AKI Biomarker 2016 Chaken

Conceptual model for AKI
KDIGO AKI Guideline 2012
Limitation of Serum
creatinine
Not specific for AKI
Typically diagnosis is 24-72 hours following AKI
Rising creatinine only when lost 50% of their
functional capacity
Surrogate marker of GFR, not tubular function
Not allow differentiation between hemodynamically
mediated changes, such as pre-renal azotemia from
intrinsic renal failure or obstructive uropathy
Lisowska-Myjak B. Blood Purif 2010;29:357365

Endre ZH, et al. Clin Biochem Rev 2011 I 121-4
Sources of creatinine
error in GFR estimation
Source of error
Factors affecting
creatinine generation
Example
Race/ethnicity
Extremes of muscle
mass
Extremes of body size
Diet and nutritional
status
- High protein diet
- Creatine
supplements
Muscle wasting
diseases
KDIGO CKD 2012. Kidney International Supplements
Ingestion of cooked
Sources of creatinine
error in GFR estimation
Source of error
Factors affecting
tubular secretion of
creatinine
Example
Decrease by drug-induced
inhibition
Trimethoprim
Cimetidine
Fenofibrate
Factors affecting extra- Dialysis
renal elimination of
Decrease by inhibition of
creatinine
gut creatininase by
antibiotics
Increased by large
volume losses of
KDIGO CKD 2012. Kidney
International
Supplements
extracellular
fluid
(2013) 3, 514
E me r g i n g
B i oma r k e
rs
Anatomical correlation of AKI biomarker
Koyner JL et al. nd critical illness. Clin J Am Soc Nephrol.

2013; 8(6): 103442
Type of AKI biomarkers

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin
Up-regulated proteins
Kidney injury molecule 1 (KIM-1)

Neutrophil gelatinase-associated lipocalin
(NGAL)
Liver-type fatty acidbinding protein (LFABP)
Interleukin 18 (IL-18)
-trace protein (BTP)
Asymmetric dimethylarginine (ADMA)
Low-molecular-weight
proteins
Serum and Urine cystatin C

N-acetyl-glucosaminidase (NAG)
Glutathione-s-transferase (GST)
Gamma-glutamyl transpeptidase (GGT)
Alanine aminopeptidase (AAP)
Lactate dehydrogenase (LDH)
Enzymes
Gene product (cell cycle

arrest)
TIMP2
IGFBP7
Adapted from Betjes MG,,et al. Clin Kidney J. 2012; 5 (2): 102-10
Cystatin C
MW: 13.3 kDa
Freely filtered by glomerulus, reabsorbed
completely by PCT, and not secreted
Early marker of impaired glomerular filtration
rather than of tubular lesion
Urinary excretion of the cystatin C : correlates
with severity of acute tubular damage
Serum cystatin C : early marker of impaired
GFR, not significantly affected by age, gender,
race, or overall muscle mass
Coca SG, et al. Kidney Int 2008; 73: 10081016.
Nguyen MT, et al. Pediatr Nephrol 2008; 23:
Serum cystatin C and

creatinine on the three days
prior on the day AKI
In 85 critically ill patients at high risk for
developing
AKI
250
200
Cystati
nC
150
100
50
0
R-Day-3
R-day-2
R-Day-1
R-Day-0
Serum cystatin C increased already

by50% 1.50.6 days earlier compared
to creatinine Adapted from Herget-Rosenthal S. Kidney Int 2004;
Urine CyC/urine creatinine (mg/g)
Urinary cystatin C as an early

biomarker of AKI following
adult cardiothoracic surgery
5.0-
**
**
4.0-
AKI with RRT
3.0-
2.01.0-
NO AKI
0.0Pre CPB
Post CPB
ICU
6 h ICU
Day 1
Urinary CyC at the time of ICU arrival and the urinary

