Once seen as distinct, acute kidney injury (AKI) and chronic kidney disease (CKD) increasingly are viewed as partners in a downward spiral toward organ failure. Formerly, AKI was considered an incident from which patients generally recover, but research has revealed it to be a major risk factor for progression toward and worsening of CKD. While the understanding of the relationship between AKI and CKD has changed, clinical tests to detect AKI in a sensitive and specific manner have not. Currently used clinical tests based on creatinine—which may not rise until 24 to 48 hours after an AKI episode—are insufficiently sensitive or specific to detect AKI. With delays in diagnosis, clinicians miss opportunities to minimize damage, and patients incur more severe AKI with subsequent greater risk of developing CKD, predisposing them to cardiovascular disease, heart attack, and stroke.

Reliable biomarkers that would better identify individuals at high risk for developing AKI, spot AKI early on, track its progression and patients' recovery, and identify those patients at higher risk for poor outcomes, are lacking in renal care and would improve it, according to nephrology researchers. Investigations are focusing on several novel biomarkers, some of which might one day provide this information. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury-molecule 1 (KIM-1) are perhaps the most promising candidate biomarkers for AKI, but investigators are actively studying many others, said researchers from the Chronic Kidney Disease Biomarkers Consortium (CKD BioCon), funded by the National Institute of Diabetes and Digestive and Kidney Diseases.

"The hope is that new biomarkers of AKI and CKD can help better identify high-risk patients so they can be targeted for more intensive monitoring, treatment, or enrollment in clinical trials," said Chi-yuan Hsu, MD, CKD BioCon researcher and professor and division chief of Nephrology at the University of California, San Francisco. He spoke at the recent 30th Annual Beckman Conference, "Novel Biomarkers of Kidney Disease: False Dawn or New Horizon?" which was co-sponsored by AACC and the American Society of Nephrology.

Paradigm Shift

In contrast to earlier assumptions that AKI generally had no long-term consequences, recent research shows that severe AKI can increase the risk of developing CKD, worsen underlying CKD, and sometimes, cause end-stage renal disease (ESRD) directly. Some studies have found that AKI's severity, duration, and frequency are important predictors of poor patient outcomes, while others indicate that reduction of renal mass and nephron number, vascular insufficiency, cell cycle disruption, and maladaptive repair mechanisms appear to be important modulators of progression in patients with and without coexistent CKD. To better clarify AKI's role in renal pathology, investigators have called for long-term follow-up after first episodes of AKI (Kidney Int 2012;82:516–24).

Research has also driven the realization that CKD can lead to heart disease, underscoring the need to identify AKI early on because of its role in hastening and worsening CKD, emphasized Harold Feldman, MD, CKD BioCon researcher, senior investigator for the National Institutes of Health's (NIH) Chronic Renal Insufficiency Cohort Study (CRIC), and chair of the department of biostatistics and epidemiology at the University of Pennsylvania Perelman School of Medicine in Philadelphia. "Nephrologists used to think of CKD principally as a risk factor for kidney failure, dialysis, and the need for transplantation. Now, CKD is also recognized as promoting cardiovascular disease. CKD needs active management not just to prevent ESRD, but also heart failure, myocardial infarction, and stroke," he said, adding that as CKD worsens, comorbidities become severe. CKD patients often don't get cardioprotective treatments because nephrologists don't know which patients are at high risk, he noted, adding that more reliable biomarkers will help to identify them.

Problems With Old Markers

Researchers pointed to a number of ways in which current kidney injury biomarkers, especially creatinine and protein in urine, are inadequate. Protein in urine is considered a sensitive marker of kidney injury and a means of determining recovery, as well as for CKD and its progression. But it is not very specific. Levels may rise with use of certain nonsteroidal anti-inflammatory medications, cancers, lupus, and rheumatoid arthritis.

Creatinine, measured by labs for more than 100 years, is used to estimate glomerular filtration rate. This analyte also helps determine the magnitude of AKI, but it provides little information about the underlying cause of kidney injuries, and is less accurate for patients with low muscle mass and unusual diets. Challenges inherent in using creatinine as an AKI marker include diagnostic delays and potentially, misclassification of actual injury status.

Neither of these old markers tells the location of kidney injury. "We need a marker that will say in real time what's happening in the tubulointerstitium, the kidney's tubules and interstitial tissue. The idea is to get a real-time assessment of kidney function during therapy," said Brad Rovin, MD, CKD BioCon researcher, professor of medicine and pathology, vice chairman of research for internal medicine, and director of the division of nephrology at The Ohio State University College of Medicine in Columbus.

Researchers also seek a marker that gives more accurate information about the degree of kidney function, especially in surgery patients, Rovin added. "It's important to know who will develop AKI and why. Biomarkers could indicate which patients need more vigilant monitoring and early measures to curtail the causes of injury," he explained. Although no drugs treat AKI specifically, reliable biomarkers that identify those patients likely to develop kidney damage and correlate with outcome could aid development of drugs that stem damage, Rovin noted.

