American Association for Clinical Chemistry
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February 2010 Clinical Laboratory News: Emerging Biomarkers for Acute Kidney Injury

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February 2010: Volume 36, Number 2

Emerging Biomarkers for Acute Kidney Injury
Is There a Winner in the Offing?
By Genna Rollins

Acute kidney injury (AKI) is a complex, increasingly common syndrome, the diagnostics and treatments for which have remained essentially unchanged for decades, to the great frustration of clinicians and researchers. However, thanks to concerted efforts by key professional organizations, governmental agencies, and numerous research teams, considerable progress has been made since 2004. Now, many experts predict that the field is poised for transformation over the next decade. Novel urine and serum biomarkers will be central to this revolution in care.

“The field of acute kidney injury rests upon development of new biomarkers,” explained Mark Okusa, MD, FASN, John C. Buchanan distinguished professor of medicine and chief of nephrology at the University of Virginia School of Medicine in Charlottesville. “We’re trying to find what cardiologists found with troponin I for acute coronary syndrome. Our goal is to find a kidney troponin I.” Okusa also is chair of the AKI advisory group for the American Society of Nephrology (ASN).

Changing Definitions, Terms

Although definitions and even the nomenclature for AKI have changed over the years, the condition generally is recognized as the abrupt loss of kidney function that leads to fluid retention, accumulation of metabolic waste products, and dysregulation of extracellular volume and electrolytes. Common causes of AKI range from decreased renal perfusion and contrast-induced nephropathy to sepsis and nephrotoxicity from medications such as aminoglycoside antibiotics and non-steroidal anti-inflammatory drugs. AKI now is the preferred term over acute renal failure, to emphasize the range of AKI disease from early injury to progressive loss of function requiring renal replacement therapy.

Two classification systems for the condition proposed in recent years are gaining acceptance. Both rely on changes in serum creatinine levels and urine output. The Acute Kidney Injury Network (AKIN) defined AKI as an reduction in kidney function within 48 hours, involving an absolute increase in serum creatinine of ≥0.3 mg/dL, a percentage increase of ≥50% or 1.5 times above baseline, or documented oliguria of less than 0.5 ml/Kg per hour for more than 6 hours. Meanwhile, the Acute Dialysis Quality Initiative issued the Risk, Injury, Failure, Loss and End-stage (RIFLE) criteria, which use graded increases in glomerular filtration rate based on serum creatinine levels and weight-dependent urine output parameters.

Nationally, AKI accounts for at least 3%–4% of all hospitalizations and may be a contributing factor in more than one-third of all admissions, at a cost of about $10 billion annually. At the same time, the incidence of AKI in the community is rising, with an estimated rate of 500 per 100,000 population in 2002, up from approximately 61 per 100,000 population in 1988. Adding to the magnitude of these figures, 20%–30% of critically ill patients develop AKI, and depending on the patient population, mortality following an episode of AKI is estimated to be between 40% and 60%. Validation of both the AKIN and RIFLE systems has underscored the important effects of small declines in glomerular filtration rate on the overall outcome of critically ill patients, according to Okusa. “Even the least severe categories, ‘R’ in RIFLE or AKIN stage I, have been associated with a mortality rate of approximately 30 percent,” he indicated.

In addition, recent research has changed the thinking about the natural course of AKI. “Previously kidney function was generally thought to completely return to normal, but information now suggests that the long-term course for a significant number of patients with AKI is high-stage chronic kidney disease,” noted Paul Kimmel, MD, senior advisor in the Division of Kidney, Urologic and Hematologic Diseases at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). “AKI seems to go on to progressive injury, and the idea now is that there is a spectrum of functional kidney responses after AKI.” Kimmel also is project scientist for the NIDDK-funded Assessment, Serial Evaluation, and Subsequent Sequelae in Acute Kidney Injury (ASSESS-AKI) research initiative.

A Lackluster Gold Standard

Even as research is elucidating the pathology of and outcomes associated with AKI, treatment advances have been hampered by lack of sensitive and specific biomarkers for the condition. Although it is still considered the gold standard, serum creatinine is “far from an ideal parameter,” according to Norbert Lameire, MD, emeritus professor of medicine at the University of Gent, Belgium, and co-chair of the Kidney Disease: Improving Global Outcomes clinical practice guidelines on AKI. “In AKI it takes at least 24 hours, and in many cases, 48 hours, before you see a significant increase in serum creatinine. So you lose this crucial, let’s say 36 hours, in which a lot of injury has gone on before you see it in your serum creatinine.” As much as 50% of kidney function can be lost by the time serum creatinine levels reach abnormal levels. Serum creatinine also is affected by non-renal factors, such as protein intake, muscle mass, age, and sex, and it is not sensitive to kidney insults that do not affect filtration. Until recently, these biological variances were compounded by analytical challenges, but over the past several years considerable industry-wide effort has gone into standardization and commutability of serum creatinine measurements.