CyC level 6 hours after ICU admission were most useful
for predicting
Koyner JL, AKI
et al. Kidney Int 2008; 74: 10591069
Serum CystacinC : prognostic

factor to death, CV death, ESRD
Michael G. Shlipak, N Engl J Med2013;369:932
Sources of cystatin C error

in GFR estimation
Source of error
Example
Factors affecting cystatin C

generation
Disorders of thyroid function

Administration of
corticosteroids
Other hypothesized factors
based on epidemiologic
associations (diabetes,
adiposity)
Factors affecting tubular

reabosrption of cystatin C
None identified
Factors affecting extra-renal

elimination of cystatin C
Increased by severe
decrease in GFR
No effect by gender, age, race or muscle

mass, unlike serum creatinine
KDIGO CKD 2012. Kidney International Supplements

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Kidney Injury Molecule-1

(KIM-1)
Type 1 transmembrane protein of renal tubular
origin
KIM-1 is not detectable in a healthy kidney
KIM-1-positive proximal tubules (90%) in
various human renal diseases)
Ichimura T, et al. Am J Physiol Renal Physiol 2004; 286:F552F563.

Waanders F, et al. Journal of Pathology 2010; 716.
Urinary KIM-1: sensitive

biomarker for early detection of
kidney injury
Plasma creatinine
KIM-1
*
4
mg/dL
ng/mL
2
1
sham
I10
I20
I30
I45
Ischemia/Reperfusion
sham
I10
I20
I30
I45
Ischemia/Reperfusion
At I20 after ischemia, there was increase in the urine Kim-1,

whereas other routinely used biomarkers measured lacked
the sensitivity.
Adapted from Vaidya
VS, et al. Am J Physiol Renal Physiol 2006; 290:F517
KIM-1 level, ng/mL
Comparison of urinary KIM-1

concentration in various
forms of renal diseases
N=7
N=7
N=9
N=9
N=8
Urinary KIM-1 levels were higher in patients with

ischemic ATN (2.920.61; N=7) compared to levels in
patients with other forms of AKI (0.630.17, P< 0.01) or
CKD (0.720.37, P <0.01).
Adapted from Kidney International, Vol. 62 (2002),
Preoperative cardiac surgery

KIM-1 predicted development of
stage 1 and 3 AKI (subclinical
proximal tubular injury) N=123
Koyner JL,et al Clin J Am Soc Nephrol. 2010 Dec;
Macrophages
(number/interstitial flield)
Alpha-SMA expression
Macrophage expression
KIM-1 and tubulointerstitial

damage
100755025-
R=0.950
p<0.001
00
1
2
3
4
KIM-1 expression (% area)
Kim-1 expression was limited to

areas with inflammation and
brosis (-smooth muscle actin)
van Timmeren et al.Am J Physiol Renal Physiol2006;291(2):F456464.
Urinary KIM-1 markers of kidney injury

can predict adverse clinical outcomes in
patients with AKI
Third quartile of KIM-1

groups: 1.4-fold (95%
CI 0.6 to 3.0)
Forth quartile of KIM-1
groups: 3.2-fold (95%
CI 1.4 to 7.4)
Predicted prbability of dialysis

requirement or hospital death
Second quartile of KIM1 groups: 1.4-fold

(95% CI 0.6 to 3.0)
Dialysis requirement or hospital

death
(P =0.034)
0.80.70.60.50.40.3-
5
10
15
20
25
Urinary KIM-1 (ng/mg creatinine)
30
Liangos O, et al. J Am Soc Nephrol 2007; 18:
KIM-1 : prognostic factor

to ESRD
KIM-1 over-expression reduces kidney fibrosis
and development of ESRD
Persistent KIM-1 elevation in blood, indicates
ongoing tubular injury, which would be risk for
CKD/ESRD
Humphreys BD, et al:. J Clin Invest 2013;123:4023-

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Neutrophil GelatinaseAssociated Lipocalin (NGAL)

NGAL is expressed by neutrophils
Major renal source of urinary NGAL is
from the TAL and collecting ducts
Uttenthal O. Clin Lab Internat 2005; 29: 3941.
NGAL gene is strongly induced after

kidney injury by ischemia, sepsis or
nephrotoxins
Neutrophil GelatinaseAssociated Lipocalin

(NGAL)
During AKI, both urine and plasma NGAL are

elevated
Apoptosis inhibition and increasing proliferation
of tubule NGAL can up-regulate heme
oxygenase-1, which preserves proximal tubule
N-cadherin, and subsequently inhibits cell
death
Urine NGAL: production from TAL and CCD 3
hrs after injury
Plasma NGAL: increased hepatic production
after injury
Yamamoto T, et al.J Am Soc Nephrol. 2007 Nov.
18(11):2894-902
TRIBE AKI
Large prospective, multicenter international
cohort of adult patients undergoing cardiac
surgery (N = 1219)
What biomarkers measured at time of first
clinical diagnosis of AKI after cardiac surgery can
potentially predict AKI severity
Koyner JL, et al. J Am Soc Nephrol. 2012 May.

