Neutrophil Gelatinase-Associated Lipocalin in Acute Kidney Injury

ADVANCES IN CLINICAL CHEMISTRY, VOL. 58

NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN IN ACUTE KIDNEY INJURY Konstantinos Makris*,1 and Nikolaos Kafkas† *Clinical Biochemistry Department, KAT General Hospital, Athens, Greece † Cardiology Department, KAT General Hospital, Athens, Greece

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Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definition and Epidemiology of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology of AKI in Hospitalized Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Pathophysiology of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Urine Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Urinary Biochemistry and Indices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Urine Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Serum Creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. RIFLE and Acute Kidney Injury Network. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Are the Characteristics of an Ideal Biomarker for AKI? . . . . . . . . . . . . . . . . . . The Biology of NGAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Structure and Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Functional Roles of NGAL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. NGAL and Antibacterial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. NGAL in Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. NGAL and Neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. NGAL in Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7. NGAL in Cardiovascular Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8. NGAL Acts as a ‘‘Stress Protein’’. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of NGAL Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NGAL as Biomarker of Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. The Physiologic Role of NGAL in AKI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. The Biologic Sources of NGAL Following AKI. . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. NGAL for AKI Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. AKI and Cardiorenal Syndrome: Potential Role of NGAL. . . . . . . . . . . . . . . .

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Corresponding author: Konstantinos Makris, e-mail: [email protected] 141

0065-2423/12 $35.00 DOI: 10.1016/B978-0-12-394383-5.00012-6

Copyright 2012, Elsevier Inc. All rights reserved.

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9.5. Further Roles of NGAL in Kidney Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6. Limitations of NGAL as Biomarker of AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations 24p3R ADQI AKI AKIN AMI ARF ATN AUC-ROC BV CAD CPB CIN CKD CRS CV CVD DGF ED ELISA EPO FENa FEUN GFR ICU MMP-9 mRNA NAC NF-kB NGAL NIH NKFKDOQI RIFLE

24p3 cell-surface receptor The Acute Dialysis Quality Initiative acute kidney injury Acute Kidney Injury Network acute myocardial infarction Acute Renal Failure acute tubular necrosis area under the ROC curve biological variation coronary artery disease cardio-pulmonary bypass contrast-induced nephropathy chronic kidney disease cardiorenal syndrome coefficient of variation cardiovascular disease delayed graft function emergency department enzyme-linked immunosorbent assay erythropoietin fractional excretion of filtered sodium fractional excretion of urea nitrogen Glomerular Filtration Rate intensive care unit matrix metalloproteinase-9 messenger RNA N-acetylcysteine nuclear factor-kB neutrophil gelatinase-associated lipocalin National Institute of Health National Kidney Foundation-Disease Outcomes Quality Initiative Risk, Injury, Failure, Loss and End-stage renal disease

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ROC SA UA VEGF

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receiver operating characteristic curve stable angina unstable angina vascular endothelial growth factor

1. Abstract Acute kidney injury (AKI) is recognized as an independent risk factor for morbidity and mortality. Unfortunately, this syndrome was historically underdiagnosed due to inconsistent definition of AKI as well as insensitive and nonspecific diagnostic tools. Recent advances in defining AKI, understanding its pathophysiology, and improving its diagnostic accuracy have an impact in disease management and clinical outcome. Prompt recognition and treatment of AKI still remains the cornerstone of clinical management of this syndrome. This chapter focuses on the recent advances in diagnosis of AKI using novel serum and urine biomarkers. The role of neutrophil gelatinaseassociated lipocalin (NGAL) in pathophysiology and diagnosis of AKI is presented. A detailed analysis of the biology of NGAL and presentation of laboratory methods of measurement is also provided. The role of NGAL as biomarker beyond the boundaries of nephrology is also presented.

2. Definition and Epidemiology of AKI AKI is a complex clinical condition triggered by several etiological factors. AKI lacks satisfactory therapeutic management and presents with increasing frequency among hospitalized patients causing enormous medical costs worldwide [1]. Conservative estimates have placed the annual healthcare expenditures attributable to hospital-acquired AKI at greater than 10 billion dollars in the United States alone [2,3]. AKI is currently recognized as the preferred nomenclature for the clinical disorder formerly known as acute renal failure (ARF). It is a common and potentially life-threatening condition that can occur in multiple clinical settings including the emergency department (ED) and the intensive care unit (ICU). AKI is defined as an abrupt (sometimes within hours) and sustained decrease in renal function resulting in the retention of nitrogenous (urea and creatinine) and nonnitrogenous waste products. Depending on the severity and duration of the renal dysfunction, this accumulation is accompanied by metabolic disturbances such as metabolic acidosis and hyperkalemia, changes in body fluid balance, and effects on many other organs.

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AKI affects approximately 5–7% of all hospitalized patients, depending on their illness and disease severity, the population studied, and the criteria of classification [4–6]. The incidence of AKI in the ICU is even higher (about 25%) with a mortality rate of 50–80%. In a recent multicenter study involving critically ill patients, the overall prevalence of AKI patients was 5.7% with a mortality rate of 50–80% [4]. In this study of the patients who survived AKI to hospital discharge, 13% remained dialysis dependent. In another prospective study involving 4622 medical and surgical patients admitted in tertiary hospital, renal insufficiency developed in 7.2%. The overall mortality rate observed in this study was 19.4% and was similar among patients with all causes of renal insufficiency [7].

3. Etiology of AKI in Hospitalized Patients Causes of AKI can be broadly divided into three categories (Figure 1). In the prerenal AKI (or as often called prerenal azotemia), where there is a reversible increase in serum creatinine and blood urea concentrations, AKI results from decreased renal perfusion, which leads to a reduction in glomerular filtration rate (GFR), without tubular damage. Postrenal AKI is due to obstruction of the urinary collection system by either intrinsic or extrinsic masses. The remaining cases constitute the intrinsic (renal) form of AKI, in which structures of the nephron, such as tubules, the glomeruli, vessels, or

FIGURE 1 CLASSIFICATION AND MAJOR CAUSES OF AKI Acute kidney injury (AKI)

Prerenal azotemia

Intrinsic renal

Postrenal

Absolute decrease in effective blood volume (hemorrhage, volume depletion) Relative decease in blood volume (ineffective arterial volume as seen in heart failure) Occlusion or stenosis of renal artery Hemodynamic from drugs (NSAID, ACE inhibitors, or angiotensin II) Vascular vasculitis, hypertension Acute glomerulonephritis, postinfectious glomerulonephritis Acute interstitial nephritis (drug associated) Acute tubular Ischemic necrosis Nephrotoxic Endogenous (ATN) Exogenous (antibiotics, cisplatin, radiocontrast agents) Obstruction of collecting system

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interstitium are affected. The major cause of AKI is tubular necrosis. This disorder is caused by ischemic or nephrotoxic injury of the kidney and is a specific histopathologic and pathophysiologic entity, which can result from several distinct renal insults. Prerenal azotemia and ischemic acute tubular necrosis (ATN) occur on a continuum of the same pathophysiologic process and together account for 75% of the AKI cases [7]. Although the terms AKI and ATN have quite different definitions, they are commonly used synonymously in the literature. Factors contributing to the development of AKI in hospitalized patients can be classified as modifiable or nonmodifiable. Nonmodifiable risk factors are mostly those that relate to renal aging and an increased number of comorbidities. Older age is also a risk factor for AKI. The most important modifiable risk factors are dehydration, hypovolemia, toxicities related to medications or contrast agents, surgery-related issues, and some factors involved in septic AKI and the cardiorenal syndrome (CRS) (Table 1). A number of studies have attempted to determine the etiology of AKI in hospitalized patients [7–10]. In several of these papers, the researchers who reviewed the charts subjectively attributed a cause without the use of specific predefined criteria, which can certainly introduce bias. A large prospective study by Nash et al. is one of the few studies which used predefined and very detailed criteria to determine the causes or mechanisms for AKI development in hospitalized patients [7]. These researchers found that the most common causes and mechanisms of AKI, in decreasing order of frequency, were decreased renal perfusion (including volume depletion, hypotension, and/or

TABLE 1 MOST COMMON CAUSES OF AKI IN HOSPITALIZED PATIENTS Nonmodifiable factors

Modifiable factors

Age-related changes in the kidney Chronic kidney disease Cardiovascular disease Hypertension Diabetes

Hypovolemia Sepsis Systemic inflammatory response syndrome (SIRS) Cardiorenal syndrome (CRS) Impaired renal microcirculation Drug-related toxicity Perioperative factors Contrast-induced nephropathy (CIN) Infection Obstructive uropathy Rhabdomyolysis Glomerulonephritis Hypotension

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congestive heart failure), medications, radiographic contrast media, postoperative factors, sepsis, transplantation-related factors (liver and heart), obstruction, and hepatorenal syndrome [7]. Another large hospital-based study that prospectively investigated the incidence and causes of ARF development in hospitalized patients also used strict criteria when determining etiology. The authors found that ARF was almost always multifactorial and determined that the most common causes were nephrotoxic drugs, sepsis, hypoperfusion, surgery, and radiocontrast media [8].

4. The Pathophysiology of AKI AKI may occur in three clinical patterns, which are as follows: 1. As an adaptive response to severe volume depletion and hypotension, with structurally intact nephrons; 2. In response to cytotoxic, ischemic, or inflammatory insults to kidney, with structural and functional damage; and 3. With obstruction to the passage of urine. Therefore, in general terms, AKI may be classified as prerenal, intrinsic, and postrenal. While these classifications are useful in establishing a differential diagnosis, many pathophysiologic features are shared among the different categories. The pathophysiology of AKI is quite complex, not very well understood, and to some extent, varies, based on the particular cause of AKI. It has two components: microvascular and tubular (Fig. 2). The microvascular can be further divided into preglomerular and outer medullary vessel components. This complex interplay between vascular and tubular processes ultimately leads to organ dysfunction. AKI is a state often characterized by enhanced intrarenal vasoconstriction; it is also associated with enhanced renal-nerve activity and increased tissue levels of vasoconstrictive agents such as angiotensin II and endothelin. On the other hand, a decreased vasodilation was observed in response to agents that are present in the postischemic kidney. With increased endothelial and vascular smooth muscle cell damage, there was enhanced leukocyte–endothelial adhesion leading to activation of the coagulation system and vascular obstruction with leukocyte activation causing increases in inflammation and providing a positive-feedback network. Inflammation produces increased levels of mediators expanding the interactions between leukocytes and endothelial cells and activating the coagulation pathways. The resultant effects on oxygen and nutrient delivery to the epithelial cells provoke damage to those cells. Further, damaged tubular cells also generate proinflammatory mediators [11–14].

