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Biomarkers for Assessment of Renal Function During Acute Kidney Injury
Acute kidney injury (AKI) is a common and serious condition, the diagnosis of which depends on serum creatinine measurements. Unfortunately, creatinine is a delayed and unreliable biomarker of AKI. The lack of reliable early biomarkers has crippled our ability to translate promising experimental therapies to human AKI. Fortunately, understanding the early stress response of the kidney to acute injuries has serendipitously revealed a number of potential non-invasive biomarkers. The discovery, translation and validation of neutrophil gelatinase-associated lipocalin (NGAL), the most widely studied novel AKI biomarker, is reviewed. NGAL is emerging as an excellent stand-alone troponin-like biomarker in the plasma and urine, as a diagnostic as well as prognostic tool in several common clinical scenarios. The current status of other promising AKI biomarkers, including kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), and interleukin-18 (IL-18) is also reviewed. The potential role of biomarker combinations, the use of novel biomarkers independent of creatinine, and the limitations of AKI biomarker studies are also explored.
Keywords
biomarker, acute kidney injury, neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, interleukin-18, liver-type fatty acid binding protein
Biomarkers of Acute Kidney Injury—an Un-Met Need
The incidence of acute kidney injury (AKI) is rising globally, and so are the associated morbidity and mortality rates. AKI afflicts 5–7% of all hospitalized patients. In critically ill patients, the overall prevalence of severe AKI requiring renal replacement therapy (RRT) is about 6%, with a mortality rate of 60%. Mortality and morbidity from AKI has not substantially improved in the past few decades despite technological advances in supportive care. AKI is largely asymptomatic, and establishing the diagnosis currently hinges on functional biomarkers such as serial serum creatinine measurements. Unfortunately, serum creatinine is a delayed and unreliable indicator of AKI for a variety of reasons. First, normal serum creatinine is influenced by several non-renal factors such as age, gender, muscle mass, muscle metabolism, medications, hydration status, nutrition status, and tubular secretion. Second, a number of acute and chronic kidney conditions can exist with no increase in serum creatinine due to the concept of renal reserve—it is estimated that greater than 50% of kidney function must be lost before serum creatinine rises. Third, serum creatinine concentrations do not reflect the true decrease in glomerular filtration rate in the acute setting, since several hours to days must elapse before a new equilibrium between the presumably steady state of creatinine production and the decreased excretion of creatinine is established. Fourth, serum creatinine production is diminished in critical illnesses such as sepsis, and measured serum creatinine is often further reduced by hemodilution resulting from standard goal-directed fluid therapies. Fifth, an increase in serum creatinine represents a late indication of a functional change in glomerular filtration rate, which lags behind important structural changes that occur in the kidney during the early damage stage of AKI. The delay in AKI diagnosis imposed by our dependence on serum creatinine changes is a problem, since animal studies have identified several interventions that can prevent and/or treat AKI if instituted early in the disease course, well before the serum creatinine begins to rise. The lack of early biomarkers has hitherto hampered our ability to translate these promising therapies to human AKI. Also lacking are reliable methods to assess efficacy of preventive or therapeutic interventions, and early predictive biomarkers of drug toxicity.
Desirable Characteristics of aki Biomarkers
First, with respect to assay characteristics, AKI biomarkers should be non-invasive and easy to perform at the bedside or in a standard clinical laboratory, using easily accessible samples such as blood or urine, with quick turn-around times. The majority of AKI biomarkers described thus far have been measured in the urine. Urinary diagnostics have several advantages, including the non-invasive nature of sample collection, the reduced number of interfering proteins, and the potential for the development of patient self-testing kits. However, disadvantages also exist, including the lack of sample from patients with severe oliguria, and potential changes in urinary biomarker concentration induced by hydration status and diuretic therapy. Plasma-based diagnostics have revolutionized many facets of medicine, as exemplified by the use of troponins for the early diagnosis of acute myocardial infarction. On the other hand, plasma biomarkers may be confounded by extra-renal sources as well as by subclinical changes in renal elimination. Thus, in the case of AKI, it is ideal to develop both urinary and plasma biomarkers.