Promising New Biomarkers

Several novel biomarkers under investigation could be useful for diagnosing AKI and CKD, and for monitoring CKD's progression. New biomarkers for kidney function include cystatin C, a small molecule that is filtered and metabolized after tubular absorption, and beta-trace protein, which some research has shown is a more sensitive indicator of glomerular filtration than serum creatinine. For diagnosis of CKD and its progression in the structure of the kidney, new markers include NGAL, a ubiquitous iron-carrying protein expressed in the tubular epithelium of the distal nephron and released following damage into the blood and urine, as well as KIM-1, a transmembrane tubular protein that is produced with kidney damage.

Researchers are also studying N-acetyl-β-o-glucosaminidase (NAG), a marker of proximal tubular damage and possibly other areas of the nephron, and liver-type fatty acid-binding protein (L-FABP), which is expressed in proximal tubular cells and is a marker of inflammation studied in diabetes, hypertension, and early CKD. Researchers are also studying many other markers of glomerular injury, endothelial dysfunction, inflammation, fibrosis, cardiovascular dysfunction, and metabolic disorders. NGAL and KIM-1, originally discovered in the setting of AKI, have subsequently garnered considerable interest as markers for CKD, said Hsu and other CKD BioCon members.

NGAL has several attractive traits, especially its rapid rise in response to kidney injury, typically within 2–4 hours. NGAL clinched status as a promising biomarker for AKI following a 2005 study at Cincinnati Children's Hospital Medical Center that showed it to be a sensitive, specific, and highly predictive early biomarker of AKI in 71 children undergoing cardiac surgery. Diagnosis with serum creatinine was only possible 1–3 days after cardiopulmonary bypass, but urine concentrations of NGAL rose from a mean of 1.6 µg/L at baseline to 147 µg/L just 2 hours after cardiopulmonary bypass, and the amount in serum increased from a mean of 3.2 µg/L at baseline to 61 µg/L during the same timeframe. Univariate analysis showed a significant correlation between acute renal injury and urine and serum concentrations of NGAL at the time of surgery, and 2 hours later. Multivariate analysis showed that the amount of NGAL in urine 2 hours following surgery was the most powerful independent predictor of acute renal injury (Lancet 2005;365:1231–8).

NGAL also may identify those CKD patients most likely to deteriorate. A recently published study led by Hsu found urine NGAL was an independent risk factor of progression among 3,386 CRIC study CKD patients. But NGAL did not substantially improve prediction of outcome events. The cohort's baseline mean estimated glomerular filtration rate (eGFR) was 42.4 mL/min per 1.73 m2, the median 24-h urine protein was 0.2 g/day, and the median urine NGAL concentration was 17.2 ng/mL. Over an average follow-up of 3.2 years, there were 689 cases in which eGFR decreased by half or incident ESRD developed. After accounting for eGFR, proteinuria, and other known CKD progression risk factors, urine NGAL remained a significant independent risk factor. Adding urine NGAL to a model that included eGFR, proteinuria, and other CKD progression risk factors led to net reclassification improvement of 24.7% over a 2 year time horizon, but the C-statistic remained nearly identical (Kidney Int 2013;83:909–14).

The Danish company BioPorto Diagnostics markets an NGAL test in Canada and the European Union, but no such test is clinically available in the U.S. The U.S. Food and Drug Administration (FDA) approval is awaiting evidence that information about NGAL concentration changes clinical care, according to Joseph Bonventre, MD, PhD, chief of the renal division at Brigham and Women's Hospital and professor of medicine at Harvard Medical School in Boston.

Novel Kidney Markers Aid Drug Safety Studies

Novel kidney markers under investigation for improved clinical care may also prove useful in the preclinical evaluation of new drugs’ safety. Nephrotoxicity resulting from drug exposure causes an estimated 19–25% of all cases of acute kidney injury (AKI) in critically ill patients. The Predictive Safety Testing Consortium (PSTC), a collaboration of the U.S. Food and Drug Administration (FDA) and the European Medicines Agency, seeks to identify kidney damage biomarkers that can be used in animals early in drug development, well before clinical studies are underway. PSTC researchers seek markers that identify kidney injury early, detect degree of toxicity, site of kidney injury, and track progression or injury and recovery.

Kidney injury molecule-1 (KIM-1) was found by the PSTC, and subsequently by the European consortium to stand out among biomarkers studied as indicators of drug toxicity, according to Joseph Bonventre, MD, PhD, chief of the renal division at Brigham and Women’s Hospital and professor of medicine at Harvard Medical School in Boston. KIM-1 was the first injury biomarker of kidney toxicity qualified by the FDA for preclinical toxicity testing and drug development and, on a case-by-case, use in humans. That qualification is based on a study by the PSTC comparing KIM-1 to other biomarkers. KIM-1 significantly outperformed serum creatinine and blood urea nitrogen (BUN) at detecting renal tubular injury in rats (Nature Biotechnology 2010;5:436–40).