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Microvascular Glomerular

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Tubular Medullary

Increased vasoconstriction renal nerves, adenosine angiotensin II, thromboxane A2 endothelin, leukotrienes

Cytosceletal breakdown

Decreased O2

Decreased vasodilatation acetylocholine, bradykinin nitric oxide, PGE2

Inflammatory vasoactive mediators

Loss of polarity Apoptosis and necrosis Desquamation of viable and necrotic cells

Increased endothelial and vascular smooth muscle cellular damage

Tubular obstruction

Increased leukocyte–endothelial adhesion

Backleak

vascular obstruction leukocyte activation and inflammation

FIG. 2. The pathophysiology of AKI. The pathophysiology of AKI may be divided into microvascular and tubular components; the former can be further divided into preglomerular and outer medullary vessel components. With AKI, there is enhanced vasoconstriction and decreased vasodilatation in response to agents that are present in the postischemic kidney. With increased endothelial and vascular smooth muscle cellular damage, there is enhanced leukocyte–endothelial adhesion leading to activation of the coagulation system and vascular obstruction with leukocyte activation and potentiation of inflammation. At the level of the tubule, there is cytoskeletal breakdown and loss of polarity followed by apoptosis and necrosis, intratubular obstruction, and backleak of glomerular filtrate through a denuded basement membrane. In addition, the tubule cells generate inflammatory vasoactive mediators that, in turn, can affect the vasculature to enhance vascular compromise. A positive-feedback mechanism ensues whereby the vascular compromise results in decreased oxygen delivery to the tubules that, in turn, generate vasoactive inflammatory mediators to enhance the vasoconstriction and the endothelial–leukocyte interactions (adapted from Ref. [11]).

Whether injury is related to oxygen deprivation, toxins, or a combination of factors (as more often is the case), there are many common features in the epithelial cell response. Insult results in rapid loss of cell polarity and cytoskeletal integrity (Fig. 3). The proximal tubule brush border sheds and there is a loss of polarity with the mislocalization of adhesion molecules and other membrane proteins (e.g., adenosine triphosphatase and b-integrins), apoptosis, and necrosis ensues. With severe injury, viable and nonviable cells are desquamated, leaving regions where the basement membrane remains the only barrier between filtrate and the peritubular interstitium. These cells and their debris combine with proteins present in the tubular lumen and they enter the lumen, forming casts that can obstruct the tubule, increase intratubular pressure, and appear in the urine of patients as a hallmark of AKI. This increased intratubular pressure results in the reduction of the glomerular transcapillary

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Normal epithelium

Calcium ROS Purine depletion Phospholipases

Necrosis

Loss of polarity

Apopresis

Cell death

Potential urinary biomarkers for early diagnosis of AKI NAG β2M α1M RBP Cystatin C KIM-1 Clusterin Microalbumin

GFR

Delayed biomarkers for kidney injury

NGAL CYR-61 IL-18 OPN FABP NHE3 Fetuin A

Serum creatinine Blood urea nitrogen

Differentiation & reestablishment of polarity

Proliferation

Sloughing of viable and dead cells with luminal obstruction

Migration, dedifferentiation of viable cells

Adhesion molecules Na+/K+-ATPase

FIG. 3. Injury-repair to the epithelial cell of the kidney with ischemia/reperfusion. Use of early and late biomarkers for recognition and intervention. Early response to kidney injury is the loss of the brush border and the polarity of the epithelial cell with mislocation of adhesion molecules and Naþ/Kþ-ATPase and other proteins. With increasing injury, there is cell death by either necrosis or apoptosis. Some of the necrotic debris is then released into the lumen, where it interacts with luminal proteins and can ultimately result in obstruction. In addition, with the mislocation of adhesion molecules, viable epithelial cells lift off the basement membrane and are found in the urine. The kidney can respond to the injury by initiating a repair process, if there are sufficient nutrients and sufficient oxygen delivery, and the basement membrane integrity has not been altered irreparably. Viable epithelial cells migrate and cover denuded areas of the basement membrane. The source of these cells appears to be from the kidney itself and not from the bone marrow. Bone marrow cells may contribute to the interstitial cellular infiltrate and may produce factors to modulate inflammation and facilitate repair. Cells replacing the epithelium may derive from differentiated epithelial cells or from a subpopulation of progenitor cells in the tubule; the cells undergo division and replace lost cells. Ultimately, the cells go on to differentiate and reestablish the normal polarity of the epithelium (adapted from Ref. [11]).

hydrostatic pressure gradient with resulting reductions in the GFR and, combined with the loss of normal epithelial barrier function, allows for backleak of the filtrate. The activation and injury of the epithelium result in the generation of inflammatory and vasoactive mediators. These have autocrine and paracrine effects on adjacent tubular epithelial cells and act on the

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vasculature to worsen vasoconstriction and inflammation. Therefore, inflammation contributes in a critical way to the pathophysiology of AKI [11–14]. The kidney can respond to the injury by initiating a repair process if there are sufficient nutrients and sufficient oxygen delivery and the basement membrane integrity has not been altered irreparably. Repair involves the replacement of lost cells in the tubule by mechanisms that at present are very well understood. The kidney can recover from an ischemic or toxic insult resulting in cell death, although it is recognized that there are longer term detrimental effects from even brief periods of ischemia. The surviving cells that remain adherent undergo repair and potentially can recover normal renal function. Also, viable epithelial cells migrate and cover the denuded areas of the basement membrane. The source of these cells appears to be the kidney itself and not the bone marrow. Proximal tubules are able to undergo repair after ischemic or nephrotoxic damage. While cell death itself is not a regenerative response, epithelial cells in the process of dying may generate signals that initiate the repair response. Cytokines may play a role in determining the fate of the epithelial cells, contribute to the generation of signals that result in neutrophil and monocyte infiltration into the tissue, and promote dedifferentiation and proliferation of epithelial cells. These cytokines may derive from the kidney tissue, epithelial and mesenchymal cells, or infiltrating cells such as macrophages.

5. Diagnosis of AKI AKI is an underrecognized illness, mainly because we lack early biomarkers for the condition, unlike myocardial infarction, in which early markers such as troponin have advanced our understanding and diagnosis of this condition. In current clinical practice, the diagnosis of AKI is made on the basis of the presence of an increased serum creatinine level, a decreased urine output, and/or increased blood urea nitrogen levels. A summary of the traditional laboratory tests used to diagnose AKI are presented in Table 2. 5.1. URINE OUTPUT Urine output is a commonly measured parameter of kidney function in AKI. Following of urine output can be advantageous because it is a dynamic gage of kidney function and is measured continuously. Urine output can be a more sensitive barometer for changes in renal hemodynamics than biochemical markers of solute clearance. Dynamic changes to urine output have been integrated into the Risk, Injury, Failure, Loss and End-stage renal disease (RIFLE) classification of AKI [15]. However, the urine output is also of

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MAKRIS AND KAFKAS TABLE 2 TRADITIONAL METHODS OF INVESTIGATION OF ACUTE KIDNEY INJURY

Test

Comment

Urinalysis Dipstick for blood, protein, or both Microscopy for casts, crystals Urine volume Urine indices Biochemistry Serial serum urea creatinine Electrolytes, Ca, P Blood gas analysis, serum bicarbonate Creatine kinase (CK), myoglobinuria C-reactive protein Hematology Full blood count Coagulation studies Radiology Renal ultrasonography

Suggest renal inflammatory process Red cell casts diagnostic in glomerulonephritis Acute anuria or severe oliguria are quite specific indicators of AKI Can help discriminate between AKI and prerenal azotemia Can help establish AKI diagnosis Important metabolic consequences of AKI include hyperkalemia, metabolic acidosis, hypocalcemia, hyperphosphatemia Markedly elevated serum CK and myoglobinuria suggest rhabdomyolysis Nonspecific marker of inflammation Eosinophilia may be present in acute interstitial nephritis, cholesterol embolism, or vasculitis Disseminated intravascular coagulation associated with sepsis Renal size, symmetry, evidence of obstruction

limited sensitivity and specificity, with patients capable of developing severe AKI, as detected by a markedly elevated serum creatinine, while maintaining normal urine output (i.e., nonoliguric AKI). Because nonoliguric AKI has been described as having a better outcome than oliguric AKI, urine output is frequently used to differentiate AKI; however, the value of this distinction is questionable and can be frequently negated by the use of diuretics [16]. Oliguria has classically been defined as urine output of approximately < 5 ml/kg/day or 0.5 ml/kg/h.

5.2. URINARY BIOCHEMISTRY AND INDICES Classic tests of urinary biochemistry and derived indices have been described and traditionally used to aid clinicians for the detection and classification of AKI into prerenal azotemia and the so-called ATN or established AKI. These tests and indices are outlined (Table 3).

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TABLE 3 LABORATORY TESTS USED TO HELP DIAGNOSE ‘‘ESTABLISHED’’ ACUTE KIDNEY INJURY Test

Prerenal azotemia

AKI

Urine sediment

Normal

Specific gravity Urine sodium (mmol/l) FENa FEUN Urine osmolality U/P creatinine ratio Urea/creatinine plasma ratio

High >1.020 Low <10 <1% <35% High >500 High >40 High

Epithelial casts ‘‘Muddy’’ brown granular casts Cellular debris Low <1.020 High >10 >1% >35% Near serum values <300 Low <10 Normal

U, urine; P, plasma.

Of these, the fractional excretion of filtered sodium (FENa) may help to differentiate established AKI from prerenal azotemia [17–19]. Filtered sodium is avidly reabsorbed in the renal tubules from glomerular filtrate in the setting of prerenal AKI, resulting in an FENa < 1%, whereas in the setting of renal tubular injury in established AKI, the resulting FENa is > 1%. However, the diagnostic utility of the FENa has been questioned, since diuretics decrease sodium reabsorption and thus increase FENa [20–23]. In contrast, the fractional excretion of urea nitrogen (FEUN) is primarily dependent on passive forces and is therefore less influenced by diuretic therapy. Therefore, FEUN has been cited as a more precise method for discriminating early AKI, in particular, if concomitant diuretic therapy has been given, with an FEUN < 35% indicating prerenal azotemia and > 35% consistent with ATN [24]. In a study by Carvounis et al., where AKI was classified as prerenal AKI by diuretic exposure or ATN, an FEUN < 35% was evident in 90%, 89%, and 4% of patients for cases of prerenal, prerenal with diuretics, and ATN, respectively [22]. This study found that FEUN was superior in sensitivity and specificity compared with FENa for classifying AKI. There are numerous additional urinary biochemical tests and derived indices that have been reported that aim to further improve our capability to discriminate prerenal AKI from established AKI. These have included urinary sodium concentration, urine to plasma creatinine ratio, urine to plasma urea ratio, serum urea to creatinine ratio, and urine uric acid to creatinine ratio fractional excretion of uric acid. Due to the paucity of studies, the significance of each of these measures remains largely unproven. In summary, considering the evidence available, the clinical utility of the urinary biochemical tests and the derived indices in the diagnosis, classification,