All-in-all, these shortcomings have had the compound effect of not only holding back treatment advances but also delaying anti-AKI drug development. A series of summits in 2004, sponsored by the ASN and with participation from NIDDK, FDA, and other professional associations, highlighted that reliance on serum creatinine was “stifling therapeutic progress,” according to Chirag Parikh, MD, PhD, associate professor of medicine at Yale University School of Medicine. “People realized we’d not made any progress in terms of patient care and morbidity and mortality of AKI. Serum creatinine was not only hindering diagnosis and treatment, but it was weakening the drug development process.” Earlier, more sensitive biomarkers would enable physicians to fine-tune the basics of AKI treatment—blood pressure management along with reperfusion and vasopression therapy—and enable them to start dialysis earlier when needed. Above all, better biomarkers ultimately would lead to development of drugs that would halt quickly the progression of AKI.

Unprecedented Enthusiasm

Since the 2004 summits, research has accelerated rapidly, to the point that some proposed biomarkers are tantalizingly close to being implemented in clinical practice. “There’s now unprecedented enthusiasm in nephrology for early AKI biomarker detection research,” observed Parikh. “There is a pipeline of development full of numerous possibilities and it is very likely a test or tests will come from that pipeline that are good enough to replace the current paradigm of care.” A review he conducted in 2008 identified 21 serum and urine biomarkers of AKI that had utility in the differential diagnosis, early detection and/or prognosis of the condition (Kidney Int 2008;73:1008–16). These markers also have been associated with injury to specific segments of the kidney nephron, such as the proximal and distal tubules, Loop of Henle, and collecting ducts, placing them in the realm of structural indicators of injury in contrast to serum creatinine’s role as a functional parameter.

Of the many potential markers, neutrophil gelatinase-associated lipocalin (NGAL) is at the top of many researchers’ lists. Also known as lipocalin-2 or siderocalin, NGAL is a protein in neutrophils that rises within 2–4 hours after kidney injury. Arguably the most studied emerging marker of AKI, NGAL has been investigated across a broad range of clinical settings, including post-cardiac surgery, critical and emergency care, as well as in adult and pediatric populations. A recent meta-analysis of 19 studies involving more than 2,500 patients found that the area under the curve/receiver operating characteristic of NGAL to predict AKI overall was 0.815, 0.775 in cardiac surgery patients, 0.728 in critically ill patients, and 0.894 following contrast infusion, respectively (Am J Kidney Dis 2009;54:1012–24). NGAL showed better predictive ability in children than adults, and it appeared to be useful in predicting renal replacement therapy and, to some extent, in-hospital mortality. “Our analysis found that NGAL appears to have diagnostic value for early AKI and prognostic value for renal replacement therapy and mortality, both overall and across a range of subgroups,” said lead author Michael Haase, MD, assistant professor of nephrology and intensive care medicine at Charité-University Medicine Berlin in Germany.

The Need for More Data

As promising as this analysis appeared to be, it highlighted the challenge facing implementation of all the AKI biomarkers under investigation. Most importantly, there has yet to be a prospective validation study in a large number of patients with different causes of AKI. The majority of studies have been in small populations or single centers examining AKI in one setting or clinical circumstance, such as post-cardiac surgery or in critically ill children. In addition, with NGAL and various other proposed biomarkers, a variety of test platforms have been used with different protocols and reference ranges. For instance, the majority of studies identified in Haase’s meta-analysis used CLIA-waived ELISAs, with NGAL reference ranges for the non-AKI control populations varying from <10 ng/mL to <550 ng/mL. ASSESS-AKI is expected to fill crucial gaps in the AKI evidence base (See Tracking the Course, below). 

Tracking the Course of Acute Kidney Injury
Landmark Study Will Evaluate New Markers

Although numerous potential biomarkers for acute kidney injury (AKI) have been identified and investigated—many with favorable performance profiles—the candidate markers need further evaluation in larger, more diverse populations for longer periods of time in order to achieve breakthroughs in AKI care. A major National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) initiative seeks to do just that.

Assessment, Serial Evaluation, and Subsequent Sequelae in Acute Kidney Injury (ASSESS-AKI) is a landmark study that will follow for a mean of 3 years a diverse population of 1,200 patients in a range of clinical settings at three participating centers. ASSESS-AKI has two primary goals: determining whether hospitalized patients with an episode of AKI are at greater risk of developing chronic kidney disease than patients without AKI, after adjusting for pre-existing levels of kidney function and potential confounders; and determining whether these AKI patients are at higher risk for death, cardiovascular and other adverse events after hospitalization than control subjects.

“Our purpose is two-fold. One is to provide more information about the natural history of AKI, particularly acute tubular necrosis in hospitalized patients,” explained ASSESS-AKI project scientist Paul Kimmel, MD, who also is senior advisor in the Division of Kidney, Urologic and Hematologic Diseases at NIDDK. “The other is to develop our understanding of how biomarkers can help in predicting outcomes, specifically long-term kidney response in patients who develop AKI.”

