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Urine Neutrophil Gelatinase-Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Associated Lipocalin Moderately Predicts Acute Kidney Injury in Predicts Acute Kidney Injury in Critically Ill AdultsCritically Ill AdultsEdward 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, MDAnand Reddy, MD

MSSM, Aug 26MSSM, Aug 26thth 2009 2009J 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 BiomarkersAKI biomarkers must:

Allow for early detection of renal injuryIdentify 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 AKIGuide timing of therapyReflect improvement and worsening of the kidney injuryBe 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 familyComposed of eight ß-strands that form a b-barrel enclosing a calyxHuman 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 kidneyExpressed by Neutrophils and various epithelial cells

Physiology of NGALRole 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 bacteriaPrevents 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 developmentIn 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 factorPlays 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-Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Associated Lipocalin Moderately Predicts Acute Kidney Injury in Predicts Acute Kidney Injury in Critically Ill AdultsCritically Ill Adults

Edward D. Siew,* Lorraine B. Ware,† Tebeb Gebretsadik,‡ Ayumi Shintani,‡Karel G. M. Moons,§ Nancy Wickersham,† Frederick Bossert,† and T. Alp Ikizler*


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 Characteristics588 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

STUDY

RESULTS

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 HrsAKI 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

(A) 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.

(B) 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 m2

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 measurementsMultiple 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 populationSerum 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

1. Pomerantz BJ, Reznikov LL, Harken AH, Dinarello CA. Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1beta. Proc Natl Acad Sci U S A. 2001;98:2871-2876.

2. Hedtjarn M, Leverin AL, Eriksson K, Blomgren K, Mallard C, Hagberg H. Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci. 2002;22:5910-5919.

3. Bonventre JV, Weinberg JM. Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol. 2003;14:2199-2210.

4. Leslie JA, Meldrum KK. The role of interleukin-18 in renal injury. J Surg Res. 2008;145(1):170-175.5. Sugawara I. Interleukin-18 (IL-18) and infectious diseases, with special emphasis on diseases induced by

intracellular pathogens. Microbes Infect. 2000;2:1257-1263.6. Parikh CR, Jani A, Melnikov VY, Faubel S, Edelstein CL. Urinary interleukin-18 is a marker of human acute

tubular necrosis. Am J Kidney Dis. 2004;43:405-414.7. Randers E, Erlandsen EJ. Serum cystatin C as an endogenous marker of the renal function—a review. Clin

Chem Lab Med. 1999;37:389-395.8. Coll E, Botey A, Alvarez L, et al. Serum cystatin C as a new marker for noninvasive estimation of glomerular

filtration rate and as a marker for early renal impairment. Am J Kidney Dis. 2000;36:29-34.9. Dharnidharka VR, Kwon C, Stevens G. Serum cystatin C is superior to serum creatinine as a marker of kidney

function: a meta-analysis. Am J Kidney Dis. 2002;40:221-226.10. Christensson A, Ekberg J, Grubb A, Ekberg H, Lindström V, Lilja H. Serum cystatin C is a more sensitive and

more accurate marker of glomerular filtration rate than enzymatic measurements of creatinine in renal transplantation. Nephron Physiol. 2003;94(2):p19-27.

11. Sarnak MJ, Katz R, Stehman-Breen CO, et al. Cystatin C concentration as a risk factor for heart failure in older adults. Ann InternMed. 2005;142:497-505.

12. ShlipakMG, SarnakMJ, Katz R, et al. Cystatin C and the risk of death and cardiovascular events among elderly persons. N Eng JMed. 2005;352:2049- 2060.

13. Poge U, Gerhardt T, Bokenkamp A, et al. Time course of low molecular weight proteins in the early kidney transplantation period—influence of corticosteroids. Nephrol Dial Transplant. 2004;19:2858-2863.

14. Mangge H, Liebmann P, Tanil H, et al. Cystatin C, an early indicator for incipient renal disease in rheumatoid arthritis. Clin Chim Acta. 2000;300: 195-202.

15. Kleber M, Cybulla M, Bauchmuller K, Ihorst G, Koch B, Engelhardt M. Monitoring of renal function in cancer patients: an ongoing challenge for clinical practice. Ann Oncol. 2007;18:950-958.

16. Knight EL, Verhave JC, Spiegelman D, et al. Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement. Kidney Int. 2004;65:1416-1421.

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Urine Neutrophil Gelatinase-Urine Neutrophil Gelatinase-Associated Lipocalin Moderately Associated Lipocalin Moderately Predicts Acute Kidney Injury in Predicts Acute Kidney Injury in Critically Ill AdultsCritically Ill AdultsEdward D. Siew,* Lorraine B. Ware,† Tebeb Gebretsadik,‡ Ayumi Shintani,‡Karel G. M. Moons,§ Nancy Wickersham,† Frederick Bossert,† and T. Alp Ikizler*


Anand Reddy, MDAnand Reddy, MD

MSSM, Aug 26MSSM, Aug 26thth 2009 2009

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