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and prognosis of AKI in hospitalized and critically ill patients who often receive diuretics, vasopressor infusions, radiocontrast media, nephrotoxic drugs, and fluid resuscitation remains questionable [21,25]. The significance of these tests was recently reviewed in the context of septic AKI [25]. This study concluded that there was no single urinary test that could reliably be used to diagnose, classify, or predict the course of septic AKI. Finally, we must recognize that prerenal azotemia and established AKI are parts of a continuum and their separation into two different clinical entities is rather arbitrary with little clinical significance since therapeutic interventions are similar. 5.3. URINE MICROSCOPY The urine sediment might also be helpful to differentiate between prerenal azotemia and AKI. An active sediment with renal tubular epithelial cells, cellular debris, and ‘‘muddy brown’’ broad tubular cell cast supports the diagnosis of ATN. Large amounts of urinary protein (> 3.0 g/24 h) and numerous red blood cell casts are indicative of AKI secondary to acute glomerulonephritis or vasculitis. The absence of cellular elements and protein in urine is most compatible with prerenal and postrenal azotemia. Presence of crystals in urine is also indicative of AKI. A recent review concluded that urine microscopy with sediment examination can be of value differentiating ATN from prerenal azotemia. They also found that the presence of renal tubular epithelial cells and renal epithelial casts and or granular casts in the urine sediment may help the diagnosis and are useful in predicting more severe kidney damage (nonrecovery of AKI and need for dialysis) [26]. 5.4. SERUM CREATININE Nitrogenous compounds accumulated in blood (blood urea nitrogen and serum creatinine) are routinely used for AKI diagnosis. In clinical practice, AKI is detected when serum creatinine concentration increases over a short period of time with or without oliguria. The measurement of serum markers for AKI may be useful in patients with severe oliguria as well as in patients under diuretic therapy and changes in hydration status. Unfortunately, serum creatinine is an unreliable marker of acute renal dysfunction in most patients for several reasons. First, increased serum creatinine is not specific for AKI and require differentiation from other prerenal or extrarenal causes of azotemia. Second, serum creatinine is not specific for renal tubular lesions, pathogenetically related to AKI development, but rather reflecting the loss of glomerular filtration function that accompany the development of AKI. Third is the delayed increase after renal insult. Indeed, studies have shown that changes in serum creatinine

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may lag behind changes in GFR by several days [27]. In addition, renal function must reach a steady state before the serum creatinine level can be of diagnostic value. The aim of achieving an early diagnosis of AKI with serum creatinine measurement is therefore impossible. Fourth, serum creatinine is a poor marker of kidney dysfunction as changes in its concentration are neither specific nor sensitive in response to slight GFR alterations and become apparent only when the kidneys have lost more than 50% of their functional capacity. Another major weakness of using serum creatinine level as a diagnostic tool is the variable rate of production of creatinine, which is highly dependent on muscle mass. This drawback is of greatest importance among older patients, since muscle mass decreases continuously with age as it is replaced by adipose tissue [28]. Serum creatinine levels may vary with intravascular volume expansion or depletion and with hemodynamic changes, while renal parenchymal structure and function remain unaffected [29]. As a result of hemodilution or hemoconcentration, the absolute value of serum creatinine can fluctuate considerably in an individual patient according to hydration status and vascular tone, regardless of renal function [29]. This problem is also important among older patients as they are particularly susceptible to the development of dehydration. Consequently, for the same degree of renal dysfunction, serum creatinine will generally tend to be much lower in elderly patients [30]. Finally, sex and nutritional status also affect serum creatinine. This problem often leads to delayed recognition of AKI and late initiation of treatment, as unfortunately many physicians still base clinical decisions on arbitrarily determined serum levels of creatinine and urea. Diagnostic criteria also include urine output in the critically ill patient. Lowered urine output tends to signal renal dysfunction before serum creatinine rises. Moreover, in contrast to ICU patients, serum creatinine is seldom drawn on a daily basis in most hospitalized patients not in the ICU, and urine output cannot be measured reliably in the absence of an indwelling catheter. These considerations make the use of conventional criteria for the diagnosis of AKI even less appealing in hospitalized patients who are not in the ICU. 5.5. RIFLE AND ACUTE KIDNEY INJURY NETWORK Another major drawback to the diagnosis and the successful implementation of new therapies is the lack of a consensus definition of AKI. The Acute Dialysis Quality Initiative (ADQI), an international interdisciplinary workgroup, found that over 30 definitions for ARF were used in the literature. The definitions varied from a 25% increase over baseline serum creatinine to the need for dialysis [31]. The term AKI is of relatively recent origin and was proposed to better account for the diverse spectrum of molecular, biochemical, and structural processes that characterize AKI [32].

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In order to better classify AKI, the ADQI developed a consensus classification scheme for defining AKI: the RIFLE (reflecting the terms risk, injury, failure, loss, and end-stage renal disease) classification system [15,33]. The proposed classification is based on serum creatinine GFR and urine output. The first three classes represent degrees of injury and the last two are outcome measures. This system is easy to utilize, has a clinical applicability that is sensitive and specific for different populations, considers serum creatinine from baseline, and is applicable for both acute and chronic kidney disease (CKD). The classification system is illustrated in Table 4. RIFLE has also shown to correlate well with mortality rates [34]. However, the use of the RIFLE criteria for the detection of AKI requires a ‘‘wait-and-watch’’ strategy because of the time needed between initial insult and increased serum creatinine. In order to further refine the definition of AKI, the Acute Kidney Injury Network (AKIN) proposed a modified version of the RIFLE classification, known as the AKIN criteria. The AKIN criteria define AKI as an abrupt (within 48 h) reduction in kidney function as measured by an absolute increase in serum creatinine  0.3 mg/dl, a percentage increase in serum creatinine  50%, or documented oliguria (< 0.5 ml/kg/h) for more than 6 h [35]. Minor modifications of the RIFLE criteria include broadening the ‘‘risk’’ category of RIFLE to include an increase in serum creatinine of at least 0.3 mg/dl in order to increase the sensitivity of RIFLE for detecting AKI at an earlier time point. In addition, the AKIN criteria sets a window on

TABLE 4 RIFLE CRITERIA (ACUTE DIALYSIS QUALITY INITIATIVE)

Stage

Serum creatinine (sCr) criteria

GFR criteria

R ¼ risk

Increase in sCr  1.5 from baseline

I ¼ injury

Increase in sCr  2.0 from baseline

F ¼ failure

Increase in sCr  3.0 from baseline OR sCr  4.0 mg/dl (in the setting of an acute rise  0.5 mg/dl)

Decrease in GFR  25% Decrease in GFR  50% Decrease in GFR  75%

L ¼ loss

Persistent loss of kidney function >4 weeks Persistent failure >3 months

E ¼ end-stage renal disease (ESRD)

Adapted from Ref. [15].

Urine output criteria <0.5 ml/kg/ h for > 6 h <0.5 ml/kg/ h for >12 h <0.3 ml/kg/ h for >24 h or anuria for >12 h

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first documentation of any criteria to 48 h and categorizes patients in the ‘‘failure’’ category of RIFLE if they are treated with renal replacement therapy, regardless of either changes in creatinine or urine output. Finally, AKIN replaces the three levels of severity R, I, and F with stages 1, 2, and 3 (Table 5) [35,36]. The major limitation of the current AKI definition is that it is still entirely based upon an increased serum creatinine or decreased urine volume. Unfortunately, creatinine is a suboptimal marker following acute injury and often nonreflective of GFR due to a number of renal and nonrenal influences on creatinine concentration. In the setting of AKI, the delay between changes in serum creatinine and changes in GFR inhibits the ability of accurately estimating the time of injury and the severity of dysfunction following injury [37]. A sudden falling GFR to a constant low level causes a gradual increase in serum creatinine until a new steady state between generation and excretion is achieved. The rate of rise of serum creatinine following AKI is dependent on many factors, including the new GFR, the rate of tubular secretion, rate of generation, and volume of distribution [37,38]. As a result, large changes in GFR may be associated with relatively small changes in serum creatinine in the first 24–48 h following AKI, resulting not only in delayed diagnosis and intervention but also in underestimation of the degree of injury. In addition, there is considerable variability among patients in the correlation between serum creatinine and baseline GFR, in the magnitude of functional renal reserve, and in creatinine synthesis rates. As a result, a renal injury of comparable magnitude may result in disparate alterations in creatinine concentration in different patients [15]. There is an urgent need for better biomarkers to permit more timely diagnosis of AKI, better prediction of injury severity, and safety assessment

TABLE 5 AKIN CRITERIA (ACUTE DIALYSIS QUALITY INITIATIVE) Stage

Serum creatinine (sCr) criteria

GFR criteria

Urine output criteria

1

Increase in sCr  1.5  from baseline or absolute increase of 0.3 mg/dl Increase in sCr  2.0  from baseline

Decrease in GFR  25% Decrease in GFR  50% Decrease in GFR  75%

<0.5 ml/kg/h for >6 h

2 3

Increase in sCr  3.0  from baseline OR sCr  4.0 mg/dl (in the setting of an acute rise  0.5 mg/dl) Adapted from Ref. [35].

<0.5 ml/kg/h for >12 h <0.3 ml/kg/h for >24 h or anuria for >12 h

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during drug development. The onset of AKI is rapid and conventional tests are often unable to identify the condition in the first 48 h when time is critical. Thus, the potential utility of a novel biomarker as early predictor of AKI has generated great interest.

6. What Are the Characteristics of an Ideal Biomarker for AKI? The quest for biomarkers is as old as medicine itself. From the earliest days of diagnostic medicine, we have been searching for measurable biological quantities that will allow us insight into the physiological workings of the human organism. In its simplest definition, a biomarker is anything that can be measured and can provide us information about a biological state or process. The National Institute of Health (NIH) Biomarkers Definitions Working Group has defined a biological marker (or biomarker) as ‘‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention’’ [39]. Biomarker discovery has relied on intimate knowledge of the pathophysiology of the diseases being studied. Biological substances that we knew were related to a disease state were investigated to see if they could serve as diagnostic markers, provide a target for therapy, or lend further insight into the etiology of the disease. While this can be tedious and relies heavily on prior knowledge of the disease mechanism, this hypothesis-driven method of research almost always provides useful scientific results, whether positive or negative. What are the ideal characteristics of a renal biomarker? To be certain, what constitutes an ideal biomarker is highly dependent upon the disease one is investigating. However, certain universal characteristics are important for any biomarker. Desirable characteristics of a clinically applicable AKI biomarker includes the following: 1. It should be noninvasive. 2. Its measurement should be inexpensive, quick and easy, and preferably on a standardized clinical assay platform. 3. It should be from readily available sources, such as blood or urine. 4. It should be easy to perform at the bed-side or in a standard clinical laboratory. 5. It should have high sensitivity allowing early detection of AKI and have no overlap in values between diseased patients and healthy controls. 6. Biomarker levels should aid in risk stratification and possess prognostic value in terms of real outcomes.