Now PSTC researchers are gearing up to test KIM-1, NGAL, and other novel nephrotoxicity markers in patients at the Brigham and Women’s Hospital and six other institutions, Bonventre noted. The study, which uses fresh urine, will test blood and urine samples from 150 cancer patients undergoing treatment with either cisplatin or aminoglycosides, two drugs known to cause injuries to the proximal tubule of the kidney. The study is notable because it will not use creatinine levels as an endpoint, but rather whether patients got these drugs, said Bonventre.

Data generated from the project is intended to advance regulatory acceptance of new kidney biomarkers appropriate for monitoring kidney safety in the clinic and improve clinical diagnoses of drug-induced kidney injury during drug development and patient therapy.

KIM-1, Combined With Others

As a marker for AKI, KIM-1 is promising because it is very specific for expression in the often injured proximal tubule of kidney and easily detectable in urine, said Bonventre, who discovered and characterized the protein. A sensitive and specific biomarker of proximal tubular injury in a variety of species including humans, KIM-1 may be most useful in combination with other markers, including NGAL, to show not only kidney damage but also predict who may develop it, and where the injury is most prominent in the kidney, he added. Research indicates that KIM-1 may also be useful in determining drug toxicity (See Sidebar, above).

A combination of KIM-1, NGAL, and NAG may allow for early detection of postoperative AKI after cardiac surgery before a rise in serum creatinine, according to a study by Thomas Jefferson University researchers. In a prospective study of 90 adult cardiac surgery patients, the investigators measured urinary KIM-1, NAG, and NGAL at five time points in the first 24 hours after surgery and normalized to the urinary creatinine concentration after cardiac surgery. Thirty-six patients developed AKI within 72 hours. The areas under the curve (AUCs) for predictions of AKI immediately and 3 hours after operation were 0.68 and 0.65 for KIM-1; 0.61 and 0.63 for NAG; and 0.59 and 0.65 for NGAL. Combining the three biomarkers enhanced the sensitivity for early detection of postoperative AKI compared with each of the biomarkers individually, with AUCs of 0.75 and 0.78 (Clin J Am Soc Nephrol 2009;4:873–82).

KIM-1 and NAG may also yield information about kidney damage in patients with less serious health issues. Levels of KIM-1 and NAG increased significantly in certain patients who got shockwave lithotripsy (SWL) and ureteroscopy (URS), minimally invasive treatment alternatives for kidney stones. Fifty kidney stone patients treated by SWL and 10 by URS gave urine samples 2–3 hours following the treatments, 2 days afterward, and 2 weeks later. Mean KIM-1 values were increased in patients with kidney stones, compared to healthy volunteers. Poor kidney function was significantly associated with increased biomarker activity both in baseline and post-SWL measurements (J Endourol 2013; doi:10.1089/end.2013.0188).

A Role for Laboratorians

Laboratorians can play an important role in adapting research assays to useful tests that function well in clinical labs, which are not as tightly controlled as research labs, Rovin noted. In addition, laboratorians can figure out how to consistently extract biomarkers and test them "in a cost-effective fashion in real time," and develop quality control procedures and standards for assay consistency. Rovin also suggested that laboratorians can help determine the most appropriate testing technologies, evaluate instrumental drift, and develop reference ranges.

Bonventre urged laboratorians to carefully evaluate research on AKI biomarkers, noting that doing so can be difficult because of what he sees as a problem of logic inherent in many studies that compare biomarkers to creatinine. "You cannot judge biomarkers as being better than creatinine, based on creatinine," he explained. "It's important to recognize that information in these markers doesn't get conveyed in large studies that use creatinine as a gold standard."

Another point to consider is that many studies use frozen urine—not fresh as clinical labs typically do—and in more tightly controlled conditions than exist in many clinical labs, said Bonventre. He noted a "need to test biomarkers on urine that has been well-stored if the assays are not performed in a timely way." That's because urine is subject to compositional changes that can interfere with assays, he explained, urging clinical chemists to "validate urine assays in real urine from CKD patients, if they wish to gain information about these patients by making urine biomarker measurements."

Despite these challenges, Bonventre is optimistic that better kidney biomarkers will eventually become available in the clinic. Noting that he and his colleagues liken their work to a search for "the troponin of the kidney," he pointed out that acceptance of troponin as a cardiac marker was a long process. "Different [kidney] markers will find different roles. Keep an open mind regarding their utility," he urged.

Deborah Levenson is a freelance writer who lives in College Park, Md. Her email address is [email protected].