ASSESS-AKI began enrolling patients in December 2009; outcomes from the trial are expected by the end of 2013. Evaluating currently used and novel biomarkers will be a key aspect of the study. At least five unique blood and urine samples will be collected over the course of 3 years from 600 patients with AKI and 600 matched controls. A minimum of 16 novel urine and 10 novel serum biomarkers will be measured, in addition to standard tests such as urine and serum creatinine, blood urea nitrogen, urine and blood albumin, calcium, and glucose, among others. “It’s quite likely, because we’ll have enough patients, statistical power, and tests, that we’ll be able to find any relationships between new biochemical assays and long-term outcomes that may exist,” said Kimmel.

ASSESS-AKI’s precise sample collection, handling and storage protocols also are expected to advance the AKI evidence base, according to Kimmel. “One of the things we’re trying to do is to put our measurements into a clinical context. So if we learn that urine has to be handled in a certain way or we lose certain markers, but find that there’s a six hour delay until it’s processed in a standardized manner in all the study’s different clinical sites, that will be very important. The utility of something that has to be collected under extremely specified conditions will be less generalizable.”

Analytics will be a key in implementing any AKI biomarkers, according to Won Han, MD, assistant professor of nephrology at Thomas Jefferson University in Philadelphia. “If you look at the assays out there, they all have different protocols, so it’s kind of hard to compare them head-to-head,” he explained. “We need to be thinking about how to standardize the assays and what substances can interfere with a given assay. Another thing is the stability of the various proteins. Without that you’re not going to have something that’s accurate.” Han was lead author of a recent study that explored the stability of NGAL and two other urinary proteins in an investigation of AKI after cardiac surgery (Clin J Am Soc Nephrol 2009;4:873–882). The researchers found that the three biomarkers were stable under a variety of storage times, temperatures, and freeze and thaw cycles, except that urinary NGAL degraded significantly after prolonged storage at –20ºC.

Other biomarkers considered by some researchers to be the most promising for AKI diagnosis, treatment and/or prognosis include urine kidney injury molecule-1(KIM-1), urine interleukin 18 (IL-18), N-acetyl-β-D-glucosaminidase (NAG) and urinary liver-type fatty acid binding protein (L-FABP) (See Table, below). KIM-1 is a renal tubular protein that has been shown to differentiate acute tubular necrosis from other types of kidney injury. IL-18 is a pro-inflammatory cytokine indicative of renal tubular inflammation that has been found in significantly higher concentrations in patients with acute tubular necrosis in comparison to a variety of other conditions. NAG is a high molecular-weight lysosomal enzyme with considerable activity in renal proximal tubular cells that has been shown to rise in urine when there is proximal tubular cell necrosis. L-FABP binds unsaturated fatty acids and lipid peroxidation products during tissue injury from hypoxia and has been shown to be significantly higher in patients with poor AKI outcomes. Pre-clinical studies also have shown renal papillary antigen 1 and 2 (RPA-1 and RPA-2) to be promising site-specific markers for drug-induced nephrotoxicity (Toxicologic Pathology 2009;37:629–643).

Proposed Biomarkers of Acute Kidney Injury

Numerous biomarkers have been proposed for the differential diagnosis, early detection and prognosis of patients with AKI.

Differential Diagnosis in Established AKI

Serum Markers

  • Carbamylated hemoglobin (carb Hb)
  • Cystatin C
  • Neutrophil gelatinase-associated lipocalin (NGAL)

Urine Markers

  • α-1 microglobulin
  • Glutathione-S-transferase (GST)
  • Interleukin-18 (IL-18)
  • Kidney injury molecule-1 (KIM-1)
  • N-acetyl-β-D-glucosaminidase (NAG)
  • NGAL
  • Sodium hydrogen exchanger 1 (NHE3)
  • Matrix metalloproteinase-9 (MMP-9)

Early Detection of AKI

Serum Markers

  • Cystatin C
  • Prohormone of atrial natriuretic peptide (ProANP)
  • Neutrophil-CD11b
  • NGAL

Urine Markers

  • α-GST
  • γ-glutamyl transpeptidase
  • π-GST
  • Alkaline phosphatase
  • GST
  • KIM-1
  • IL-18
  • Lactate dehydrogenase (LDH)
  • NAG
  • NGAL
  • MMP-9

Prognosis of AKI

Serum Markers

  • Cystatin C
  • IL-6
  • IL-8
  • IL-10
  • NGAL

Urine Markers

  • α-1 microglobulin
  • α-GST
  • β-2 microglobulin
  • Cystatin C
  • γ-glutamyltransferase
  • IL-18
  • KIM-1
  • LDH
  • NAG
  • NGAL
  • Retinol-binding Protein

Adapted from Coca, SG, Yalavarthy, R, Concata, J, and Parikh, CR, Kidney Intl 2008;73:1008-16.