Second, with respect to diagnostic properties, AKI biomarkers should be sensitive to facilitate early detection, with a wide dynamic range that allow for risk stratification. They should also be highly specific for AKI, enable the identification of AKI sub-types and differentiate AKI from chronic kidney disease (CKD). Ideally, biomarkers are also needed to identify the primary location of injury (proximal tubule, distal tubule, interstitium, or vasculature), and discern AKI etiologies (ischemia, toxins, sepsis, or a combination).
Third, with respect to prognostic abilities, AKI biomarkers should allow for risk stratification (duration and severity of AKI), prediction of hard clinical outcomes (need for renal replacement therapy, length of hospital stay, mortality) and monitoring the response to AKI interventions. Biomarkers associated with clear biologic plausibility and known pathophysiologic mechanisms in AKI are most likely to satisfy the desired diagnostic and prognostic characteristics.
Given the limitations of serum creatinine, the search for improved biomarkers of AKI is of intense contemporary interest. During the past decade, an improved understanding of the early pathophysiologic response of the kidney to stress has uncovered a number of genes and proteins that are rapidly induced in the kidney. They have been implicated in the regulation of novel pathways and mechanisms that modulate the kidney injury. Serendipitously, some of these kidney proteins are also detected in the urine and/or plasma, and are emerging as early non-invasive biomarkers of AKI and its clinical outcomes. Table 75.1 lists the desirable characteristics of AKI biomarkers in general, and illustrates the current status of the four most promising novel AKI biomarkers whose bench-to-bedside translation is chronicled in this chapter. Table 75.2 summarizes the biological characteristics of these and other proposed AKI biomarkers. Since NGAL represents the most extensively studied of the novel biomarkers, it will be the primary focus of this chapter.
Table 75.1
Desirable Characteristics of Acute Kidney Injury Biomarkers
| Property | NGAL | KIM-1 | IL-18 | L-FABP |
|---|---|---|---|---|
| Noninvasive (measured in urine or blood) | Yes | Yes | Yes | Yes |
| Rapid, standardized clinical platforms available | Yes | Yes | No | No |
| Sensitive to establish an early diagnosis of AKI | Yes | Yes | Yes | Yes |
| Results available while damage is limitable | Yes | Yes | Yes | Yes |
| High gradient to allow severity prediction | Yes | Yes | Yes | Yes |
| Specific to intrinsic AKI (versus pre-renal AKI) | Yes | Unknown | Unknown | Unknown |
| Discerns AKI from chronic kidney disease | No | No | No | No |
| Predicts hard clinical outcomes | Yes | Yes | Yes | Yes |
| Predicts response to therapies | Yes | Unknown | Unknown | Unknown |
| Associated with a known mechanism | Yes | Yes | Yes | Yes |
AKI: acute kidney injury; NGAL: neutrophil gelatinase-associated lipocalin; KIM-1: kidney injury molecule-1; IL-18: interleukin 18; L-FABP: liver type fatty acid binding protein.
Table 75.2
Biological Characteristics of Promising Acute Kidney Injury Biomarkers
| Biomarker | Sample | Origin | Biological Function |
|---|---|---|---|
| NGAL | Urine | Distal tubule, collecting duct | Regulates iron trafficking, promotes tubule cell survival and proliferation, limits tubule cell apoptosis |
| NGAL | Blood | Liver, lung, neutrophils | Acute phase reactant, marker of organ cross-talk following acute kidney injury |
| KIM-1 | Urine | Proximal tubule | Promotes epithelial regeneration, regulates tubule cell apoptosis |
| IL-18 | Urine | Proximal tubule | Initiates and promotes tubule cell apoptosis and necrosis |
| L-FABP | Urine | Proximal tubule | Endogenous antioxidant, suppresses tubulointerstitial damage |
| NAG | Urine | Proximal tubule | Marker of proximal tubule lysosomal enzyme release as a result of damage to proximal tubule |
| β2-MG | Urine | Systemic and Proximal tubule | Marker of altered glomerular permeability and/or decreased proximal tubular reabsorption due to damage |
| Albumin | Urine | Systemic and Proximal Tubule | Marker of altered glomerular permeability and/or decreased proximal tubular reabsorption due to damage |
NGAL: neutrophil gelatinase-associated lipocalin; KIM-1: kidney injury molecule-1; IL-18: interleukin 18; L-FABP: liver type fatty acid binding protein; NAG: n-acetyl glucosaminidase; β2-MG; beta2-microglobulin.