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7. It should have a high specificity, being greatly upregulated (or downregulated) specifically in the diseased samples and unaffected by comorbid conditions. 8. Biomarker levels should vary rapidly in response to treatment. 9. It should exhibit strong biomarker performance on statistical analysis, including accuracy testing by receiver-operating characteristic curves. 10. It should be biologically plausible and provide insight into the underlying disease mechanism. In addition to aiding in the early diagnosis and prediction, it should be highly specific for AKI and enable the identification of AKI subtypes and etiologies. AKI is traditionally diagnosed when kidney’s major function, glomerular filtration, is affected and indirectly measured by change in serum creatinine. However, prerenal factors such as volume depletion, decreased effective circulating volume, or alterations in the caliber of the glomerular afferent arterioles all cause elevation in serum creatinine. Also postrenal factors such as urinary tract obstruction similarly result in serum creatinine elevations. Finally, a wide range of intrinsic renal diseases may result in abrupt rise in serum creatinine, especially in hospitalized patients. Other tests to distinguish these various forms of AKI such as microscopic urine examination for casts and determination of FENa have been imprecise and have not enabled efficient clinical trial design. A marker that can distinguish prerenal and postrenal conditions from true intrinsic AKI would be a great improvement. Biomarkers may serve several other purposes in AKI (Table 6). Thus, biomarkers are also needed for (i) identifying the primary location of injury (proximal tubule, distal tubule, interstitium, or vasculature), (ii) pinpointing the duration of kidney failure (AKI, CKD or ‘‘acute-on-chronic’’ kidney injury), (iii) identifying AKI etiologies (ischemia, toxins, sepsis, or a combination), (iv) risk stratification and prognostication (duration and severity of

TABLE 6 IN AKI, BIOMARKERS ARE NEEDED TO DETERMINE 1 2 3 4 5 6 7 8

Location of injury Duration of AKI AKI subtypes AKI etiologies Differentiate from other forms of acute kidney disease Risk stratification and prognostication Defining course of AKI Monitoring interventions

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AKI, need for dialysis, length of hospital stay, mortality), and (v) monitoring the response to AKI interventions. Further, AKI biomarkers may play a critical role in expediting the drug development process. The Critical Path Initiative first issued by the Food and Drug Administration in 2004 stated that ‘‘Additional biomarkers (quantitative measures of biologic effects that provide informative links between mechanism of action and clinical effectiveness) and additional surrogate markers (quantitative measures that can predict effectiveness) are needed to guide product development’’. Collectively, it is envisioned that biomarkers will play an indispensable role in personalizing nephrologic care, by providing a more precise determination of disease predisposition, diagnosis and prognosis, earlier preventive and therapeutic interventions, a more efficient drug development process, and a safer and more fiscally responsive approach to medicine. Not surprisingly, the pursuit of improved biomarkers for the early diagnosis of AKI and its outcomes is an area of intense contemporary research. For answers, we must turn to the kidney itself. Indeed, understanding the early stress response of the kidney to acute injuries has revealed a number of potential biomarkers. The biomarker development process has typically been divided into five phases [40,41] as is shown in Table 7. The preclinical discovery phase requires high-quality, well-characterized tissue, or body fluid samples from carefully chosen animals or human models of the disease under investigation. Usually, tissue analysis utilizes genomic approaches, whereas body fluids are best analyzed by proteomic techniques. Identifying biomarkers in the serum or urine is most desirable since these samples are easy to obtain and allow for

TABLE 7 PHASES OF BIOMARKER DISCOVERYa Phase

Terminology

Action step

1

Preclinical discovery

2

Assay development

3

Retrospective studies

4

Prospective screening

5

Disease control

          

a

Created from Refs. [2,3].

Discover biomarkers in tissues or body fluids Confirm and prioritize candidates Develop and optimize clinically useful assay Test on existing samples of established disease Test biomarker in completed clinical trials Test if biomarker detects the disease early Evaluate sensitivity and specificity Use biomarker to screen population Identify extent and characteristics of disease Identify false referral rate Determine impact of screening on reducing disease burden

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noninvasive testing. Urine is more likely to contain biomarkers arising from the kidney, more applicable for easy patient self-testing, and easier to proteomic screening due to the limited number of protein species present in normal urine. The major limitations of urine samples are that they are more prone to protein degradation, and biomarker concentrations are confounded by changes in urine flow rate. On the other hand, serum samples are readily available even in anuric patients and serum biomarkers exhibit better stability. However, serum biomarkers may reflect systemic response to a disease process rather than specific organ involvement, and the presence of a large number of proteins usually present in the serum makes proteomic approaches difficult. Technologies such as functional genomics and proteomics have accelerated the rate of novel biomarker discovery; for example, microarrays or cDNA chips allow investigators to search thousands of genes simultaneously. Such gene expression profiling studies have identified several genes whose protein products are investigated as potential AKI biomarkers [42,43]. However, microarray-based methods cannot be used for the direct analysis of body fluids, and usually confirmation by proteomic techniques is required before clinical use. Proteomics is the study of both structure and function of proteins by a variety of methods such as gel electrophoresis, immunoblotting, mass spectrometry, and enzymatic or metabolic assays. Each method provides different type of information and each has its own strengths and limitations Several promising candidates for clinical use as biomarkers in AKI are under investigation (phases 3 and 4), and some have already been approved for clinical use in many countries (Table 8). The current status of NGAL as an AKI biomarker is further discussed below.

7. The Biology of NGAL 7.1. STRUCTURE AND EXPRESSION Lipocalins constitute a family of over 20 small secreted proteins defined on the basis of their three-dimensional structure, that is characterized by eight b strands that form a b-barrel, which forms an enclosing calyx. The calyx is capable of binding and transporting a wide variety of low molecular weight molecules [44], which are thought to define the biological activity of the lipocalin. This unique structure renders lipocalins as efficient shuttles and transporters for diverse substances such as retinoids, arachidonic acid, prostaglandins, fatty acids, pheromones, steroids, and iron. To mention a few examples, retinol-binding protein binds and transports vitamin A [45], the lipocalin a1-microglobulin scavenges heme [46].

TABLE 8 MAJOR URINARY AND SERUM BIOMARKERS USED THAT WERE EVALUATED IN STUDIES FOR THE EARLY DETECTION OF ACUTE RENAL INJURY a/a

Biomarker

Origin

Laboratory measurement

Clinical value

N-acetyl-b-Dglucosaminidase (NAG)

Lysosomal enzyme involved in the breakdown metabolism of glucoproteins

Enzymatic method, spectrophotometrically

a and p GlutathioneS-transferase (GST)

Cytoplasmic enzymes found in proximal and distal tubular epithelial cells, respectively Brush border membrane enzyme

Enzymatic method, spectrophotometrically – ELISA Enzymatic method, spectrophotometrically

Increased activity may suggest tubular cell injury or increased lysosomal activity without cell disruption Increased urinary excretion implies cellular necrosis

Expressed on the surface of all nucleated cells, filtered freely by glomerulus, and completely reabsorbed but not secreted by proximal tubular cells Synthetized in the liver, filtered freely by glomerulus, and completely reabsorbed and catabolized (but not secreted) by proximal tubular cells Produced by all nucleated cells, filtered freely by glomerulus, and completely reabsorbed but not secreted by proximal tubular cells Filtered freely by glomerulus and completely reabsorbed but not secreted by proximal tubular cells

PENIA–PETIA–ELISA

Urinary tubular enzymes

Alkaline phosphatase (AP) g-Glutamyl-transpeptidase (g-GT) Alanine aminopeptidase Urinary low molecular weight proteins 1

b2-Microglobulin (b2M)

2

a1-Microglobulin

3

Cystatin C

4

Retinol-binding protein

PENIA–PETIA–ELISA

Increased urinary secretion implies injury to the brush border membrane with loss of microvillous surface

Increased urinary b2M excretion has been observed to be an early marker of tubular damage in a number of clinical settings Increase in tubular damage

PENIA–PETIA–ELISA

With impaired renal tubular function levels rising up to 200-fold

PENIA–PETIA–ELISA

Even minor tubular dysfunction leads to increased excretion

AKI biomarkers specifically produced by the kidney (1) Protein products of genes specifically related to AKI 1 Kidney injury molecule 1 Membrane protein expressed and (KIM-1) upregulated in epithelial cells of proximal tubules after ischemic or toxic renal injury 2 Neutrophil gelatinaseExpressed in several human tissues. In associated lipocalin postischemic kidney, upregulated in (NGAL) several nephron segments and accumulated in proximal tubules to colocalize with proliferating cells 3 Cysteine-rich protein 61 Cysteine-abundant heparin-binding (CYR 61) protein is induced in proximal tubular cells as a consequence of renal ischemia

WB–ELISA

Presence in urine associated with greater risk for AKI. More studies needed to determine validated cut-off points

WB–ELISA–PETIA– chemiluminescence

Independent predictor of AKI in various clinical settings

WB

Detected in urine 3–6 h after renal ischemia. Not detected after volume depletion. More studies needed to validate its utility

(2) Urinary cytokines and chemokines 1

Urinary interleukin 18 (u-IL18)

Mediator of inflammation and ischemic tissue injury in many organs. Detection in proximal tubular cells and urine after renal injury (3) Structural and functional proteins of the renal tubule

ELISA

Increased levels in urine allow early diagnosis and severity of AKI in various clinical settings

1

NHE-3

Immunoblot

Increased urinary excretion may be regarded as a specific marker of acute tubular lesion—may differentiate ischemic or toxic AKI from other kidney diseases or prerenal azotemia. More studies needed to determine validated cut-off points

Sodium transporter localized in the apical membrane and subapical endosomes of proximal tubular cells, responsible for 60–70% of reabsorption of the filtered sodium and bicarbonate

PENIA; particle enhanced nephelometric immunoassy, PETIA; particle enhanced turbidimetric immunoassay, WB; western blot.

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NGAL (also known as lipocalin 2, siderocalin, uterocalin, proteinase-3, 24p3, and neu-related lipocalin) is a member of the lipocalin family that has captured the interest of medical world for the past few years. Human NGAL was originally identified as a protein isolated from the secondary granules of human neutrophils [46], and subsequently it was demonstrated to be a 25-kDa glycoprotein covalently linked to neutrophil gelatinase (matrix metalloproteinase-9, MMP-9) [44]. Mature peripheral neutrophils lack NGAL messenger RNA (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 human tissues including the kidney, bone marrow, prostate, uterus, salivary gland, stomach, colon, lung, and the liver [45,47]. Several of these tissues are prone to exposure to microorganisms and consequently express the NGAL protein at low levels. It is strongly expressed in adenomas and inflamed epithelia of the bowel [48], adenocarcinomas of the breast [49], and urothelial carcinomas [50]. The promoter region of the NGAL gene contains binding sites for a number of transcription factors, including the nuclear factor-kB (NF-kB) [45]. This could explain the constitutive, as well as inducible, expression of NGAL in several of the nonhematopoietic tissues. 7.2. FUNCTIONAL ROLES OF NGAL Besides MMP-9, the major ligands of NGAL are siderophores. These are small iron-binding molecules [51]. NGAL binds with the siderophores, transporting them within cells after interacting with specific membrane receptors. In particular, a membrane protein called 24p3 cell-surface receptor (24p3R) represents the most important cellular target of NGAL. The interaction of NGAL with this receptor leads to the internalization of the complex NGAL– siderophore, producing a significant increase in cytoplasmic iron [52,53]. This mechanism governs the numerous effects attributed to NGAL including antibacterial activity, embryogenesis, and neoplastic growth. 7.3. NGAL AND ANTIBACTERIAL ACTIVITY Enterochelin was identified as another significant ligand for NGAL. Enterochelin is a siderophore that binds iron with extremely high affinity. Bacteria produce siderophores in order to scavenge iron from the extracellular space and use specific transporters to recover the siderophore–iron complex, ensuring their iron supply. NGAL’s ability to capture and deplete siderophores renders it as a bacteriostatic agent that prevents growth of those bacterial strains by depleting their intracellular iron stores [51,54,55]. The biological

