Because the treatment is relatively toxic, the remain stable. NGAL and NAG levels are elevated
specificity of any predictive test would have to be in patients with IgA nephropathy and significant
very high to justify its use. It is possible that a suitable tubulointerstitial disease. IL-6 is a cytokine ex-
panel of biomarkers may be developed that will allow pressed by antigen-presenting cells that has been
for this degree of certainty. shown to have elevated levels in urine of patients
Apart from predicting relapses, another potential with active lupus nephritis. Similarly, elevated uri-
benefit of biomarkers would be to noninvasively deter- nary IL-6 levels have been noted in patients with
mine which class of lupus is present in patients with progressive IgA nephropathy relative to controls,
known lupus nephritis. Currently, this is done by suggesting ongoing intrarenal inflammation. Cyto-
examining the urine sediment and quantifying protein- kines including MCP-1 also have been shown to
uria. The presence of red blood cell casts suggests have increased levels in patients with progressive
class III or IV nephritis, whereas heavy proteinuria is disease.
more suggestive of membranous lupus nephritis. This Antineutrophil cytoplasmic antibody (ANCA) dis-
is not an exact science and it is not unusual for a ease is a pauci-immune crescentic vasculitis that
kidney biopsy specimen to be unexpectedly abnormal commonly affects the kidneys and is associated
in a patient with relatively benign urine sediment. A with a high risk of relapse. Active urinary sediment
number of biomarkers are being investigated for their and increasing ANCA titer are traditional biomark-
potential in identifying lupus class. A recent report ers of relapse, but these can be unreliable. Recently,
examined urine metabolomic profiles of patients with a proteome analysis of urine from patients with
different classes of lupus and found that patients with ANCA disease and matched controls found that a
class III/IV lupus had significantly lower taurine lev- panel of 47 biomarkers could reliably distinguish
els than controls and patients with other classes of ANCA disease from other causes of kidney dysfunc-
lupus. Patients with class V lupus had significantly tion and also correlated with disease activity and
lower urinary citrate levels. Urinary CXCR3, inter- response to therapy. Most of these biomarkers were
feron-producing protein 10, transforming growth fac- related to breakdown products of hemoglobin or
tor ␤, and vascular endothelial growth factor also albumin.
were able to differentiate class IV lupus from other The diagnosis and treatment of membranous ne-
classes, with urinary interferon-producing protein 10 phropathy may be aided in the future by the discov-
performing the best. None of these tests are reliable ery that ⬃80% of patients with primary membra-
enough currently to take the place of kidney biopsy at nous nephropathy have IgG4 antibodies to the
present. M-type phospholipase A2 receptor in their serum
IgA nephropathy is the most common glomerulo- that is not present in healthy controls. Levels of this
nephritis and a frequent cause of end-stage renal antibody correlate with disease activity and re-
disease. The clinical course of IgA nephropathy is sponse to treatment. Interestingly, the presence of
very variable; the presence of crescents on a kidney these antibodies in patients prior to transplantation
biopsy specimen suggests a more aggressive dis- does not necessarily predict recurrence, suggesting
ease with a poorer prognosis and mandates the use that there is an interplay of risk factors that predis-
of aggressive immunosuppression. Currently, pa- poses to this disease. Other autoantibodies, includ-
tients are stratified according to the presence of ing antibodies to bovine serum albumin and super-
specific histologic features on kidney biopsy speci- oxide dismutase, are being evaluated for their roles
mens and degree of proteinuria at presentation. in the pathogenesis of idiopathic membranous ne-
Many biomarkers are being investigated to deter- phropathy. Other biomarkers that may have a role
mine their utility in predicting which patients will in understanding the pathogenesis, clinical identifi-
progress to chronic kidney failure and earlier stages cation, and management of patients with nephrotic
of CKD and the overall response to therapy without syndrome include circulating soluble urokinase re-
resorting to follow-up biopsies. One study of pa- ceptor in focal segmental glomerulosclerosis and
tients with crescentic IgA found that combining the podocyte-secreted angiopoietin-like-4 in minimal
number of preserved glomeruli on biopsy with change disease.

