NGAL

1. Acute Kidney InjuryAcute Kidney Injury
Current status & emerging scopeCurrent status & emerging scope

2. Introduction to AKIIntroduction to AKI
Delay in diagnosis and initiation of therapy that
may prevent nephron loss
AKI secondary to ischemic injury
Common & potentially devastating problem
A persistently high rate of mortality despite
significant advances in supportive care
Outcome
Why?

3. AKI – diagnostic criteriaAKI – diagnostic criteria
Mehta et al. Critical Care 2007 11:R31

4. AKI – Clinical stagingAKI – Clinical staging
Mehta et al. Critical Care 2007 11:R31

5. current
simple test
for renal
status
AKI - MarkersAKI - Markers
Contemporary markersEffective
tests but
inadequate
for early
inference
Emerging Biomarkers
Albumin Urea Creatinine
Cystatin C KIM-1 IL-18 NGAL
eGFR

6. What is the current simpleWhat is the current simple
test?test?
 In individuals with diabetes:In individuals with diabetes:
– ““Spot” urine albumin to creatinine ratioSpot” urine albumin to creatinine ratio
 In others at risk:In others at risk:
– ““Spot” urine albumin to creatinine ratio ORSpot” urine albumin to creatinine ratio OR
standard dipstickstandard dipstick (Bouleware, et al., 2003)(Bouleware, et al., 2003)
– Estimate GFR from serum creatinine usingEstimate GFR from serum creatinine using
the MDRD prediction equation.the MDRD prediction equation.
*24 hour urine collections are NOT needed. Diabetics should be tested once a
year. Others at risk testing less frequently as long as normal.
back

7. AKI - CreatinineAKI - Creatinine
 Does not rise until ~50% of renal
function is lost, typically 48-72 hrs
following acute injury.
– Creatinine is correlated with muscle mass and
nutrition
– MDRD equation has not been validated for:
– elderly (over 70 years of age)
– pregnant women
– serious comorbid conditions
– extremes of body size, muscle mass, or nutritional status
Nature Clinical Practice Nephrology, 27 November 2007

8. AKI - CreatinineAKI - Creatinine
 Does not rise until ~50% of renal
function is lost, typically 48-72 hrs
following acute injury.
– Creatinine based GFR equations are
imprecise when GFR>60
– Acutely ill have greater variability in results
– Inpatients have shown poorer agreement
than outpatients between eGFR and
measured GFR
Nature Clinical Practice Nephrology, 27 November 2007

9. AKI - PossibilityAKI - Possibility
 Possibility to identify biomarkers of AKI
that can enable detect / impact high risk
patients -
1. To diagnose AKI earlier, prior to overt
azotemia development when majority of
nephron loss may have already occurred.
2. To reduce risk for a disastrous outcome, &
3. To prevent progression with early
intervention
Nature Clinical Practice Nephrology, 27 November 2007

10. AKI - Reasons for focusAKI - Reasons for focus
 AdvantagesAdvantages
– Patients, physicians, industryPatients, physicians, industry
 BeliefBelief
– Direct relationship between duration and mortality
– Early forms of AKI are often reversible and
potentially effective preventive and therapeutic
measures are frequently delayed due to lack of early
diagnostic markers
– Early diagnosis → timely institution of measures →
prevention of progression [animal and human
studies reveal a narrow “window of opportunity”]
– A biomarker test that is simple, non-invasive and ofA biomarker test that is simple, non-invasive and of
early diagnostic and prognostic value.early diagnostic and prognostic value.

11. AKI -AKI - Ischaemic kidney responseIschaemic kidney response
Multifaceted
response of
the kidney
to
ischaemia
1
Persistent vasoconstriction
2
Tubular
obstruction
3
Cellular structural
changes
4
Metabolic
alterations
5Inflammatory
response

12. In ischaemic kidney, the
proximal tubule cells
undergo a complex
temporal sequence of
events, including
Ischaemic kidneyIschaemic kidney manifestationsmanifestations
2
Cell death due to
apoptosis and
Necrosis
3
Dedifferentiation
& proliferation of
viable cells
4
Reestablishment
of the epithelial
phenotype
1Loss of cell
polarity
Interventions for innovative and effective therapy
must:
1. oppose tubule cell death and / or
2. enhance the recovery phase

