This document discusses biomarkers for the early diagnosis of acute kidney injury (AKI). It notes that current diagnostic criteria based on serum creatinine and urine output can lead to delayed diagnosis in intensive care unit patients. Emerging urine and plasma biomarkers like neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), cystatin C, and the combination of tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) can provide earlier detection of AKI before functional changes occur. The combination of TIMP-

1. BIOMARKERS IN ACUTE
KIDNEY INJURY
Dr MÜGE AYDOĞDU
GAZİ UNIVERSITY MEDICAL FACULTY
PULMONARY DISEASES DEPARTMENT AND CRITICAL CARE UNIT
13.10.2014

2. Acute Kidney Injury (AKİ):
Epidemiology
• AKI occurs in 5% of hospitalised patients and in
30-50% of critical care patients * ;
• Incidence is increasing with extensive ICU
procedures!!!!
• Mortality among the ICU patients with AKI
requiring RRT is; > %50*.
• Among the ICU patients receiving RRT; CKD or
ESRD develops with in 3-5 years*
*Cole vd., 2000; Gursel ve Demir, 2006; Hoste vd., 2006; KDIGO, 2012;
Uschino vd., 2005

3. AKI-Why Early Diagnosis?
• AKI can be reversible when diagnosed earlier.
• With eary diagnosis;
• It will be possible to start treatment earlier
• Nephrotoxic procedures or medications can be avoided

4. AKI-Why Early Diagnosis?
Hoste et al. Crit Care 2006; 10(3): R73

5. AKI- Problems of Diagnosis in ICU
• Etiology of AKI is multifactorial (nephrotoxic
drug use, sepsis, ischemia etc)
RIFLE ve AKIN diagnostic criteria depend on
serum creatinin and urine output levels
These will lead to misdiagnosis or delayed
diagnosis in ICU

7. AKI: Problems of Early Diagnosis
Serum Creatinin Caused Delayed Diagnosis

8. AKI: Problems of Early Diagnosis
Serum Creatinin Caused Delayed
Diagnosis

9. AKI: Problems of Early Diagnosis
 Factors decreasing
creatinin levels:
 Muscle mass and activity
 Hepatic failure
 Sepsis
 Body weight
 Age
 Sex
 Gender
 Protein intake
 Excess volume
 Pregnancy
 IADH
 Blood transfusion
 Factors increasing
creatinin levels :
 Trauma, rhabdomyolisis
 Excessive protein intake
 Fever
 Immobilisation
 Drugs (TMP, Cimetidine,
Triamterene, Probenecide,
Spironolacton, Amiloride)

10. AKI: Problems of Early
Diagnosis
• BUN
• Low molecular weight,water soluble by-product of protein
metablolism
• Serum marker of uremic solute retansion and elimination.
• Not a sensitive marker of renal injury
• Increased BUN levels with extrarenal factors :
• High protein diet
• Critical illness (sepsis, burn, trauma)
• GI bleeding
• Glucocorticoid treatment and tetracycline use
• Decreased BUN levels with extrarenal factors
• Chronic hepatic disease
• Low protein intake

11. AKI: Problems of Early Diagnosis
Oliguria
• At least 6 hours are necessary for detection of oliguria
• In most of the patients excretory function of kidneys are
not disturbed during AKI
• Misdiagnosis can be made by just looking oliguria in acute
tubular injury
• Trend should be followed in ICU
• Has low sensitivity and specificity

12. AKI-Clinical Continuity

13. Normal
İnjury ????
GFR↓
SCr ↑
Renal
Failure

14. AKI Biomarkers :Why Necessary?
• To make earlier diagnosis of structural abnormalities during
renal injury before funtional abnormalities take place
• To make differential diagnosis of reversible AKI and CKD
• Risk classification and triage
• To start treatment and to take preventive measure as soon as possible
• To define indications and criteria for future therapies.
• To identify prognosis (dialysis, death, ICU and hospital stay)
• Similar to Troponin and BNP
• Monitorization of treatment response