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significance of this finding has recently been highlighted in NGAL-deficient mice, which develop a marked sensitivity to Gram-negative bacterial infections and an increased susceptibility to death from sepsis [56]. Therefore, NGAL comprises a critical component of innate immunity to exogenous bacterial infections. This is consistent with its normal expression in a number of human tissues that are typically exposed to the external environment, including the respiratory, gastrointestinal, and urinary tracts. 7.4. NGAL IN EMBRYOGENESIS On the other hand, siderophores produced by eukaryotes participate in NGAL-mediated iron trafficking, which is critical to various cellular responses, such as proliferation and differentiation [54]. This property provides a potential 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 [57,58]. However, NGAL expression is also markedly induced in injured epithelial cells, including the kidney, colon, liver, and lung. This is likely mediated via NF-kB, which is known to be rapidly activated in epithelial cells after acute injuries [59], and plays a central role in controlling cell survival and proliferation [60]. In the context of an injured mature organ, such as the kidney, the biological role of NGAL induction is one of the marked preservation of function, attenuation of apoptosis, and an enhanced proliferative response [61]. 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. 7.5. NGAL AND NEOPLASIA NGAL seems to have more complex activities than its antimicrobial effect. Recent evidence indicates that NGAL is induced in a number of human cancers where it often represents a predictor of poor prognosis [62–65]. A number of related lipocalins are overexpressed in a variety of human cancers including breast colorectal ovarian and pancreatic cancers, and lipocalin ligands have been shown to regulate proliferation, differentiation, and protease activities. A heterogeneous expression of NGAL was first documented in a subset of subjects with primary breast carcinoma at both the mRNA and protein levels; the NGAL protein was found within the breast carcinoma cells but not in the normal ductal epithelium. While the significance of these findings is not yet fully elucidated, preliminary evidence suggest that NGAL expression may represent a predictor of poor prognosis

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in primary human breast cancer. The overexpressed NGAL protein binds to MMP-9, thereby preventing MMP-9 degradation and increasing MMP-9 enzyme activity. In turn, MMP-9 activity promotes cancer progression by degrading the basement membranes and extracellular matrix, liberating vascular endothelial growth factor (VEGF), and thus enabling angiogenesis, invasion, and metastasis. 7.6. NGAL IN ANEMIA It has been shown that there is a close link between human lipocalin and several white blood cell disorders [66]. NGAL plays a key role in tissue invasion by leukemia clones and in the mechanisms underlying the suppression of normal hematopoiesis through the induction of apoptosis. This is confirmed by the finding that patients with chronic myeloid leukemia have higher NGAL blood levels than healthy subjects [67]. However, the relationship between NGAL and leucocytes does not appear to be exclusive. Recent studies have demonstrated that this protein also plays a key role in the physiology and pathophysiology of red blood cells, particularly in anemia. NGAL represents a key factor in the regulation of erythrocyte growth due to its ability to inhibit the maturation and differentiation of bone marrow erythroid precursors [68–70]. The regulatory effect of NGAL on erythrocyte maturation suggests that the protein may be involved in the diseases affecting red blood cells, such as anemia. When a condition of primary anemia occurs, especially when acute, the tissues have a dual response to NGAL. In the bone marrow, the production decreases, whereas there is an increase in the peripheral production in order to counteract the hypoxic stress [71]. Several systemic diseases are associated with the presence of secondary anemia (CKD, heart failure, chronic inflammation, etc.). These have been shown to induce an increase in the circulating levels of serum NGAL. Because of the ability of NGAL to suppress erythropoiesis [69,70], it is reasonable to suggest that this molecule may play a role in the induction or in the worsening of anemia. Experimental models have demonstrated that chronic inflammatory states are able to induce NGAL overexpression [72]. This was confirmed by high serum-NGAL levels found in patients with osteoarthritis, chronic inflammatory bowel disease, vasculitis, and systemic lupus erythematosus [73–77]. It is known that, in patients with CKD, anemia is caused from the progressive reduction of the endogenous production of erythropoietin (EPO). Whatever the primary etiology of CKD (diabetes, hypertension, glomerulonephritis, polycystic kidney disease), these patients show high serum and urine levels of NGAL, making this molecule not only a promising marker of CKD progression but also a helpful tool in the assessment and

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management of iron deficiency in these patients [78,79]. The relevance of NGAL in anemia associated with heart failure has not been investigated yet. Further studies are needed to verify whether the high NGAL systemic levels found in these conditions play a role in causing or worsening the secondary anemia associated with these conditions. 7.7. NGAL IN CARDIOVASCULAR DISEASE The relevance of NGAL to cardiovascular disease (CVD) remains primarily unknown. Systemic inflammation participates in atherosclerosis evolution from the early development of endothelial dysfunction, to formation of mature atheromatic plaques, to the ultimate endpoint, rupture, and thrombotic complications [80]. Atherosclerosis is a chronic inflammatory disease and the acute clinical manifestations represent acute or chronic inflammation. Inflammatory cells, involving activated neutrophils, are more frequently found in plaques vulnerable to rupture [81]. Neutrophil activation has been reported in unstable angina (UA) and acute myocardial infarction (AMI) but not in patients with stable angina (SA) [82–88]. This activation seems to precede myocardial injury in patients with AMI [89]. Therefore, biomarkers of neutrophil activation could be of prognostic and even diagnostic importance. Elevated plasma NGAL levels were associated with atherosclerosis and were implicated as a predictor for cardiovascular mortality after cerebrovascular ischemia, possibly because of activation of blood leukocytes [90–92]. Although recent reports has shown that NGAL is present in atherosclerotic plaques and in human abdominal aortic aneurisms, raising the possibility that expression of NGAL can be induced in vascular cells during atherogenesis, the underlying mechanism for the induction of NGAL in vascular cells, remains unknown [93,94]. In further analysis, the main source of NGAL was found to be neutrophils, probably recruited in the vascular wall by platelet activation [94]. Recent studies have shown that gelatinase B, also known as MMP-9, an endopeptidase capable of degrading the extracellular matrix, is thought to be associated with atherosclerosis and plaque rupture [95,96]. Therefore, MMP9 is considered to be an important mediator of vascular remodeling and plaque instability. The MMP-9 action is enhanced by NGAL. The formation of a complex with NGAL and MMP-9 is crucial for atherosclerotic plaque erosion and thrombus formation [93]. NGAL is considered to have a protective effect on MMP-9, and enhancing its proteolytic activity could be considered an important factor indirectly contributing to the progression of aneurism as well as involving in the physiologic and pathologic remodeling of vessel walls. This view is further supported by the observation that similar neutrophil NGAL/MMP-9 overexpression can be found in atherosclerotic plaques, particularly those with intramural

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hemorrhagic debris and central necrosis [93,97]. The above evidence supports the clinical observations that high circulating leucocyte (particularly neutrophil) counts are independent predictors of recurrent ischemic attacks. This may be explained by their presence in the necrotic core of unstable plaques and by their proteolytic activity toward atherosclerotic tissue and secondary mobilization of thromboembolic fragments [98]. The evidence derived from these experimental studies, showing the close link between neutrophils, their products, and the natural history of atherosclerosis and its complications, generated clinical studies that investigated the clinical utility of serum-NGAL measurements. In two recent studies was found that serum levels of NGAL were significantly elevated in patients with angiographically confirmed coronary artery disease (CAD) compared to those with normal arteries or controls [99,100]. Another recent study found evidence of increased systemic and myocardial expression of NGAL in clinical and experimental heart failure [101]. In this study, Yndestad et al. found increased serum levels of NGAL in both acute and chronic heart failure, significantly correlating with disease severity (assessed by clinical and neurohormonal parameters). They also found that the source of this NGAL was mainly the failing myocardium and that it was not solely a product of activated neutrophils. These data support a role of the innate immune system in the pathogenesis of heart failure. Also, recent studies showed that NGAL is highly expressed in adipose tissue and its secretion is highly regulated via activation of inflammation or infection [102]. Circulating NGAL levels are increased in obese animals as well as in human subjects with type 2 diabetes [103,104]. These results show a potential role of NGAL in insulin resistance. 7.8. NGAL ACTS AS A ‘‘STRESS PROTEIN’’ NGAL is hyperproduced by various cell types in response to exposure to adverse conditions, probably in order to activate iron-dependent response pathways [57]. Therefore, several inflammatory conditions, including those of the respiratory, gastrointestinal, and urinary tract, are associated with significant increase in the local and the systemic expression of NGAL [72]. In specific conditions, for example, in kidney diseases, NGAL levels in serum and urine seem to be of great diagnostic importance as this protein represents an early biomarker of organ stress [105].

8. Methods of NGAL Measurement A variety of methods for NGAL measurement have been used in published studies. In initial studies, both urine and serum-NGAL estimations were carried out by Western blot technique [106]. Subsequent clinical studies have

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utilized immunoblotting or research enzyme-linked immunosorbent assay (ELISA)-based techniques using a commercially available monoclonal NGAL antibody (Antibodyshop, Gentofte, Denmark) [107]. The time-consuming (over 10 h analytical time) immunoblot was quickly abandoned in favor of much quicker (2–4 h analytical time) ELISAs. Commercial ELISAs are based on sandwich enzyme immunoassay technique. A monoclonal antibody specific for NGAL has been precoated onto a microplate. Standards and samples (cell culture supernates, urine, serum, plasma) are pipetted into the wells and any NGAL present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for NGAL is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of NGAL bound in the initial step. The color development is stopped, the intensity of the color is measured, and the NGAL concentration in the sample is determined by interpolation on a calibration curve created from the standards. Many commercial kits (Bioporto, R&D, Biovendor, etc.) are currently in production. A point-of-care test, the Triage-NGAL test (Biosite Inc., San Diego, CA, USA), has been developed for the rapid measurement of plasma neutrophil gelatinase-associated lipocalin. Specimens should be either whole blood collected in EDTA tubes or plasma. The Triage-NGAL test is a fluorescence-based immunoassay used in conjunction with the Triage Meter (Biosite Inc.). The assay device is a single-use plastic cartridge that contains an NGAL-specific monoclonal antibody conjugated to a fluorescent nanoparticle, NGAL antigen immobilized on a solid phase, and stabilizers. The device is integrated with control features incorporating negative and positive control immunoassays, which ensure that the test performs properly and that the reagents are functional. The test is performed by inoculating several drops of whole blood or plasma into the sample port where the specimen moves through an integrated filter that separates cells from plasma. The plasma reconstitutes the detection nanoparticles that contain the fluorescent antibody and flows down the diagnostic lane via capillary action. NGAL present in the sample prevents binding of the fluorescent detection particles to the solid phase immobilized in the detection zone such that the analyte concentration is inversely proportional to the fluorescence detected. Separate solid phase zones are located along the same diagnostic lane for the control assay systems. The device is then inserted into Triage meter (a portable fluorescence photometer) and quantitative measurement of NGAL concentration in the range of 60–1300 ng/ml is displayed on the meter screen in approximately 15 min. Calibration is performed by the instrument via a lot-specific chip.