13. Emerging biomarkersEmerging biomarkers
in renal diseasein renal disease

14. Emerging biomarkersEmerging biomarkers
 Multidisciplinary functional approach to -
1. identify the most relevant biomarker
because
 current markers rise after function is already lost
and window of benefit from intervention is
closed or closing.
1. detect renal tubular injury and / or
dysfunction at an early stage
 before a decline in GFR is detected by
increased serum creatinine levels.
 Some promising biomarkers include:
KIM-1
IL-18
NGAL
Cystatin-C

15. Emerging biomarkersEmerging biomarkers
 Tubular enzymes
 Neutrophil gelatinase-associated lipocalin
 Kidney injury molecule 1
 Low-molecular weight urinary proteins –alpha1-and
beta2-microglobulin–retinol-binding protein–
adenosine deaminase-binding protein–cystatinC
 cysteine-rich protein 61
 urinary interleukins/adhesion molecules,
 management of the patient at risk for ARF.
 glomerular filtration
 proatrialnatriuretic peptide (1-98)
 Cystatin C
KIM-1
IL-18
NGAL
Cystatin-C

16. Our understandingOur understanding
Emerging biomarkersEmerging biomarkers

17. Cystatin C – what is it?Cystatin C – what is it?
 Single chain basic protein, non-glycosylated,
13,360 kD that acts as an inhibitor of cysteine
proteases
 Synthesized by all nucleated cells
 Constant production rate (gene of
housekeeping type)
– not influenced by acute phase reaction
– not influenced by endogenous or analytical factors
– not influenced by muscle mass, food intake, or body
surface
KIM-1
IL-18
NGAL
Cystatin-C

18. Cystatin C - characteristicsCystatin C - characteristics
 Cleared by free glomerular filtration, and
not secreted by tubules or eliminated by
any extra-renal route
 A marker of kidney function that may alsoA marker of kidney function that may also
be associated with inflammationbe associated with inflammation
– Reflective of mildly impaired kidney function
with increased inflammation..
KIM-1
IL-18
NGAL
Cystatin-C

19. Cystatin C - StudyCystatin C - Study
 85 patients at high risk of ARF
– 44 patients developed ARF
– 41 served as controls.
 Serum creatinine & cystatin C were
determined daily.
 ↑ cystatin C significantly preceded that of
creatinine.
 At 1.5+0.6 days, cystatin C ↑ by 50%,
earlier compared to creatinine.
KIM-1
IL-18
NGAL
Cystatin-C
Kidney Int.2004 Sep;66(3):1115-22

20. Cystatin C - SummaryCystatin C - Summary
 Serum cystatin C is a useful marker &
may detect ARF 1-2 days earlier than
creatinine.
 Over 20 years, most studies show that
cystatin C is a better predictor of GFR
than creatinine
– Meta-analysis - 49 studies, 4,492 individuals
 Dharnidharka et al. AJKD 2002
 Cystatin C’s advantage over creatinine as
a GFR measure is probably greatest in
the elderly and non-CKD persons
KIM-1
IL-18
NGAL
Cystatin-C

21. KIM-1 – what is it?KIM-1 – what is it?
 Kidney Injury Molecule – 1 is a Type 1
transmembrane protein whose
ectodomain is shed from cells upon
injury.
 Expression is markedly up-regulated in
the proximal tubule in the post-ischemic
rat kidney.
 Early studies suggest that urinary KIM-1
levels may serve as a sensitive method
for detecting early kidney injury.
KIM-1
IL-18
NGAL
Cystatin-C

22. KIM-1 studyKIM-1 study
 The most widely cited study in the field is oneThe most widely cited study in the field is one
by Han et al, studying KIM-1 for elevation ofby Han et al, studying KIM-1 for elevation of
KIM-1 in the urine, as a biomarker for ARF.KIM-1 in the urine, as a biomarker for ARF.
– 23 patients with ARF,23 patients with ARF,
– 9 with chronic renal failure (CRF), and9 with chronic renal failure (CRF), and
– 9 normal subjects9 normal subjects
 This KIM-1 study used azotemia to identify ARFThis KIM-1 study used azotemia to identify ARF
patients and the results showed the following:patients and the results showed the following:
– Urinary KIM-1 was elevated in patients with ARFUrinary KIM-1 was elevated in patients with ARF
secondary to ischemic ATN,secondary to ischemic ATN,
– No elevations seen in CRF patients & patients withNo elevations seen in CRF patients & patients with
ARF due to other causes, as well as normal subjectsARF due to other causes, as well as normal subjects
– No sensitivity, specificity, or ROC data was provided.No sensitivity, specificity, or ROC data was provided.
KIM-1
IL-18
NGAL
Cystatin-C