23(5):905-14.
plasma NGAL performing the best

(category-free net reclassification
improvement of 0.69, P < 0.0001).
Plasma NGAL on day of AKI
diagnosis improve risk stratification
and identify patients at higher risk
for progression of AKI and worse
patient outcomes
Koyner JL, et al. J Am Soc Nephrol. 2012 May.
23(5):905-14.
NGAL expression in
ischmia-reperfusion mice
Neal Paragas, et al Nat Med. 2011 Feb; 17(2):
Volume depletion failed

to activate NGAL
expression
Neal Paragas, et al Nat Med. 2011 Feb; 17(2):
20 children (28%) developed acute renal injury,

but diagnosis with serum creatinine was only
possible 1-3 days after cardiopulmonary
bypass
J. Mishara et al. Lancet 2005; 365: 12313
NGAL in urine at 2 h after cardiopulmonary

bypass was the most powerful independent
predictor of acute renal injury
J. Mishara et al. Lancet 2005; 365: 12313
Major studies reporting use of

NGAL as an early biomarker in
AKI
Soni SS, et al. Blood Purif 2009;28:165174
Major studies reporting use of

NGAL as an early biomarker in
AKI
Soni SS, et al. Blood Purif 2009;28:165174
Early diagnosis of cardiac surgeryassociated AKI : a meta-analysis

24 studies from
2004-2016
diagnostic
accuracy of
NGAL in early
(within 12 h
postoperatively
) diagnosis of
CSA-AKI
Zhou F,. Eur J Cardiothorac Surg 2016;49:74655.
NGAL as a Biomarker in
AKI
Reliable early biomarker for

AKI
Correlation with risk factors
for AKI
Reliable predictor of severity
and duration of AKI, length of
hospital stay, RRT
requirement and mortality
Dent CL, et al. Crit Care 2007; 11:R127.

Bachorzewska H, et al. Nephrol Dial Transplant 2007; 22: 295296.
Wagener G, et al. Anesthesiology 2006;105: 485491.
Xin C, : et al. Ren Fail 2008; 30: 904913.
NGAL and Progression

of CKD
Bolignano D,. Clin J Am Soc Nephrol 2009;4:337-344
Factors Influencing
NGAL Measurement
Urinary NGAL levels were
signicantly elevated in all patients
(with or without AKI)
initiates inflammation and activation of
neutrophils leading to increased NGAL
levels.
NGAL concentrations may be

influenced by a number of
coexisting variables such as
systemic or urinary tract infections.
Wagener G, et al. Am J Kidney Dis 2008; 52: 425433.

Mitsnefes M, et al. Pediatr Nephrol 2007;22:1018.

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Liver-Type Fatty Acid-Binding Protein

(L-FABP)
14-kD protein expressed in proximal tubules
Urinary L-FABP increase immediately and peak
within 6 h rafter tubular injury and correlate
strongly with renal ischemic time
L-FABP increase in patients with known renal
disease risk factors of hypertension and
diabetes in the absence of overt kidney damage
The exact function of L-FABP is yet to be fully
elucidated.
Considered as renal protective protein
(antioxidant properties)
Tan H.L. et al Blood Purif 2016;41:144-150
Urinary LFABP in AKI: A Meta-analysis
P.Susantitaphong,Am J Kidney Dis. 2013 Mar; 61(3):

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Interleukin-18 (IL-18)
22-kD pro-inflammatory cytokine formed in proximal
tubular cells.
Urinary IL-18 is elevated following renal injury
Caspase-1 activation, resulting in IL-18 maturation.
Mature IL-18 mediates inflammatory response through
upregulating NF-kB pathway including TNF-, iNOS,
chemokines attracting microphage and neutrophils
IL-18 worsens tubular necrosis in ischemic-reperfusion
and nephrotoxin animal models
AKI patients with high urine IL-18 concentration could
potentially benefit from anti-IL-18 therapy
Tan H.L. et al Blood Purif 2016;41:144-150