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The assay was found to correlate well with research ELISA. Its clinical application was validated in a study of 120 patients undergoing cardiopulmonary bypass (CPB) [108]. Recently, two tests were developed for automated chemistry and immunochemistry analyzers. The first is a urine only test, based on chemiluminescent microparticle immunoassay technology, and was developed by Abbott (Abbott Diagnostics, Illinois, USA), for its Architect series of immunochemistry analyzers. The assay is a two-step (sandwich) assay using high-affinity antibodies toward distinct epitopes on NGAL. Assay standards (for the calibration of the assay) were prepared in-house using human recombinant NGAL. The assay was found to correlate well with research ELISA. Its clinical application was validated in a study of 196 patients undergoing CPB [109]. The second (the NGAL-TestTM) is a particle-enhanced turbidimetric immunoassay for the quantitative determination of NGAL in human urine and EDTA plasma on automated clinical chemistry analyzers. Briefly, a sample of human urine or EDTA plasma is mixed with reaction buffer. After a short incubation, the reaction is started by the addition of an immunoparticle suspension (polystyrene microparticles coated with mouse monoclonal antibodies to NGAL). NGAL in the sample causes the immunoparticles to aggregate. The degree of aggregation is quantified by the amount of light scattering measured as absorption of light. The NGAL concentration in the sample is determined by interpolation on an established calibration curve. The clinical value of this assay is currently under investigation. However, several analytical and preanalytical issues remain to be clarified. Very few data exist on sample storage conditions. Urine samples seem to remain stable at 2–8  C for short periods of time (up to 48 h). However, longterm storage requires a temperature of  70  C for optimum sample stability. No published data exist on storage conditions for other types of sample [110,111]. Standardization of all these assays is an issue since neither universally accepted reference method exists for the measurement of NGAL nor primary reference material for the calibration of such method. Therefore, all commercially available methods are not traceable to an internationally accepted reference method and calibrators that accompany commercially available kits are produced and have values assigned ‘‘in-house.’’ This may limit the transferability of values from lab to lab. Previous studies with NGAL, as well as with other analytes, have shown that the choice of antibodies has a great impact on the clinical performance of the assay [112,113]. This is more evident with NGAL because several molecular forms were identified in serum and urine of various patient groups. NGAL exists as a 25-kDa monomer or as a 45-kDa disulfide-linked homodimer and finally as a 135-kDa heterodimer when it is covalently conjugated

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with MMP9 [44]. The source of these different molecular forms has not been fully elucidated yet. In a recent study, Cai et al. [114] reported the existence of several molecular forms of NGAL (monomeric, dimeric) in urine of AKI patients after cardiac surgery. They also reported the ratio of dimeric to monomeric NGAL changed after the operation. In a more recent study by the same group, they managed to identify the possible sources of these various molecular forms and their effect on the performance of various assays using different combinations of monoclonal and polyclonal antibodies with different epitope specificities. Their results showed that monomeric (and to some extent, the heterodimeric) NGAL is produced by the tubular epithelial cells whereas the dimeric form seems unique to neutrophils. The presence of neutrophils in urine may explain the presence of dimeric NGAL. Another important finding was that the choice of antibodies that were used in the assays for the quantitation of NGAL influenced greatly the result. This suggests differences in molecular structure of these different forms of NGAL. These differences in molecular structure may explain the inability of NGAL originating from tubular epithelial cells to form dimmers. This molecular diversity has to be taken into account for the development of an assay for the quantification of NGAL in urine [112,114,115]. Biological variation (BV) has been studied only in a small group of healthy people and only in random urine collections [116]. In this study, BV was calculated as a coefficient of variation (CV) of ‘‘absolute’’ NGAL value and as ratio to urine creatinine in first morning urine and in a random afternoon collection. The CV is ranged from 81% to 124%. The use of the ratio significantly improved the intraindividual variation observed in NGAL measurements. These preliminary data show that BV is considerable in human urine of healthy people and that values must be at least doubled in follow-up studies in order to determine a significant increase of NGAL. There is also a controversy whether ‘‘absolute’’ NGAL values or the ratio to creatinine is to be used. In a recent study, Waikar et al. criticized the use of ratio in situations of AKI since the production of creatinine is not stable in these subjects and the normalization of any biomarker to urinary creatinine concentration may result in under- or overestimation of this marker [117].

9. NGAL as Biomarker of Kidney Injury 9.1. THE PHYSIOLOGIC ROLE OF NGAL IN AKI Recent advances in cellular and molecular biology of ischemic renal injury have revealed that proximal tubule cells undergo a complex temporal sequence of events. These include loss of cell polarity, cell death as a result

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of cell apoptosis and necrosis, dedifferentiation and proliferation of viable cells, and reestablishment of the epithelial phenotype [118,119]. Understanding of the early cell injury and repair mechanisms is critical for application of effective therapy. Identification of interventions that may oppose tubule cell death and/or enhance the recovery phase is of considerable interest. The molecular basis of early renal responses has been identified with the use of novel techniques. Using cDNA microarray techniques, researchers identified the gene that encodes NGAL to be expressed by kidney cells [106,120]. This gene has been found to undergo upregulation in response to renal ischemia in experimental animal models of AKI [106]. NGAL is upregulated in tubular epithelial cells that undergo proliferation [106]. Recent evidence suggests that NGAL can enhance epithelial phenotype. During kidney development, NGAL is expressed by the penetrating ureteric bud and triggers nephrogenesis by stimulating the conversion of mesenchymal cells into kidney epithelia [121]. NGAL may play a renoprotective role in ischemic AKI. In the postischemic mature kidney, NGAL is markedly upregulated predominantly in proximal tubules but also in distal nephron segments. In the proximal tubule, NGAL colocalizes at least in part with proliferating epithelial cells [106]. These findings suggest that NGAL may be expressed by the damaged tubule to induce reepithelialization. In support of this hypothesis is the recent identification of NGAL as an iron-transporting protein during nephrogenesis [122]. It is well known that the delivery of iron into cells is crucial for cell growth and development, and this is presumably also critical to renal regeneration after nephrotoxic injury. Because NGAL can be endocytosed by the proximal tubule [122], the protein could potentially recycle iron into viable cells, thereby stimulating regeneration of renal epithelial cells after ischemic injury. An alternative hypothesis is that NGAL may serve as a reservoir for iron that is released from tubule cells that are damaged by nephrotoxic injury. This might remove iron, a reactive molecule, from the site of tissue injury, thereby limiting iron-mediated cytotoxicity. It is possible that both mechanisms are operative in the postischemic kidney [61]. 9.2. THE BIOLOGIC SOURCES OF NGAL FOLLOWING AKI The genesis and the sources of plasma and urinary NGAL following AKI are not yet fully elucidated. The kidney does not appear to be the major source of plasma NGAL. In animal studies, direct ipsilateral renal vein sampling after unilateral ischemia indicates that the NGAL synthesized in the kidney is not introduced efficiently into the circulation, but is abundantly present in the ipsilateral ureter [54]. However, studies in humans and animal models have demonstrated that AKI has a significant effect on the function

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of extrarenal organs and results in a dramatically increased NGAL mRNA expression in distant organs, especially the liver and lungs [123]. This overexpressed NGAL protein is released into the circulation and may constitute a distinct systemic pool. Additional contributions to the systemic pool in AKI may derive from the fact that NGAL is an acute-phase reactant and may be released from neutrophils, macrophages, and other immune cells. Further, any decrease in GFR resulting from AKI would be expected to decrease the renal clearance of NGAL, with subsequent accumulation in the systemic circulation. The relative contribution of these mechanisms to the rise in plasma NGAL after AKI remains to be determined. Although plasma NGAL is freely filtered by the glomerulus, it is largely reabsorbed in the proximal tubules by efficient megalin-dependent endocytosis [54]. Direct evidence for this theory is derived from systemic injection of labeled NGAL, which becomes enriched in the proximal tubule but does not appear in the urine of animals [124]. Thus, any urinary excretion of NGAL is likely only when there is a concomitant proximal renal tubular injury that precludes NGAL reabsorption and/or increased de novo NGAL synthesis. However, 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 [54]. The resultant synthesis of NGAL protein in the distal nephron and secretion into the urine appears to comprise the major fraction of urinary NGAL. Supporting clinical evidence is provided by the consistent finding of a high fractional excretion of NGAL reported in human AKI studies [54,124]. The overexpression of NGAL in the distal tubule and rapid secretion into the lower urinary tract is in accord with its teleological function as an antimicrobial strategy. It is also consistent with the proposed role for NGAL in promoting cell survival and proliferation, given the recent documentation of abundant apoptotic cell death in distal nephron segments in several animal and human models of AKI [125,126].

9.3. NGAL FOR AKI PREDICTION Proteomic analyses revealed that NGAL is one of the most highly induced proteins in the kidney after ischemic or nephrotoxic AKI in animal models [106,124,127]. The finding that NGAL protein was easily detected in the urine soon after AKI in animal studies has initiated many human studies to evaluate NGAL as a novel noninvasive marker in human AKI. A number of human studies have now implicated NGAL as an early diagnostic marker for AKI in several common clinical situations as we analyze in the following paragraphs.

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9.3.1. NGAL and the Prediction of AKI After Cardiac Surgery CPB is one of the most frequent major surgical procedures performed in hospitals nowadays. AKI requiring dialysis represents the strongest independent risk factor for death in these patients [128]. Even a minimal change in baseline serum creatinine (i.e., 0.2–0.3 mg/dl) is associated with a significant increase in mortality after cardiac surgery [129]. In addition, AKI after cardiac surgery is associated with adverse outcomes, such as prolonged intensive care and hospital stay, dialysis dependency, and increased longterm mortality [130]. The pathogenesis of cardiac surgery-associated AKI is complex and multifactorial [131]. It involves several major injury pathways that are largely nonmodifiable. Mechanisms include ischemia–reperfusion injury (caused by low mean arterial pressures and loss of pulsatile renal blood flow), exogenous toxins (caused by contrast media, nonsteroidal antiinflammatory drugs, and aprotinin), endogenous toxins (caused by iron released from hemolysis), and inflammation and oxidative stress (from contact with bypass circuit, surgical trauma, and intrarenal inflammatory responses). These mechanisms of injury are likely to be active at different times with different intensities and may act synergistically. In several prospective studies involving children who underwent elective cardiac surgery, AKI occurred 1–3 days after surgery [132–134]. In these studies, AKI was defined as a 50% increase in serum creatinine from baseline. By contrast, measurements of NGAL by ELISA, revealed a 10-fold or more increase in the urine and plasma, within 2–6 h of the surgery, in those patients who subsequently developed AKI. Both urine and plasma NGAL were excellent independent predictors of AKI, with an area under the curve (AUC) of the receiver operating characteristic curve (ROC) of over 0.9 for the 2–6 h urine and plasma NGAL measurements. These findings have been confirmed in prospective studies involving adult patients who developed AKI after cardiac surgery and in whom urinary and/or plasma NGAL was significantly elevated by 1–3 h after the operation [135–142]. However, the area under the ROC curve (AUC-ROC) for prediction of AKI has been rather disappointing when compared with pediatric studies and has ranged widely from 0.61 to 0.96. The somewhat inferior performance in adult populations may be reflective of confounding variables, such as older age groups, preexisting kidney disease, prolonged bypass times, chronic illness, and diabetes [136,143]. Children often lacked the comorbidities that accompany adults. The predictive performance of NGAL also depends on the definition of AKI employed as well as on the severity of AKI [142]. Further, the predictive value of urinary NGAL for AKI after cardiac surgery varied with baseline renal function with optimal discriminatory performance in patients with normal preoperative renal function [144]. However, a recent meta-analysis

