Journal Club Presentation

Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Predicts Acute Kidney Injury in Critically Ill Adults Edward D. Siew,* Lorraine B. Ware,† Tebeb Gebretsadik,‡ Ayumi Shintani,‡ Karel G. M. Moons,§ Nancy Wickersham,† Frederick Bossert,† and T. Alp Ikizler* *Vanderbilt University Medical Center, Department of Medicine, Division of Nephrology, Nashville, Tennessee; †Vanderbilt University Medical Center, Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Nashville, Tennessee; ‡ Vanderbilt University Medical Center, Department of Biostatistics, Nashville, Tennessee; §Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands Anand Reddy, MD MSSM, Aug 26 th 2009 J Am Soc Nephrol 20: 1823-1832, 2009

Urgent need for early biomarkers in the management of AKI
AKI associated with significant morbidity and mortality.
Traditional blood (creatinine, blood urea nitrogen [BUN]) and urinary markers of kidney injury (urinary casts, fractional excretion of sodium) do not allow for early detection of AKI.
Change in SCr does not discriminate the time and type of renal insult nor the site and extent of glomerular or tubular injury.
Absence of sensitive and specific biomarkers for the early detection of AKI impairs progress in the diagnosis and treatment.
Dialysis remains the only Food and Drug Administration (FDA)-approved treatment option for established AKI

AKI Biomarkers
AKI biomarkers must:
Allow for early detection of renal injury
Identify the types & severity of AKI
Discerning AKI subtypes (prerenal, intrinsic renal, or postrenal);
Identifying AKI etiologies (ischemia, toxins, sepsis, or a combination);
Provide a rationale for risk stratification in clinical studies, including the identification of patients at risk for AKI
Guide timing of therapy
Reflect improvement and worsening of the kidney injury
Be amenable to quick and reliable measurement at the bedside or clinical laboratory.

Urinary Biomarkers for Kidney Injury in Humans

Promising biomarkers for AKI in humans

Kidney Injury Molecule-1 (KIM-1)
KIM-1 is a type 1 transmembrane glycoprotein
Undetectable in normal kidney tissue or urine, but is expressed at high levels in dedifferentiated proximal tubule epithelial cells from human and rodent kidneys after ischemic or toxic injury, and in renal cell carcinoma. [8-11]
KIM-1 levels were found within 12 hours after an initial ischemic insult and prior to the appearance of granular casts in the urine
Urinary KIM-1 had an AUC-ROC of 0.57 at 2 hours, 0.83 at 12 hours, and 0.78 at 24 hours after pediatric surgery. [29]

Interleukin-18 (IL-18)
Proinflammatory cytokine that is involved in mediating inflammation in many organs
Inflammationplays an important role in the pathophysiology of AKI associatedwith ischemia, sepsis, and many nephrotoxicants.
M RNA levels are significantly upregulated in the proximal tubules followingischemia-reperfusion injury, autoimmune nephritis, and cisplatin-induced nephrotoxicity. [16]
Urinary IL-18 was significantly increased in the urine of patients with AKI and increased within 24 hours after kidney transplantation in patients with delayed allograft dysfunction. [18]
Urinary IL-18 had an AUC-ROC of 0.95 for patients with established AKI and delayed graft function. [18]

Cystatin C (Cys-C)
Nonglycosylated basic protein that is synthesized at a relatively constant rate and released into the plasma by all nucleated cells.
Serum Cys-C is freely filtered by the glomerulus and catabolized in the proximaltubules.
Several studies have demonstrated that a change in serum Cys-C is more sensitive than a change in SCr as a marker of change in glomerular filtration.
Single-center study of 85 ICU patients athigh risk to develop AKI, a 50% increase in serumCys-C predicted AKI 1-2 days prior to an elevation in SCr, with AUCs of 0.97 and 0.82, respectively.[23]
? Effect of Age, wt, Gender on Serun cys levels.