15. How Biomarkers Are Developed?
• Phase 1-Discovery Phase
• To find the candidate biomarkes and to evaluate the
biological competence by using scientific technologies
• Gene analysis (cDNA microarray ve NextGen
sequencing)
• Protein analysis (Proteomics)
• Phase 2-Translational Phase
• To define and develop the assay methods, tests for
candidate biomarkers.
• Phase 3-Validation Phase
• To evalute the prognostic value and clinical applicability
of the biomarker with large clinical trials.
Devarajan, Biomarkers Med 201; 4: 265-80

16. How can be the ideal biomarker?
1. Noninvasive
2. Easy, rapid result, cheap, can be studied in an already
present sample like urine
3. High sensitivity and specificity
4. Rapid and reliable rise secondary to renal injury
5. Correlated with the level of renal injury
6. Provide prognostic information and risk classification
7. Specific to kidneys
8. Can be applied to differen populations.
9. Can show possible causes of renal injury (Prerenal,
intrarenal, postrenal)
10. Stabile during time, at different temperatures and pHs
11. Not affected from other drugs

18. AKI –Biomarker Studies
In recent years many studies have being performed for
AKI early diagnosis, both from urine and plasma
samples.
[ serum Cystatin C, urine IL-18 and neutrophil gelatinase
associated lipocalin (NGAL), urine KIM-1(kidney injury marker-1)
and urine L-FABP (liver fatty acid binding protein) ]

20. AKI Biomarkers

22. Neutrophil gelatinase associated
lipocalin-2 (NGAL)
• 25 kDa protein, covalenty bound to neutrophil
gelatinase
• Expressed at low levels in lungs, stomach, colon
and renal proximal tupular epithelial cellls
• It was found to be increasing so rapidly with in 2 hours
after an ischemic insult in (Mishra et al, 2003)
• In 2005, Mori et al identified that in AKI developing in
ICU patients, plasma NGAL levels increase 10 times,
and urine NGALlevels increase 100 times when
compared with non AKI controls.

24. Woo. K. Han Currrent Biomarker Findings 2012

25. Woo. K. Han Currrent Biomarker Findings 2012

26. Extrarenal Factors for NGAL Rise:
 Systemic stress
 CKD
 Acute bacterial infection, sepsis
 Gastric malignity
 Pancreas carcinoma
 Endometrial hyperplasia
 Down Syndrome
 Pelvic inflamatory disease, inflam. Bowel disease
 Glioma
 Esential thrombocytemia, policytemia vera
 CML with molecular remission
 ST segment elevation MI, as a prognostic indicator
 Acute vs stabile coronary arterial disease

27. NGAL
 Valuable for detecting subclinical AKI
 Does sublinical AKI affects long term prognosis????
 Not an ideal biomarker due to low specificity

29. Serum Cystatin C
• Cystatin C; low molecular weight endogenous systein
proteinase inhibitor.
• Cystatin C; can be synthesized and released to plasma at a
stabile rate from all nucleated cells in the body
• Not affected from the age, gender, muscle mass and diet
protperties of the patients .
• Superior to serum creatinine in predicting GFR and in follow
up of renal functions. *
*Dhamidharka VR et al. Am J Kidney Disease 2002; 40: 221-6
*Coll E et al. Am J Kidney Disease 2000; 36: 29-34

30. Serum Cystatin C
• Has better performance in predicting GFR in some
special patient groups;
• Elderly
• Children
• Renal transplant patients
• Cirhosis
• Malnutrition
• Serum Cystatin C, when compared with creatinin more
sensitive to mild and earlier renal dysfunction

31. Serum Cystatin C
• In a small cohort study of critically ill patiens; rise in
Cystatin C levels can be identified 1-2 days before the rise
in serum creatinine (> 50% of the v-baseline) in AKI
patients.
• Due to developing standart immunonephelometric assays
serum Cystatin C is being used more commonly in our
daily practice and gives rapid results.
*Herget-Rosenthal S. et al. Kidney Int 2004; 66: 115-1122