23. IL-18 - studyIL-18 - study
 Biomarker of human acute tubular necrosis.
 In the most popular IL-18 study, Parikh et al
examined urinary levels of IL-18 in
– 11 healthy controls,
– 14 patients with ATN,
– 8 with pre-renal azotemia,
– 5 with UTI, and
– 12 with chronic renal failure.
 Again, as noted for the KIM-1 study, study
subjects were diagnosed using azotemia as a
criterion.
KIM-1
IL-18
NGAL
Cystatin-C

24. IL-18 - summaryIL-18 - summary
 The results showed urinary IL-18 to be a
good marker for renal failure secondary
to ATN, as compared to all other causes
in the study,
 ROC analysis showed a sensitivity of
95%, and a specificity of 82%.
KIM-1
IL-18
NGAL
Cystatin-C

25. FocusFocus
NGALNGAL

26. NGAL – what is it?NGAL – what is it?
 Neutrophil gelatinase–associated
lipocalin (NGAL) is a 25-kDa
epithelial protein that is covalently
bound to gelatinase from human
neutrophils.
 178 aa disulfide-bridged polypeptide
chain
 Calculated molecular mass: 22 kDa
 Apparent molecular mass: 25 kDa
(glycosylation)
 Forms complex with 92-kDa matrix
metalloproteinase-9 (MMP-9;
gelatinase B) - capable of protecting
from degradation by interacting with this
protein.
KIM-1
IL-18
NGAL
Cystatin-C

27. NGAL – when is it found?NGAL – when is it found?
 NGAL has also been linked to
apoptosis in reproductive
tissues.
 Epithelial cells of the involuting
mammary gland and uterus
express high levels of NGAL,
temporally coinciding with a
period of maximal apoptosis.
 Thus, it is likely that a subset of
epithelial cells may utilize this
mechanism to regulate their
own demise
KIM-1
IL-18
NGAL
Cystatin-C

28. NGAL – why is it found?NGAL – why is it found?
 Induced in response to apoptotic
stressors and may be a
compensatory response that
involves cell defence pathways and
thus play a role in cell survival.
 Most up-regulated gene in the
kidney after AKI in animal models
and especially high levels after renal
ischaemia
 Levels increase within minutes of
injury
 Spills into blood and urine and
released in inflammation from
several epithelia.
KIM-1
IL-18
NGAL
Cystatin-C

29. NGAL – where is it found?NGAL – where is it found?
 Expression is markedly induced in
stimulated epithelia and expressed in:xpressed in:
– Secondary granules of neutrophilsSecondary granules of neutrophils
– Certain epithelial cells (gut, lung, uterus,Certain epithelial cells (gut, lung, uterus,
mammary gland, kidney)mammary gland, kidney)
 Elevated in the serum of patients with
acute bacterial infections, in the
sputum of patients with asthma or
COPD, and the bronchial fluid from
the emphysematous lung.
 NGAL counters the morphologic
response of renal tubular epithelial
cells to ischemia.
KIM-1
IL-18
NGAL
Cystatin-C

30. NGAL - AvailabilityNGAL - Availability
NGAL may be expressed by the damaged tubule to induce
re-epithelialization and proliferation
NGAL is expressed by the penetrating ureteric bud and
triggers nephrogenesis by stimulating the conversion of
mesenchymal cells into kidney epithelia
Renal
research
findings
suggest
During
kidney
development

31. NGAL - RoleNGAL - Role
NGAL
[Neutrophil
Gelatinase
Associated
Lipocalin
5
Tilts the overall balance of proximal tubule cell fate toward cell survival after
ischemic injury
1
Upregulated in the post-
ischemic kidney, in
tubular epithelial cells
that are undergoing
proliferation
2
Can enhance the epithelial
phenotype, based at least in part
on its ability to ameliorate tubule
cell apoptosis and enhance tubule
cell proliferation
3
Functions as an iron-
transporting protein
during nephrogenesis
4
Plays a
renoprotective
role in ischemic
ARF