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of published studies in all patients after cardiac surgery revealed an overall AUC-ROC of 0.78 for prediction of AKI, when NGAL was measured within 6 h of initiation of CPB and AKI was defined as an increase of > 50% from baseline in serum creatinine [145]. 9.3.2. NGAL and the Prediction of AKI After Kidney Transplantation AKI due to ischemia–reperfusion occurs frequently after transplantation of renal allografts (either from deceased or live donors) [146]. This leads to varying degrees of renal dysfunction. AKI leading to delayed graft function (DGF) complicates 4–10% of live donor and 5–50% of deceased donor kidney transplants. In addition to the two well-known complications of kidney transplantation (AKI and dialysis), DGF is the major cause of both acute and chronic rejection, suboptimal graft function at 1 year after transplantation, and increases the risk of chronic allograft nephropathy and loss [147]. NGAL has been evaluated as a biomarker of AKI and DGF (defined as dialysis requirement within the first postoperative week) in patients undergoing kidney transplantation. Protocol biopsies of kidneys obtained 1 h after vascular anastomosis revealed a significant correlation between NGAL staining intensity in the allograft and the subsequent development of DGF [148]. In a prospective multicenter study of children and adults, urine NGAL levels in samples collected on the day of transplant identified those who subsequently developed DGF (which typically occurred 2–4 days later), with an AUC-ROC of 0.9 [149]. This has now been confirmed in a larger multicenter cohort. Urine NGAL measured within 6 h of kidney transplantation predicted subsequent DGF with an AUC-ROC of 0.81 [150]. Plasma NGAL has also been correlated with DGF following kidney transplantation from donors after cardiac death [151]. 9.3.3. NGAL in Contrast-Induced Nephropathy Contrast-induced AKI (also named contrast-induced nephropathy, CIN) is the third most common cause of hospital-acquired AKI, accounting for approximately 11% of cases of AKI [152]. Approximately half of these cases are in subjects undergoing cardiac catheterization and angiography, and approximately a third follow computed tomography [153]. Technological advances in diagnostic and interventional imaging techniques have contributed to the increase of the number of individuals being exposed to iodinated contrast media [154]. The reported incidence of CIN varies widely across the literature, depending on the patient population and the baseline risk factors. Moreover, as with any clinical condition, the incidence varies depending on the criteria used to define it. Generally, CIN is defined as an

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increase in serum creatinine concentration of > 0.5 mg/dl (> 44 mmol/l) or 25% above baseline or fall of GFR by > 25% from baseline, after systemic contrast administration in the absence of other causes. Typically, CIN occurs within 24–48 h of exposure, serum creatinine peaks in 3–5 days, and renal function returns to baseline within 7–21 days [155]. If renal function does not return to baseline, other possible causes of renal injury should be suspected. The AKIN definition requires a rise in serum creatinine > 0.3 mg/dl. The pathogenesis of CIN is complex, with a cascade of contributing factors that are not fully understood. Alterations in renal hemodynamics and direct tubular toxicity are believed to be the primary pathways responsible for CIN. After injection of contrast media, renal blood flow increases transiently, followed by a more prolonged decrease in renal blood flow, particularly at the corticomedullary junction of the kidney, suggesting that renal ischemia is a major factor in the pathogenesis of CIN [153]. The outer medulla is particularly susceptible to ischemic injury because of its high metabolic activity and low prevailing oxygen tension. Associated with the decrease in renal blood flow is a decrease in GFR due to afferent arteriolar vasoconstriction which is calcium dependent. Vasoconstriction is caused by the release of adenosine, endothelin, and other renal vasoconstrictors triggered by iodinated contrast. The concentration of iodinated contrast in the renal tubules and collecting ducts of the kidney allows for direct cellular injury and death of renal tubular cells. The degree of cytotoxicity to renal tubular cells is directly related to the length of exposure those cells have to iodinated media; hence, the importance of high urinary flow rates before, during, and after the contrast procedures. The sustained reduction in renal blood flow to the outer medulla leads to medullary hypoxia, ischemic injury, and death of renal tubular cells. Reactive oxygen species formed as a result of postischemic oxidative stress can lead to AKI through their direct effects on renal endothelial cells, which include apoptotic cell death. The possible benefit of N-acetylcysteine (NAC) and sodium bicarbonate in the prevention of CIN is hypothesized due to the ability of these compounds to mitigate oxidative injury [156]. Any superimposed insult such as sustained hypotension, the use of intra-aortic balloon counterpulsation, or a bleeding complication can amplify the injury process occurring in the kidney. Virtually every report describing risk factors for CIN lists abnormal baseline serum creatinine, low GFR, or CKD as risk factors. Other risk factors include diabetes mellitus, hypertension, volume depletion, nephrotoxic drugs, hemodynamic instability, and other comorbidities [155]. Several investigators have examined the role of NGAL as a predictive biomarker of AKI following contrast administration [157–160]. In a prospective study of children undergoing elective cardiac catheterization with contrast administration, both urine and plasma NGAL predicted CIN (defined as a

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50% increase in serum creatinine from baseline) within 2 h after contrast administration, with an AUC-ROC of 0.91–0.92 [160]. In several studies of adults administered contrast, an early rise in both urine (4 h) and plasma (2 h) NGAL was documented, in comparison with a much later increase in plasma cystatin C levels (8–24 h after contrast administration), providing further support for NGAL as an early biomarker of contrast nephropathy [157– 159]. A recent meta-analysis revealed an overall AUC-ROC of 0.894 for prediction of AKI, when NGAL was measured within 6 h after contrast administration and AKI was defined as an increase in serum creatinine of over 25% [145]. 9.3.4. NGAL for AKI Prediction in the Heterogeneous Population of ICU AKI is a frequent complication in critically ill patients with hospital mortality of 45–60% [160,161]. This patient population is extremely heterogeneous, and the etiology and timing of AKI are often unclear. Up to 60% of patients may have already sustained AKI on admission to the ICU [162]. Sepsis accounts for 30–50% of all AKI encountered in critically ill patients and generally is a cause of poorer prognosis with lower survival [163]. The combination of AKI and sepsis is associated with 70% mortality as compared with 45% mortality among patients with AKI alone. Other etiologies for AKI in this setting include exposure to nephrotoxins, hypotension, kidney ischemia, mechanical ventilation, and multiorgan disease. Each of these etiologies is associated with distinct mechanisms of injury that are likely to be active at different times with different intensities and may act synergistically. Urine and plasma NGAL measurements have been demonstrated to represent early biomarkers of AKI in a heterogeneous pediatric intensive care setting, being able to predict this complication approximately 2 days prior to the rise in serum creatinine, with high sensitivity and AUC-ROCs of 0.68–0.78 [164,165]. Several studies have also examined plasma and urine NGAL levels in critically ill adult populations [166–171]. Severe trauma is commonly associated with AKI, which almost doubles the risk of ICU mortality and increases the duration of mechanical ventilation and length of hospital stay [163,166]. In a large observational multicenter study, 18% of all major trauma patients admitted to ICU developed AKI within the first 24 h [163]. The value of NGAL as a prognostic marker in multitrauma patients has been investigated in one study. In this study, urine NGAL, obtained on admission, predicted subsequent AKI in multitrauma patients with an outstanding AUC-ROC of 0.98 [166]. In that study also, NGAL levels were elevated at the time of ICU admission in patients who developed AKI and persisted elevated for the following 2 days compared to those who did not develop AKI. These early results are promising and

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support the need for additional studies to fully understand diagnostic and prognostic value of NGAL in critically ill patients. However, in a more mixed population of all critical care admissions, the urine NGAL on admission was only moderately predictive of AKI with an AUC-ROC of 0.71 [167]. In studies of adult intensive care patients, plasma NGAL concentrations on admission constituted a very good to outstanding biomarker for development of AKI within the next 2 days, with AUC-ROC ranges of 0.78–0.92 [168,170]. In subjects undergoing liver transplantation, a single plasma NGAL level obtained within 2 h of reperfusion was highly predictive of subsequent AKI, with an AUC-ROC of 0.79 [171]. Finally, in a study of adults in the ED setting, a single measurement of urine NGAL at the time of initial presentation predicted AKI with an outstanding AUC-ROC of 0.95 and reliably distinguished prerenal azotemia from intrinsic AKI and from CKD [107]. Thus, NGAL is a useful early AKI marker that predicts development of AKI, even in heterogeneous groups of patients with multiple comorbidities and with unknown timing of kidney injury. However, it should be noted that patients with septic AKI display the highest concentrations of both plasma and urine NGAL when compared with those with nonseptic AKI [167], a confounding factor that may add to the heterogeneity of the results in the critical care setting. In another prospective observational study that aimed to identify a biomarker panel to predict organ dysfunction in ED patients with suspected sepsis, NGAL was optimal in predicting severe sepsis within 72 h of ED admission [172]. A recent meta-analysis revealed an overall AUC-ROC of 0.73 for prediction of AKI, when NGAL was measured within 6 h of clinical contact with critically ill subjects, and AKI was defined as a > 50% increase in serum creatinine [145]. 9.4. AKI AND CARDIORENAL SYNDROME: POTENTIAL ROLE OF NGAL Another clinical concept whose definition has been refined in recent years is the CRS. There is a close association between renal and cardiac function in both acute and chronic diseases. CVD causes over 50% of deaths in patients with renal failure, while poor renal function increases mortality in patients with heart failure [173–175]. The term ‘‘cardiorenal syndrome’’ had been loosely used in the past to describe the relationship between renal and cardiac function but it was not until 2004 that the National Heart, Lung, and Blood Institute defined CRS as a condition in which therapy to relieve congestive symptoms of heart failure is limited by a decline in renal function as manifested by a reduction in GFR. Although this definition suggests a one-way effect of renal on cardiac function, further work has shown the interactions to work in both directions and in a variety of clinical conditions [176]. In 2008, the ADQI suggested the use of CRS to identify a

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disorder of the heart and kidneys whereby acute or chronic dysfunction in one organ may induce acute or chronic dysfunction in the other organ [177]. The ADQI report had goals similar to that of the RIFLE and AKIN initiatives in encouraging comparison between epidemiological and interventional studies as well as the development of diagnostic tools for the prevention and management of the different syndromes. Five different subtypes of syndrome were identified (Table 9). Acute CRS (type I): Acute worsening of heart function leading to kidney injury and/or dysfunction. Up to 40% of patients with acute decompensated heart failure develop AKI and fall into this category [178,179]. AKI was defined as the secondary event using RIFLE–AKIN criteria. Chronic CRS (type II): Chronic abnormalities in heart function leading to kidney injury and/or dysfunction. Up to 63% of hospitalized patients with congestive heart failure fall into this category [180,181]. Renal dysfunction was defined as the secondary event using KDOQI criteria [180]. Acute reno-cardiac syndrome (type III): Acute worsening of kidney function (AKI) leading to heart injury and/or dysfunction. AKI was defined as the primary event using RIFLE–AKIN criteria. Chronic reno-cardiac syndrome (type IV): CKD leading to heart injury, disease, and/or dysfunction.