32. Serum Cystatin C
• Among a large cross sectional study with 8058 patients some
other factors were found to be associated with increased
serum Cystatin C levels*;
• Old age,
• Male gender,
• Being tall and overweight,
• Active smoking,
• Increased CRP levels
• Cystatin C affected at the same time from**;
• Thyroid functional abnormalities
• Immunosuppressive treatment
• Systemic inflammation
*Knight EL et al. Kidney Int 2004; 66: 1115-22
**Manetti L et al. Clim Chim Acta 2005; 356:227-228
Manetti L et al. J Endocr Invest 2005; 28:346-349

33. Urine Cystatin C
• Cystatin C; also identified in urine of AKI patients. This
can be used for evaluating the severity of tubular injury..
• In a small prospective study among the critically ill
patients, increased urine Cystatin C levels were identified
to be more successfull in predicting the need for RRT
than other urinary biomarkers*
* Herget Rosenthal S et al. Clin Chem 2004; 50:552-558

35. Woo. K. Han Currrent Biomarker Findings 2012

37. Woo. K. Han Currrent Biomarker Findings 2012

48. AKI Biomarkers
• TIMP-2;
• Matrix metalloproteinase inhibitor
• Inhibits endothelial cell proliferation directly .
• IGFBP7:
• Stimulates cell adhesion, plays a role in cell cycle arrest

49. AKI Biomarkers
TIMP2 ve IGFBP7
•IGFBP7 (Insulin Like Growth Factor
Binding Protein-7) & TIMP-2 (Tissue
Inhibitor of Metalloproteinase-2);G1 cell
cycle arrest markers showing early
cellular injury
•After injury renal tubular cells enter a
short period of G1 cell cycle arrest
•G1 cell cycle arrest is a kind of
protective mechanismi preventing the
division and multiplication of injured
DNA
•IGFBP7 & TIMP-2 at the same time
plays a role of “alarm” released by
otocrine and paracrine ways from the
site of injury Kashani et al. Critical Care 2013, 17:R25

50. If no new injury occurs
and if sepsis treated
properly cyclin/CDK4
complex will allow cell
cycle to continue into
G1/S, and will lead to renal
In the earlier period of
sepsis , p53 ve p21
proteins will stop cell
cycle, and together with
released IGFBP-7 ve TIMP-2
cause the G1 cell cycle
cellular regeneration
arrest.

51. AKI Biomarkers
Urine IGFBP-7 ve TIMP-2 first studied in;
 Early diagnosis of urinary system malignancies
 Animal studies
 Sapphire Study of Kashani K et al. in identifying AKI in early
period of AKI in septic ICU patients
*Are g g e r F, Ue hling e r DE, Wito ws ki J, Brunisho lz RA, Hunz ike r P, Fre y FJ, Jö rre s A. 2 0 1 3 .
“Id e ntific a tio n o f IGFBP-7 by urina ry p ro te o m ic s a s a no ve l p ro g no s tic m a rke r in e a rly a c ute
kidne y injury ”. Kid ne y Inte rna tio na l; 8 5 : 9 0 9 -9 1 9
**Biho ra c A, Chawla L, Shaw AD, AL-Kha fa ji A, Da vis o n DL, De Muth GE, e t a l. 2 0 1 4. “Va lid a tio n o f
c e ll c y c le a rre s t bio m a rke rs fo r a c ute kidne y injury us ing c linic a l a d jud ic a tio n”. Am J o f Re s p ira to ry
a nd Critic a l Ca re Me d ic ine ; 1 8 9 (8 ): 9 3 2 -9 3 9 .
***Ka sha ni K, Kha fa ji AA, Ard ile s T, Artig a s A, Ba g shaw SM, Be ll M, e t a l. 2 0 1 3 . “Dis c o ve ry a nd
va lid a tio n o f c e ll c y c le a rre s t bio m a rke rs in hum a n a c ute kidne y injury ”. Crit Ca re ; 1 7 : R2 5