32. NGAL – Iron transportNGAL – Iron transport
NGAL
Based iron
mechanisms
operative in
the post-
ischemic
kidney
5
Delivery of iron into cells is crucial for cell growth and renal regeneration after
nephrotoxic injury
1
Because NGAL can be
endocytosed by the
proximal tubule, the
protein could potentially
recycle iron into viable
cells
2
May serve as a reservoir for iron
that is released from tubule cells
that are damaged by nephrotoxic
injury
3
Removes iron, a reactive
molecule, from the site of
tissue injury, thereby limiting
iron-mediated cytotoxicity
4
May also facilitate
the removal of
excess intracellular
iron, thereby
limiting oxidant
mediated apoptosis

33. NGAL in kidney - explanationNGAL in kidney - explanation
KIM-1
IL-18
NGAL
Cystatin-C A B
The forest fire therapy for kidney function: these figures represent 2 patients
with both having 60% viable nephrons as green trees and 40% damaged
nephrons due to ischaemia. However, patient B has much stronger response
than patient A. It is proposed that sreum creatinine or GFR is indicative of the
viable nephron while renal NGA indicates the extent of active lesion.
Viable nephron Damaged nephron Active lesion

34. NGAL studyNGAL study
 The most promising study to date is the NGAL
study by Mishra et al. who followed a
population of children coming off
cardiopulmonary bypass for the development of
ARF and correlation to rises in urine and serum
NGAL.
 Of the 71 study subjects, 20 developed ARF
(diagnosed by rise in serum creatinine).
 Serum and urine NGAL were shown to rise
within 2 hours off cardiopulmonary bypass, a
full 24-36 hours before the development of
azotemia.
KIM-1
IL-18
NGAL
Cystatin-C

35. NGAL - ConclusionsNGAL - Conclusions
 Using a cut-off of 25 ug/ml, urine NGAL
had a sensitivity of 100% and specificity
of 98%, whereas serum NGAL had a
sensitivity of 70% and specificity of 94%.
 Furthermore, ROC analysis showed urine
NGAL to have and area under the curve
of 0.998, and serum NGAL had a
corresponding value of 0.906, showing
excellent characteristics as markers for
ARF in this setting.
KIM-1
IL-18
NGAL
Cystatin-C

36. NGAL – Another studyNGAL – Another study
 In an established murine model of renal
ischemia-reperfusion injury, IV NGAL given
before, during, or after ischemia resulted in
marked amelioration of the morphologic and
functional consequences, as evidenced by -
– A significant decrease in the histopathologic damage to tubules
– A significant decrease in serum creatinine measurements.
– A reduction in the number of apoptotic tubule cells, and
– An increase in proliferating PCT after ischemic injury.
Amelioration of Ischemic Acute Renal Injury by Neutrophil Gelatinase–Associated
Lipocalin by JAYA MISHRA et al, J Am Soc Nephrol 15: 3073–3082, 2004.

37. NGAL response to renalNGAL response to renal
ischaemia - serumischaemia - serum
KIM-1
IL-18
NGAL
Cystatin-C

38. NGAL response to renalNGAL response to renal
ischaemia - urineischaemia - urine
KIM-1
IL-18
NGAL
Cystatin-C

39. NGA- Scope aheadNGA- Scope ahead

40. The futureThe future
AKI biomarkersAKI biomarkers

41. Cardiac biomarkersCardiac biomarkers
KIM-1
IL-18
NGAL
Cystatin-C

42. AKI Panel markers - perception
KIM-1
IL-18
NGAL
Cystatin-C
Cystatin C
IL-18
KIM-1
NGAL
????

43. Questions needing clarityQuestions needing clarity
 When is AKI being diagnosed currently?When is AKI being diagnosed currently?
 How sensitive is NGAL relatively?How sensitive is NGAL relatively?
 How specific is NGAL relatively?How specific is NGAL relatively?
 When does NGAL secretion begin?When does NGAL secretion begin?
 What is the normal level of NGAL?What is the normal level of NGAL?
 Is NGAL diagnostic or prognostic?Is NGAL diagnostic or prognostic?
 How does NGAL compare with IL-18?How does NGAL compare with IL-18?
 How does NGAL compare with cystatin CHow does NGAL compare with cystatin C
or KIM-1?or KIM-1?