NGAL (Neutrophil Gelatinase–Associated Lipocalin)
Protein of the lipocalin family
Composed of eight ß-strands that form a b-barrel enclosing a calyx
Human NGAL consists of a polypeptide chain of 178 amino acids with a molecular mass of 25 kDa.
Gene expression is demonstrated in human tissues like uterus, prostate, salivary glands, lung, trachea, stomach, colon, and kidney
Expressed by Neutrophils and various epithelial cells

Physiology of NGAL
Role in iron metabolism
NGAL is also called siderocalin as it is the first mammalian protein to bind and transport a bacterial siderophore [1]
Role in innate immunity to bacteria
Prevents the growth of the bacterial strains that depend on siderophores for iron supply [1]
NGAL KO mice more sensitive to Escherichia coli infection leading to sepsis and death more readily than WT mice. [3,4]
Role in kidney development
In vitro studies in rats suggest that NGAL may be involved in kidney development by virtue of its role in iron metabolism [5,6]
Role as a growth factor
Plays an important role in renal regeneration and repair after ischemic injury [7]

NGAL after CABG Bennett et al, Clin J Am Soc Nephrol 3: 665-673, 2008

Nickolas, T. L. et. al. Ann Intern Med 2008;148:810-819 Kidney injury biomarkers versus clinical outcome N-acetyl- ß -D-glucosominidase (NAG) 1-microglobulin (A1M) 1-acid glycoprotein (AAG)

NGAL as a biomarker

Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Predicts Acute Kidney Injury in Critically Ill Adults Edward D. Siew,* Lorraine B. Ware,† Tebeb Gebretsadik,‡ Ayumi Shintani,‡ Karel G. M. Moons,§ Nancy Wickersham,† Frederick Bossert,† and T. Alp Ikizler* *Vanderbilt University Medical Center, Department of Medicine, Division of Nephrology, Nashville, Tennessee; †Vanderbilt University Medical Center, Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Nashville, Tennessee; ‡ Vanderbilt University Medical Center, Department of Biostatistics, Nashville, Tennessee; §Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands J Am Soc Nephrol 20: 1823-1832, 2009

Purpose of the study
Prospectively look at uNGAL as the ability to predict both the development and severity of AKI in mixed ICU patients
Added & conjoint predictive ability of uNGAL beyond panel of prior selected clinical predictors.

Study Characteristics
NIH-sponsored Validation of biomarkers in Acute Lung Injury Diagnosis (VALID) study.
Multi-ICU prospective cohort study (Vanderbilt University Medical Center)
Primary purpose is to investigate panels of new and existing plasma, serum, or urine protein biomarkers to both diagnose ALI/ARDS in at-risk patients and to identify patients with ALI/ARDS

Patient Characteristics
588 patients enrolled in the ongoing NIH-sponsored Validation of biomarkers in Acute Lung Injury Diagnosis (VALID) study.
All adult (> 18 yr of age) patients admitted to one of four ICUs (Medical, Cardiac, Surgical, Trauma) at Vanderbilt University Medical Center (VUMC) who remained in the ICU at day 2 were eligible for enrollment
General severity-of-illness scoring systems including APACHE II and SAPS II were collected on enrollment day.
APACHE II ("Acute Physiology and Chronic Health Evaluation II") SAPS II ("Simplified Acute Physiology Score II")

AKIN staging criteria for AKI

Results

S T U D Y R E S U L T S

Biomarker Values & Outcome

Association between uNGAL and AKI Development Within 24 Hrs, AKI 64 patients (median uNGAL 313 [interquartile range (IQR): 59 to 2355] ng/mg) No AKI 386 (median uNGAL 56 [interquartile range (IQR): 16 to 194] ng/mg) ( P < 0.001) Within 48 Hrs AKI 86 patients (median uNGAL 190 [interquartile range (IQR): 32 to 995] ng/mg) No AKI 305 (median uNGAL 57 [interquartile range (IQR): 17 to 203] ng/mg) ( P < 0.001)

Association between uNGAL and AKI Development
AUC-ROCs for AKI development within 24 h (black line) and 48 h (gray line) of enrollment. A sensitivity analysis restricted to patients with an estimated GFR at enrollment of 75 ml/min/1.73m2 was also performed (red line). The AUC values and 95% CIs are listed within the figure.
The AUC-ROCs for sustained AKI within 24 h (black line) and 48 h (gray line) of enrollment. The AUC values and 95% CIs are listed within the figure.

Sensitivity Analyses of Baseline Renal Function
No baseline renal function known in 52%
Two sensitivity analysis were done to explore this issue
Method One :
Restricted analysis to patients with eGFR at enrollment to 75 ml ml/min/1.73 m 2
Results :
Within 24 Hrs: AKI 18; No AKI 257
Median uNGAL for subjects developing AKI was 203[IQR: 102 to 401] ng/mg and 44[IQR: 14 to 102] ng/mg for the controls ( P 0.001).