52. AKI Biomarkers
• Kashani K et al., evaluated the predictive value of
TIMP2.IGFBP-7 in AKI development with in 12 hours of
admission among the urine samples obtained in first 24
hours of admission to ICU.
• An additive affect was identified when urine TIMP2 and
IGFBP7 were evaluated together; AUC was 0.80
(This was 0.76 ve 0.79 when these parameters
were evaluated by themselves)).
Kashani et al. Critical Care 2013, 17:R25

53. AKI Biomarkers
Urine NGAL (ng/mL) [TIMP-2]•[IGFBP7] ((ng/mL)2 /
1000)
Kashani et al. Critical Care 2013, 17:R25

54. AKI Biomarkers
Urine KIM-1 (ng/mL) [TIMP-2]•[IGFBP7] ((ng/mL)2 / 1000)
Kashani et al. Critical Care 2013, 17:R25

55. AKI Biomarkers
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Risk for AKI (KDIGO Stage 2 -3)
A
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.01 0.1 1 10 100
Risk for MAKE30
[TIMP-2]•[IGFBP7] ((ng/mL)²/1000)
B
KDIGO 2-3 in
12h
MAKE30 (30 days) MAKE30
Death
RRT
Persistently elevated sCr
(2x over baseline)
Kashani et al. Critical Care 2013, 17:R25

56. AKI Biomarkers
• With the combination of two tests TIMP2-
IGFBP7 (NEPHROCHECK® Test) early
dignostic tool was developed and a
threshold level was searched for the early
diagnosis of AKİ;
• <0.3 (ng/ml)2/1000 low risk for (KDIGO 2-3) AKİ
• 0.3-2 (ng/ml)2/1000 high risk,
• > 2 (ng/ml)2/1000 highest risk
• < 0.3 (ng/ml)2/1000 cutt of level has a
sensitivity of 0.88 and a negative predictive
value of 0.94 for the exclusion of KDIGO 2-
3 AKI development with in first 12 hours.
Kashani et al. Critical Care 2013, 17:R25

57. AKI Biomarkers
IGFBP-7 in normal, sepsis ve
septic AKİ groups
TIMP-2 in normal, sepsis and ve
septic AKI groups
Normal Sepsis Sepsis+AKI
25
20
15
10
5
0
IGFBP-7 2,4 5,8 23,1
IGFBP - 7 ng/ml
Normal Sepsis Sepsis+AKI
700,00
600,00
500,00
400,00
300,00
200,00
100,00
0,00
TIMP-2 593,00 478,00 527,00
TIMP - 2 pg/ml
Aydogdu M ve ark. Septik Akut Böbrek Yetmezliği Erken Tanısında İdrar Hücre Siklusu Duraklama
Belirteçleri:TIMP-2 ve IGFBP-7'nin Yeri. 11.DCYB Kongresi Sözlü Sunum(SS001)

58. Figure 3- ROC curve for the
prediction of AKI with urine
IGFBP-7 (AUC: 0.83)
IGFBP-7; for 3.5ng/ml cut off level;
sensitivity %80, specificty %76
Area Under the Curve
Test Result Variable(s): ýgfbp7
Area Std. Errora
Asymptotic
Asymptotic 95% Confidence
Interval
Sig.b Lower Bound Upper Bound
,830 ,046 ,000 ,741 ,920
The test result variable(s): ýgfbp7 has at least one tie between the
positive actual state group and the negative actual state group. Statistics
may be biased.
Under the nonparametric a. assumption
b. Null hypothesis: true area = 0.5
Aydogdu M ve ark. Septik Akut Böbrek
Yetmezliği Erken Tanısında İdrar Hücre Siklusu
Duraklama Belirteçleri:TIMP-2 ve IGFBP-7'nin
Yeri. 11.DCYB Kongresi Sözlü Sunum(SS001)

60. TAKE HOME MESSAGES…
• Early diagnosis of AKI is so important inorder to prevent
morbidity and mortality.
• There are many biomarkers being studied, but no ideal
biomarker has yet been defined.
• New cell cycle arrest markers are promising but the
studies are not enough
• Whatever biomarker is preferred, it should be evaluated
together with the clinic of the patient…

61. Thank You…