Am J Kidney Dis. 2013;62(1):165-178 177

 McMahon and Waikar

Additional Readings » Wei C, El Hindi S, Li J, et al. Circulating urokinase receptor

as a cause of focal segmental glomerulosclerosis. Nat Med.
» Avihingsanon Y, Phumesin P, Benjachat T, et al. Measure-
2011;17(8):952-960.
ment of urinary chemokine and growth factor messenger
» Zhang X, Nagaraja HN, Nadasdy T, et al. A composite urine
RNAs: a noninvasive monitoring in lupus nephritis. Kidney
biomarker reflects interstitial inflammation in lupus nephritis
Int. 2006;69(4):747-753.
biopsies. Kidney Int. 2012;81(4):401-406.
» Bazzi C, Rizza V, Raimondi S, Casellato D, Napodano P,
D’Amico G. In crescentic IgA nephropathy, fractional excre-
tion of IgG in combination with nephron loss is the best FUTURE CHALLENGES
predictor of progression and responsiveness to immunosup- The convergence of basic science investigations
pression. Clin J Am Soc Nephrol. 2009;4(5):929-935.
» Brunner HI, Bennett MR, Mina R, et al. Non-invasive renal
and modern clinical epidemiologic techniques has
protein biomarkers are associated with histological features ushered in an era of discovery and early validation of
of lupus nephritis. Arthritis Rheum. 2012;64(8):2687-2697. a number of novel biomarkers of kidney disease. A
» Haubitz M, Good DM, Woywodt A, et al. Identification and number of challenges face clinicians and scientists
validation of urinary biomarkers for differential diagnosis and before the widespread adoption of novel biomarkers
evaluation of therapeutic intervention in anti-neutrophil cyto-
plasmic antibody-associated vasculitis. Mol Cell Proteomics.
of kidney disease in the clinic. These include the need
2009;8(10):2296-2307. for larger studies with clinically important end points,
» Herrmann SM, Sethi S, Fervenza FC. Membranous nephrop- comparisons of conventional versus novel biomarkers
athy: the start of a paradigm shift. Curr Opin Nephrol for their added clinical utility, correlation of severity
Hypertens. 2012;21(2):203-210. of disease with biomarker levels and duration of
» Pitashny M, Schwartz N, Qing X, et al. Urinary lipocalin-2 is
associated with renal disease activity in human lupus nephri-
elevation, clarification of the treatment implications
tis. Arthritis Rheum. 2007;56(6):1894-1903. from measurement of novel biomarkers, and develop-
» Romick-Rosendale LE, Brunner HI, Bennett MR, et al. ment of reliable assays and point-of-care testing if
Identification of urinary metabolites that distinguish membra- appropriate.
nous lupus nephritis from proliferative lupus nephritis and
focal segmental glomerulosclerosis. Arthritis Res Ther. 2011;
ACKNOWLEDGEMENTS
13(6):R199.
» Rubinstein T, Pitashny M, Levine B, et al. Urinary neutrophil Support: Dr Waikar is supported by grants DK093574,
gelatinase-associated lipocalin as a novel biomarker for DK085660, and DK075941 from the National Institute of Diabetes
disease activity in lupus nephritis. Rheumatology (Oxford). and Digestive Kidney Diseases (NIDDK).
2010;49(5):960-971. Financial Disclosure: Dr Waikar has served as a consultant to
» Stangou M, Alexopoulos E, Papagianni A, et al. Urinary CVS Caremark, BioTrends Research Group, and Takeda; provided
levels of epidermal growth factor, interleukin-6 and mono- expert testimony for GE Heathcare, Northstar Rx, and Salix; and
cyte chemoattractant protein-1 may act as predictor markers received grants from the NIDDK, Otsuka, Merck, Genzyme, and
of renal function outcome in immunoglobulin A nephropathy. Satellite Healthcare. Dr McMahon declares that he has no relevant
Nephrology (Carlton). 2009;4(6):613-620. financial interests.

178 Am J Kidney Dis. 2013;62(1):165-178