Sensitivity Analyses of Baseline Renal Function
Method Two :
Using multiple imputation methodologies to estimate baseline renal function in patients with missing baseline data.
This method accounts for other known patient data (DM,CKD,PVD,CHF,HTN etc)
The median serum creatinine in the group without known baseline function was estimated to be 0.7 [IQR: 0.6 to 0.9] mg/dl.
With the above method, AKI within 24Hrs of enrollment with an AUC of 0.61 (95% CI:0.46 to 0.76)

Association between uNGAL and Dialysis or Mortality Within 28 d of enrollment, Dead : 83 patients with uNGAL levels 223 [IQR:36 to 1074] ng/mg) Survived : 407 Patients with uNGAL levels 56 [IQR:16 to 195] ng/mg)( P 0.001) Dialysis : 17 uNGAL Levels 548 [IQR:156 to 4665] ng/ mg) No Dialysis : 473 with uNGAL levels 61 [IQR:17 to 232] ng/mg)( P 0.001)

AKIN staging criteria for AKI

uNGAL and AKIN Staging

Relative Contribution of uNGAL to a Clinical Prediction Model

Discussion
uNGAL found to be independently associated with and exhibited moderate discrimination for the prediction of subsequent AKI.
Less robust compared with fixed mechanism of injury such as in CABG or DGF.
As a stand-alone marker using modest creatinine-based cutoffs, uNGAL seems to have limited utility beyond a conventional clinical risk-prediction model.

Limitation of the Study
Creatinine measurements were made based on clinical decision making and were not protocol-driven.
Single-point time assessment of uNGAL rather than serial measurements
Multiple urine biomarkers not done (different mechanisms of injury)
52% of the patients in the study did not have the baseline creatinine.
Did not compare discriminative power of uNGAL between patients in different ICUs.
Predominant caucasian population
Serum NGAL may be elevated in severely critically ill confounding uNGAL levels.

Future Directions

References
Goetz DH, Holmes MA, Borregaard N et al (2002) The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10:1033–1043
Yan L, Borregaard N, Kjeldsen L et al (2001) The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem 276:37258– 37265
Berger T, Togawa A, Duncan GS et al (2006) Lipocalin 2- deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc Natl Acad Sci U S A 103:1834–1839
Flo TH, Smith KD, Sato S et al (2004) Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432:917–921
Gwira JA, Wei F, Ishibe S et al (2005) Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro. J Biol Chem 280:7875– 7882
Yang J, Goetz D, Li JY et al (2002) An iron delivery pathway mediated by a lipocalin. Mol Cell 10:1045–1056
Mishra J, Ma Q, Prada A et al (2003) Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14:2534–2543
Ichimura T, Bonventre JV, Bailly V, et al. Kidney InjuryMolecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. J Biol Chem. 1998;273:4135-4142.
Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV. Kidney Injury Molecule-1 (KIM-1): A novel biomarker for human renal proximal tubule injury. Kidney Int. 2002;62:237-244.
Ichimura T, Hung CC, Yang SA, Stevens JL, Bonventre JV. Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. Am J Physiol. Renal Physiol. 2004;286:F552-F563.
Han WK, Alinani A, Wu CL, et al. Human kidney injury molecule-1 is a tissue and urinary tumor marker of renal cell carcinoma. J Am Soc Nephrol.2005;16:1126-1134.
Jordan JA, Guo RF, Yun EC, et al. Role of IL-18 in acute lung inflammation. J Immunol. 2001;167:7060-7068.

References …Contd
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THANK YOU

Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Predicts Acute Kidney Injury in Critically Ill Adults Edward D. Siew,* Lorraine B. Ware,† Tebeb Gebretsadik,‡ Ayumi Shintani,‡ Karel G. M. Moons,§ Nancy Wickersham,† Frederick Bossert,† and T. Alp Ikizler* *Vanderbilt University Medical Center, Department of Medicine, Division of Nephrology, Nashville, Tennessee; †Vanderbilt University Medical Center, Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Nashville, Tennessee; ‡ Vanderbilt University Medical Center, Department of Biostatistics, Nashville, Tennessee; §Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands Anand Reddy, MD MSSM, Aug 26 th 2009

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