44. NGAL compared to IL-18NGAL compared to IL-18
KIM-1
IL-18
NGAL
Cystatin-C
NGAL and IL-18: Biomarkers in tandem Parikh et al, Kidney Int70:199-
203, 2006

45. NGAL compared to Cystatin CNGAL compared to Cystatin C
KIM-1
IL-18
NGAL
Cystatin-C
Vandevoorde and Devarajan, J Am Soc Nephrol 2006, 17:404A

46. Postmortem of the studiesPostmortem of the studies
 The KIM-1 and IL-18 studies are both
plagued by the fact that urine levels of
these markers were determined after the
diagnosis of ARF was certain (by
azotemia), and therapy initiated at such a
time may not result in improved clinical
outcomes as compared to current
standards of care.
KIM-1
IL-18
NGAL
Cystatin-C

47. Postmortem of the studiesPostmortem of the studies
 These tests may be of clinical utility to
identify patients with ARF secondary to
ischemic ATN (in the case of KIM-1), or
ATN in general (in the case of IL-18), for
potential targeted therapy of these
disease processes in the years ahead.
KIM-1
IL-18
NGAL
Cystatin-C

48. Postmortem of the studiesPostmortem of the studies
 However the NGAL study succeeds in
this respect since NGAL measurements
were made during the development of
disease, showing a robust rise in serum
and urine NGAL 24-36 hours before a
rise in serum creatinine, when the
initiation of therapy may avoid or
diminish overt renal failure.
KIM-1
IL-18
NGAL
Cystatin-C

49. Who should be tested &Who should be tested &
treated?treated?
 With diabetes:With diabetes:
– With urine albumin/creatinine ratios more than 30mgWith urine albumin/creatinine ratios more than 30mg
albumin/1 gram creatininealbumin/1 gram creatinine
 Without diabetes:Without diabetes:
– With urine albumin/creatinine ratios more than 300mgWith urine albumin/creatinine ratios more than 300mg
albumin/1 gram creatinine corresponding to about 1+albumin/1 gram creatinine corresponding to about 1+
on standard dipstickon standard dipstick
 Any patient:Any patient:
– With estimated GFR less than 60 mL/min/1.73 m2With estimated GFR less than 60 mL/min/1.73 m2

50. Therapy to prevent progressionTherapy to prevent progression
 Intensive glycemic control lessens progressionIntensive glycemic control lessens progression
from microalbuminuria in type 1 diabetesfrom microalbuminuria in type 1 diabetes
– DCCT, 1993DCCT, 1993
 Antihypertensive therapy with ACE InhibitorsAntihypertensive therapy with ACE Inhibitors
lessens proteinuria and progressionlessens proteinuria and progression
– Giatras, et al., 1997Giatras, et al., 1997
– Psait, et al., 2000Psait, et al., 2000
– Jafar, et al., 2001Jafar, et al., 2001
 Low protein diets lessen progressionLow protein diets lessen progression
– Fouque, et al., 1992Fouque, et al., 1992
– Pedrini, et al., 1996Pedrini, et al., 1996
– Kasiske, et al., 1998Kasiske, et al., 1998
Meta-Analyses
Meta-Analyses

51. Update on the ImmunocalinsUpdate on the Immunocalins
LipocalinsLipocalins

52. ImmunocalinsImmunocalins
 γγ-microglobulin-microglobulin
 ββ-microglobulin-microglobulin
 αα22-microglobulin-microglobulin
 αα11-microglobulin-microglobulin
 Ig L-chainsIg L-chains
 ββ22-microglobulin-microglobulin
 RBPRBP [RETINOL BINDING PROTEIN][RETINOL BINDING PROTEIN]
 αα11-microglobulin,-microglobulin,
Protein HCProtein HC
αα11-microglobulin-microglobulin
 Recognition in 1985:Recognition in 1985: αα11-m + RBP + others =-m + RBP + others =
new protein superfamily = Lipocalinsnew protein superfamily = Lipocalins
New LMW-proteins isolated by BerggNew LMW-proteins isolated by Berggåård et al.rd et al.

53. The ImmunocalinsThe Immunocalins

54. The ImmunocalinsThe Immunocalins

55. ConclusionConclusion
 Biomarkers detected in urine or serum shortly
after AKI, have been suggested to contribute to
prediction of ARF.
 However, it needs to be borne in mind that
excretion of these biomarkers may also
increase after reversible and mild renal
dysfunction and may not necessarily be
associated with persistent or irreversible
damage.
Shock 2006; Sep 26(3):245-53