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Urinary Enzymes of Renal

Origin
Enzymes released from damaged
tubular cells and excreted into
urine as markers of kidney damage
Distinct locations in the cell
internal structure
Cytoplasm
Lysosomes
Cell membrane
Urinary Enzymes of Renal

Origin
Cytoplasmic enzymes: Isoenzymes of glutathione
S transferase (GST):
GST- : proximal tubule
GST- : distal tubule
Lysosomes of proximal tubular cells: N-acetyl- D-glucosaminidase (NAG)

Brush border enzyme: Alkaline phosphatase, glutamyl transpeptidase, and alanine
aminopeptidase
Proximal renal tubular epithelial antigen (HRTE-1)
N-acetyl- -D
-glucosaminidase (NAG)
Lysosomal enzyme of proximal
tubular epithelial cells
Specic urinary marker for the
tubular cells
Relatively high molecular weight
(>130 kDa)
Increase in urinary NAG activity
Damage to tubular cells
Increased lysosomal activity
without cellular damage
Tubular enzymuria facilitates early

detection of acute renal impairment in
the intensive care unit
NAG index
1.0
Sensitivity
Indexed to urinary
creatinine
concentration, (GT),
alkaline
phosphatase,and
NAG were higher in
the AKI group on
admission (P<0.05).
0.8
0.6
0.4
0.2
ROC plot analysis

0.0
0
0.2
0.4
0.6
0.8
1.0
showed NAG had
1-Specificity
excellent
discriminating power
for AKI (AUC 0.845).
Westhuyzen J, et al. Nephrol Dial Transplant (2003) 18: 543
Urinary NAG
increased in
tandem with
APACHE II and
Multiple Organ
Failure scores.
Urinary NAG
markers of
kidney injury can
predict adverse
clinical outcomes
in patients with
Predicted prbability of dialysis

requirement or hospital death
Urinary NAG Activity Are

Associated with Adverse
Outcomes in AKI
0.80.70.60.50.40.30
50
100
150
200
250
300
Urinary NAG (ng/mg creatinine)
N-acetyl- -D
-glucosaminidase (NAG)
More sensitive diagnostic marker
than tubular proteinuria
Enzymes of renal tubules for the
detection and monitoring of kidney
lesions in patients with AKI in the
earliest phase of the disease
Limitation
Very low thresholds for release of
tubular enzymes
Technical difficulty in the

Biomarker Type
Biomarker
Functional marker
Serum creatinin
Serum cystatin C
Urine albumin

(NGAL)
proteins
Urine cystatin C
Enzymes

arrest)
TIMP2
IGFBP7
Urinary Tissue Inhibitor of Metalloproteinase-2

Insulin-Like Growth Factor-Binding Protein 7
TIMP-2 and IGFBP-7 are inducers of G1 cell
cycle arrest of renal tubular cells, which occurs
during the early period of cell injury caused by
ischemic or inflammatory processes
A. Sharfuddin et al. Nat Rev Nephrol 2011;7:1
TIMP-2 and IGFBP-7
A. Sharfuddin et al. Nat Rev Nephrol 2011;7:189-200
SAPPHIRE study :
methodology
728 ICU pts: 35 clinical sites in North America
and Europe
critical illness and without evidence of AKI at
enrollment
1oendpoint
stage 2-3 AKI by KDIGO within 12

hours of sample collection.
9 novel marker tested compare with traditional
markers
Kianoush Kashani et al Crit Care. 2013; 17(1): R2
SAPPHIRE STUDY
[TIMP-2][IGFBP7]
identified risk
Improved risk
stratification for
AKI well ahead of
Cr, UOP
These markers
performed well in
patients with
sepsis (with area
under receiver
operating
characteristics
curve [AUC] of
0.82)
postsurgery (AUC
0.85)
SAPPHIRE STUDY
Risk for major
adverse kidney
events (MAKE)
- Death
- Dialysis
- Persistent renal
dysfunction within
30 days (MAKE30)
elevated sharply for
[TIMP-2][IGFBP7]
above 0.3 and
doubled when values
In September 2014 US FDA allowed the

marketing of a commercial test,
NephroCheck, to help determine
critically ill patients are at risk of
developing moderate to severe acute
AKI in 12 hrs after testing
(TIMP-2) (IGFBP7) Levels Are Associated with