TABLE 9 CLASSIFICATION OF CARDIORENAL SYNDROME Class

Type

Description

Example

I

Acute cardiorenal syndrome

Abrupt worsening of cardiac function leading to acute kidney injury (AKI)

II

Chronic cardiorenal syndrome

III

Acute reno-cardiac syndrome

IV

Chronic renocardiac syndrome Secondary cardiorenal syndrome

Chronic abnormalities of cardiac function leading to chronic kidney disease (CKD) Abrupt worsening of kidney function leading to acute cardiac dysfunction CKD leading to chronic cardiac dysfunction

Hemodynamically mediated AKI secondary to acute heart failure or acute coronary syndrome CKD in patients with chronic heart failure

V

Adapted from Ref. [177].

Systemic disorders causing both cardiac and renal dysfunction

Arrhythmias or acute pulmonary edema in patients with AKI Cardiac hypertrophy and adverse cardiovascular events in patients with CKD Sepsis, leukemia, amyloidosis, etc.

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Excess cardiovascular deaths associated with increasing renal dysfunction have been estimated at over 50% [182]. Renal dysfunction was defined as the secondary event using KDOQI criteria [180]. Secondary CRS (type V): Systemic conditions (e.g., sepsis, systemic lupus erythematosus, diabetes mellitus, amyloidosis, or other chronic inflammatory conditions) leading to simultaneous injury and/or dysfunction of heart and kidney. AKI was defined as one of the possible secondary events using RIFLE–AKIN criteria. As can be seen, identification of AKI is key to the definitions of types 1, 3, and 5 CRS. Adoption of the RIFLE–AKIN criteria enables common dialog between the cardiology and nephrology communities but the reliance of the RIFLE and AKIN systems on serum creatinine measurement and its limitations discussed earlier remains. There is a need for new specific biomarkers that identify kidney injury early and that can replace serum creatinine in both the definition of AKI for epidemiological and study purposes as well as in guiding individual patient management. The role of NGAL in the diagnosis of type 1 CRS was investigated in a recent study [183]. Type 1 CRS was defined, in this study, as an increase in the creatinine levels of at least 0.3 mg/dl or 50% from baseline. CRS was developed within 48–72 h in 11.8% of studied patients. Using a 170 ng/ml NGAL cut-off, they were able to define CRS with a remarkable sensitivity (100%) and high specificity (86.7%).

9.5. FURTHER ROLES OF NGAL IN KIDNEY DISEASES 9.5.1. NGAL and CKD Although the primary role of NGAL is considered as a biomarker for AKI, accumulating evidence suggest that it may also have a role in patients with CKDs. Whatever the primary disease process, the rate of decline of kidney function is recognized as strictly influenced by several secondary components. Although hypertension, proteinuria, hyperlipidemia, and inflammation represent some important modifiable risk factors, by themselves, these elements are not sufficient to properly explain renal outcomes in patients affected by CKD [184,185]. Recent observations have pointed out the crucial role of the renal tubule in the genesis and progression of CKD; independently of the primary disease and the eventual presence of superimposed damaging conditions, the pathogenic mechanisms causing progressive renal destruction converge on a common tubulo-interstitial pathway characterized by tubular atrophy and hypoxia, peritubular capillary injury, and interstitial fibrosis, ultimately explaining the irreversible evolution to terminal uremia [186]. In accordance with this point of view, it is now widely accepted that in some

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CKD-associated diseases, such as diabetic nephropathy, the rate of deterioration in renal function and the overall renal long-term outcome, are more accurately associated with the degree of renal tubulo-interstitial impairment than with the severity of glomerular lesions. In CKD patients, Bolignano et al. [7,44,54,105–187] demonstrated increased NGAL which reflected residual renal function. Further, NGAL was significantly correlated with serum creatinine, GFR, and proteinuria. In addition to predicting CKD, NGAL may also predict the risk of CKD progression. [78,188]. Preliminary research in patients with macroproteinuria showed that high levels of NGAL at baseline were associated with worsening renal function within 1 year, compared to those with lower baseline levels of NGAL. In another study [189], it has been shown that, in patients with nonadvanced CKD, NGAL could predict the progression of disease. They measured plasma and urinary NGAL levels at baseline and during a median follow-up of 18.5 months. At baseline, NGAL levels were inversely and independently related to eGFR. Disease progression was observed in 32% of the patients who had elevated levels of NGAL at baseline compared with those whose disease did not progress. These findings could help to screen patients with CKD to determine their risk of disease progression as well as their requirement for more aggressive treatments. 9.5.2. NGAL and Response to Therapy Due to its high predictive properties for AKI, NGAL seems to be a promising early biomarker for monitoring the efficacy of therapy in clinical trials. A reduction in urine NGAL has been used as an outcome variable in clinical trials demonstrating the improved efficacy of a modern hydroxylethyl-starch preparation over albumin or gelatin in maintaining renal function in cardiac surgery patients [190–192]. In another study, the effect of treatment with intravenous immunoglobulin (Ig) on NGAL levels in 15 proteinuric patients with normal renal function was investigated [189]. At baseline, both urinary and plasma levels of NGAL were elevated in patients with proteinuria compared with controls. Infusion of a single, high-dose bolus of Ig resulted in an immediate decline from baseline in the levels of both plasma and urinary NGAL. These findings suggest a potential role of NGAL in monitoring the efficacy of treatments for renal diseases. 9.5.3. NGAL as a Marker for the Prognosis of AKI A number of studies have demonstrated the utility of early NGAL measurements for predicting the severity and clinical outcomes of AKI. In a study involving children undergoing cardiac surgery, early postoperative plasma NGAL levels strongly correlated with duration and severity of AKI, length of hospital stay, and mortality [108]. In a similar study, early

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postoperative urine NGAL levels highly correlated with duration and severity of AKI, length of hospital stay, dialysis requirement, and death [109]. In a multicenter study of children with diarrhea-associated hemolytic uremic syndrome, urine NGAL, obtained early during the hospitalization, predicted the severity of AKI and dialysis requirement with high sensitivity [193]. Early urine NGAL levels were also predictive of duration of AKI (AUC-ROC 0.79) in a heterogeneous cohort of critically ill pediatric subjects [164]. In adults undergoing CPB, those who subsequently required renal replacement therapy were found to have the highest urine NGAL values soon after surgery [135–141]. Similar results were documented in the adult critical care setting [68,107,166–171]. Collectively, the published studies revealed an overall AUC-ROC of 0.78 for the prediction of subsequent dialysis requirement, when NGAL was measured within 6 h of clinical contact [145]. Further, a number of studies conducted in the cardiac surgery and critical care populations have identified early NGAL measurements as a very good mortality marker [107,135–137,167,168]. 9.6. LIMITATIONS OF NGAL AS BIOMARKER OF AKI Although studies so far have revealed that NGAL is a powerful biomarker for AKI prediction and outcome, it appears to be more sensitive and specific in homogenous patient populations. Also in published studies, age seems to be an effective modifier of NGAL’s performance as an AKI biomarker. NGAL has better predictive ability in children (overall AUC-ROC 0.93) than in adults (AUC-ROC 0.71). Plasma NGAL measurements may be influenced by a number of coexisting variables including CKD, chronic hypertension, systemic infections, inflammatory conditions, anemia, hypoxia, and malignancies. In the CKD population, NGAL levels correlate with the severity of renal impairment. However, it should be noted that the increased plasma NGAL in these situations is generally much less than those typically encountered in AKI. In addition, NGAL has been demonstrated to be expressed in human atherosclerotic plaques, as well as abdominal aortic aneurysms, which may also influence plasma NGAL measurements. There are also important limitations that exist in the published NGAL literature. First, the majority of the studies reported were from single centers that enrolled small numbers of subjects. Validation of the published results in large multicenter studies will be essential. Second, most studies reported to date did not include patients with CKD. This is problematic, not only because it excludes a large proportion of subjects who frequently develop AKI in clinical practice but also because CKD, in itself, can result

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in increased concentrations of NGAL, thereby representing a confounding variable. Third, many studies reported only statistical associations (odds ratio or relative risk), but did not report sensitivity, specificity, and AUCs for the diagnosis of AKI; these are essential to determine the accuracy of the biomarker. Fourth, only a few studies, with a relatively small number of cases, have investigated biomarkers for the prediction of AKI severity, morbidity, and mortality. Finally, the definition of AKI in the published studies varied widely, and it was based largely on increased serum creatinine, which is not the best outcome variable to analyze the performance of a novel assay. These studies may have yielded different results if there was a true ‘‘gold standard’’ for AKI. Instead, using AKI definitions by a change in serum creatinine sets up the biomarker assay for lack of accuracy due to either false positives (true tubular injury but no significant change in serum creatinine) or false negatives (absence of true tubular injury, but elevations in serum creatinine caused by prerenal effects or any of a number of confounding variables that affect this measurement). In future studies, it will be crucial to understand the clinical outcomes of subjects who may be prone to AKI and are ‘‘NGAL-positive’’ but ‘‘creatinine-negative,’’ since this will determine whether the biomarker is overtly sensitive. Since the gold standard for true AKI (tissue biopsy) is highly unlikely to be feasible, it is vital that future studies are large enough, and demonstrate the association between biomarkers and hard outcomes, such as dialysis, cardiovascular events and death, and that randomization to a treatment for AKI, based on high biomarker levels, results in an improvement in kidney function and a reduction of clinical outcomes. This should be the next priority in the field.

10. Conclusions AKI may occur in multiple clinical settings. Conventional biomarkers that are available today do not assist in a quick and accurate diagnosis. Early recognition of AKI is critical to enable intervention with appropriate therapies, which can be introduced earlier in the course of the disease and may be more effective in preventing or reversing tubular injury (Fig. 2). NGAL, as an AKI biomarker, has successfully passed through the preclinical assay development and initial clinical testing stages of the biomarker development process. It seems to be helpful in a variety of clinical settings and research has now entered the phase of prospective studies (Fig. 4). Evidence-based medicine will continue to be vital to demonstrate the value of NGAL in improving patient outcome.

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Implications of NGAL measurement in AKI

Diagnosis · Diagnose AKI early · Consult nephrologist early · Admit or delay discharge from hospital or ICU

Therapy · · · · ·

Intervene timely in relevant hypotension or hypovolemia Optimize hemodynamics Avoid nephrotoxic medication Commence or stop RRT earlier Noninvasive monitoring after renal transplantion

Prognosis

• Predict clinical outcomes (Renal replacement Therapy, delayed graft function, mortality)

FIG. 4. Potential uses of NGAL in diagnosis, therapy, and prognosis of AKI (modified from a presentation by C. Ronco).

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