Neutrophil Gelatinase-Associated Lipocalin (NGAL) as an AKI Biomarker
NGAL Physiology and Pathophysiology
Human NGAL was originally identified as a novel protein isolated from secondary granules of human neutrophils, and subsequently shown to be a 25-kDa protein covalently bound to neutrophil gelatinase. Mature peripheral neutrophils lack NGAL mRNA expression, and NGAL protein is synthesized at the early myelocyte stage of granulopoiesis during formation of secondary granules. NGAL mRNA is normally expressed in a variety of adult human tissues, including bone marrow, prostate, salivary gland, stomach, colon, trachea, lung, liver, and kidney. Several of these tissues are prone to exposure to microorganisms, and constitutively express NGAL protein at low levels. The promoter region of the NGAL gene contains binding sites for a number of transcription factors, including NF-κB. This could explain the constitutive as well as inducible expression of NGAL in several of the non-hematopoietic tissues. Like other lipocalins, NGAL forms a barrel-shaped tertiary structure with a hydrophobic calyx that binds small lipophilic molecules. The major ligands for NGAL are siderophores, small iron-binding molecules. Teleologically, NGAL comprises a critical component of innate immunity to bacterial infection. Siderophores are synthesized by bacteria to scavenge iron from the surroundings, and use specific transporters to recover the siderophore-iron complex, ensuring their iron supply. The siderophore-chelating property of NGAL therefore renders it as a bacteriostatic agent. Experimental evidence for this role is derived from mice genetically modified to lack the NGAL gene, which renders them more susceptible to Gram-negative bacterial infections and death from sepsis.
On the other hand, siderophores produced by eukaryotes participate in NGAL-mediated iron shuttling that is critical to various cellular responses such as proliferation and differentiation. This property provides a molecular mechanism for the documented role of NGAL in enhancing the epithelial phenotype. During kidney development, NGAL promotes epithelial differentiation of the mesenchymal progenitors, leading to the generation of glomeruli, proximal tubules, Henle’s loop, and distal tubules. However, NGAL expression is also markedly induced in injured epithelial cells, including the kidney, colon, liver and lung. This is likely mediated via NF-κB, which is known to be rapidly activated in epithelial cells after acute injuries, and plays a central role in controlling cell survival and proliferation. In the context of an injured mature organ such as the kidney, the biological role of NGAL induction is one of marked preservation of function, attenuation of apoptosis, and an enhanced proliferative response. This protective effect is dependent on the chelation of toxic iron from extracellular environments, and the regulated delivery of siderophore and iron to intracellular sites. Not surprisingly, gene expression studies in AKI have demonstrated a rapid and massive upregulation of NGAL mRNA in the distal nephron segments—specifically in the thick ascending limb of Henle’s loop and the collecting ducts. The resultant synthesis of NGAL protein in the distal nephron and secretion into the urine comprises the major fraction of urinary NGAL. Although plasma NGAL is freely filtered by the glomerulus, it is largely reabsorbed in the proximal tubules. Thus, any urinary excretion of NGAL is likely only when a kidney disease precludes proximal tubular NGAL reabsorption, and/or induces distal tubular de novo NGAL synthesis. With respect to plasma NGAL, the kidney itself does not appear to be a major source. NGAL protein released into the circulation from distant organs such as the liver and lung constitute a distinct systemic pool. Additional contributions to the systemic pool may derive from activated neutrophils, macrophages, and other immune cells. Furthermore, any decrease in GFR would decrease the renal clearance of NGAL, with subsequent accumulation in the systemic circulation in patients with CKD.
Preclinical transcriptome profiling studies identified Ngal (also known as lipocalin 2 or lcn2 ) to be one of the most upregulated genes in the kidney very early after acute injury in animal models. Downstream proteomic analyses also revealed NGAL to be one of the most highly induced proteins in the kidney after ischemic or nephrotoxic AKI in animal models. The serendipitous finding that NGAL protein was easily detected in the urine soon after AKI in animal studies has inspired a number of translational human studies, and NGAL has emerged as an excellent biomarker in the urine and plasma for early diagnosis, therapeutic monitoring, and prediction of prognosis in common clinical AKI scenarios. The deployment of standardized clinical platforms for the rapid and accurate measurement of NGAL in urine and plasma has further facilitated the widespread use and validation of NGAL as a biomarker.
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