Long-Term Outcomes in Patients with AKI
At 9th month
TIMP2 IGFBP7
levels > 0.3
associated with
death or RRT only
in subjects who
developed AKI
Jay L. Koyner, J Am Soc Nephrol 26:, 2015
Biomarkers of AKI and cardiac

surgery
Biomarkers of AKI and outcomes in

critically ill

cardiorenal syndrome

kidney transplantation
Future of biomarker-assisted
approaches for AKI management
Conrm proposed expansion of diagnostic AKI criteria
Determine mechanistic pathways
Dene prognostic value of combined use of functional
and damage markers in sequential measurements
Combination of damage and functional markers can
improve recognition of AKI in the setting of CKD
Large population-based studies
Establish standard techniques for collection, handling
and presentation of biomarker data that permits
appropriate interpretation across settings.
Patrick T. Murray, et al, Kidney Int. 2014 March ; 85(3):
Proposed new AKI

criteria
Functional
criteria
Damage criteria
Patrick T. Murray, et al, Kidney Int. 2014 March ; 85(3): 513
Modied conceptual
model of AKI
Patrick T. Murray, et al, Kidney Int. 2014 March ; 85(3): 51
Thank you for your

attention

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Conceptual model for AKI

KDIGO AKI Guideline 2012

Lisowska-Myjak B. Blood Purif 2010;29:357365

Anatomical correlation of AKI biomarker

Koyner JL et al. nd critical illness. Clin J Am Soc Nephrol.

Type of AKI biomarkers

Kidney injury molecule 1 (KIM-1)

Serum and Urine cystatin C

Gene product (cell cycle

Serum cystatin C and

Serum cystatin C increased already

Urine CyC/urine creatinine (mg/g)

Urinary cystatin C as an early

AKI with RRT

Urinary CyC at the time of ICU arrival and the urinary

Serum CystacinC : prognostic

Michael G. Shlipak, N Engl J Med2013;369:932

Sources of cystatin C error

Factors affecting cystatin C

Disorders of thyroid function

Factors affecting tubular

Factors affecting extra-renal

No effect by gender, age, race or muscle

Type of AKI biomarkers

Kidney injury molecule 1 (KIM-1)

Gene product (cell cycle

Kidney Injury Molecule-1

Ichimura T, et al. Am J Physiol Renal Physiol 2004; 286:F552F563.

Urinary KIM-1: sensitive

At I20 after ischemia, there was increase in the urine Kim-1,

KIM-1 level, ng/mL

Comparison of urinary KIM-1

Urinary KIM-1 levels were higher in patients with

Adapted from Kidney International, Vol. 62 (2002),

Preoperative cardiac surgery

Koyner JL,et al Clin J Am Soc Nephrol. 2010 Dec;

KIM-1 and tubulointerstitial

Kim-1 expression was limited to

Urinary KIM-1 markers of kidney injury

Third quartile of KIM-1

Predicted prbability of dialysis

Second quartile of KIM1 groups: 1.4-fold

Dialysis requirement or hospital

Liangos O, et al. J Am Soc Nephrol 2007; 18:

KIM-1 : prognostic factor

Humphreys BD, et al:. J Clin Invest 2013;123:4023-

Type of AKI biomarkers

Kidney injury molecule 1 (KIM-1)

Gene product (cell cycle

Neutrophil GelatinaseAssociated Lipocalin (NGAL)

Uttenthal O. Clin Lab Internat 2005; 29: 3941.

NGAL gene is strongly induced after

Lisowska-Myjak B. Blood Purif 2010;29:357

Neutrophil GelatinaseAssociated Lipocalin

During AKI, both urine and plasma NGAL are

Koyner JL, et al. J Am Soc Nephrol. 2012 May.

plasma NGAL performing the best

Neal Paragas, et al Nat Med. 2011 Feb; 17(2):

Volume depletion failed

Neal Paragas, et al Nat Med. 2011 Feb; 17(2):

20 children (28%) developed acute renal injury,

J. Mishara et al. Lancet 2005; 365: 12313

NGAL in urine at 2 h after cardiopulmonary

J. Mishara et al. Lancet 2005; 365: 12313