Biomarkers in acute kidney injury

Acute kidney injury is a common condition associated with high morbidity and short-term mortality. Its pathophysiology varies according to the numerous conditions associated with its genesis. Biomarkers allow detecting changes at the level of kidney function; therefore, they play an important role in the prevention, early diagnosis, therapeutic response and prognosis of acute kidney injury. The search for biomarkers for acute kidney injury began over 15 years ago; initially, only serum creatinine was available for diagnosis. However, throughout history, great advances have been made in research, which have allowed the fi nding of new biomarkers in order to improve the health and quality of life of patients. A narrative review of the literature is carried out on the basis of available scientifi c evidence to clarify the role and importance of biomarkers in the context of acute renal injury.


Introduction
of AKI; reason why its de inition evolved based on the established criteria for each speci ic time. Commencing with the RIFLE criteria (risk, injury, failure, loss, end-stage kidney disease), followed by the AKIN (acute kidney injury network) and more recently with a focus on the international guidelines based on KDIGO (kidney disease improving global outcomes), which include changes in serum creatinine, urine output within seven days and the need of renal replacement therapy (RRT). This is concluding that a decrease in serum creatinine, although not de ining AKI, should incite healthcare personnel to have a high suspicion for it and could be useful when the baseline creatinine is unknown [3]. Based on this de inition, the development of AKI is associated with an increase in morbidity and mortality, with an increase in the length of stay and healthcare costs [4].
The search for AKI biomarkers started more than 15 years ago; it was not until 2006 when the Food and Drug Administration (FDA), the European Medicine Agency (EMA) and the Japanese Agency for Pharmaceutical Devices and Physicians joined several pharmaceutical companies to form the Predictive Security. This focused primarily on the identi ication of sensitive and speci ic biomarkers for nephrotoxicity in animal models [5]. In 2008, 7 biomarkers of nephrotoxicity were authorized by the FDA for its use in animals and humans, including urinary KIM-1, beta-2 microglobulin, Cystatin C, Trefoil Factor 3 (TFF3), Clusterin, urinary NGAL (Neutrophil Gelatinase-Associated Lipicalin), albumin, total protein, urinary interleukin-18 and liver fatty acid-binding protein (L-FABP), among others. These biomarkers generally play an essential role in regards to their positive and negative predictive values. These were compared with changes in the serum creatinine levels, given that there were no exact parameters for the detection of AKI. Using serum creatinine measurements as a gold standard for the diagnosis of AKI, these biomarkers have had a fundamental role in regards to their negative predictive value; nevertheless, their positive predictive value has been far less [5].
Only until 2016, the FDA and the National Institute of Health (NIH) in the USA summoned a council to establish the terms that de ined AKI's diagnostic biomarkers. Speci ically, the council established the BEST (Biomarkers, EndpointS, and other Tools) resources to improve and harmonize the terms utilized in all the pre-existing literature. The council de ined "biomarker" as a quanti iable tool that is an indicator of biological and pathological processes, as well as a response to an intervention. Similarly, biomarkers were divided into categories depending on their role in the pathophysiology of AKI [2]. This literature review evaluates the current concepts and the importance of the use of biomarkers in the diagnosis of AKI.

Biomarkers in AKI
AKI is condition currently associated with increased short-term morbidity and mortality [6]. Its incidence has increased exponentially in recent years, given factors such as aging and multiple comorbidities [7].
Given that AKI continues to be a global problem and that its etiology varies, it is fundamental for clinicians to detect it in an early manner and implement preventative and therapeutic strategies. It is there were the biomarkers become essential [8]. The different categories of biomarkers are capable of providing information about diagnosis, prognosis, risk prediction and response to therapy [2].
Early detection of AKI biomarkers can signi icantly reduce renal damage, improve the results in the long-term and prevent the progression to chronic kidney disease (CKD). These biomarkers could also help identify patients that can bene it from early intervention, even before the AKI becomes clinically detectable [2]. The use of these biomarkers in AKI has been studied thoroughly in recent times. Nevertheless, recent studies have generated some controversy regarding the utility of these [8,9]. Despite its clinical de inition, identi ication and strati ication of new AKI biomarkers, it is essential to know the pathophysiological mechanisms of AKI to implement an adequate use of these biomarkers [5].

Pathophysiology of AKI
The pathophysiology of AKI differs depending on the numerous conditions associated with its development [3]. Renal perfusion requires around 20% of the cardiac output, a considerable percentage of the total intravascular volume. Once in the kidney, it is distributed in a particular matter throughout the renal arteries, which will ramify into glomerular arterioles and eventually form a network of capillaries responsible for glomerular iltration, which is in charge of iltering waste substances and other molecules such as proteins [10]. Glomerular iltration is based on the elimination of body residues. One of these molecules is creatinine, a residual product of creatine, which is found in muscle tissues. Creatinine has been a biological biomarker traditionally used to assess renal function, but the latter is affected by age, sex, and muscle mass. This is why its interpretation has to be patient-speci ic [11,12].
The renal system ilters 5000 mg of proteins as an average per day in each individual, 4950 mg of which is reabsorbed in the proximal tubule via endocytosis. Low molecular weight proteins are amongst the reabsorbed proteins; these are freely iltered in the glomerulus, given its weight, and are undetectable in normal physiologic processes. The reason why its detection in urine will only appear once there is tubular epithelial injury. These include, but are not limited to, beta-2 microglobulin, lysozyme, alpha-1 microglobulin, and retinol-binding protein 4 (RBP4). These molecules serve as biomarkers of kidney injury [13].
In normal conditions, renal blood low corresponds to 5-6 mls/g/min with a pressure of 60-100 mmHg, which is necessary to maintain normal renal function [10]. Renal blood low is primarily regulated by multiple factors that involve extrarenal processes such as vascular tone, neurohormonal processes and vasodilator/ vasoconstrictor substances, among others. Hence, failure in any of these mechanisms will lead to hypoxia of the organ, which can be spontaneous or not depending on its magnitude. This also depends on the compensatory mechanisms, such as afferent arteriolar dilatation and efferent arteriolar constriction, which can supply the oxygen needs at the time of need. At this time, there is a secretion of pro-in lammatory mediators due to tissue damage, which serve as biomarkers for early kidney injury detection. These include NGAL and KIM-1. IL-8 is secreted by in lammatory cells, such as macrophages are neutrophils that enter the kidneys during the in lammatory phase [13]. These molecules express themselves primarily in distal and proximal epithelial tubular cells, respectively [2,6]. When there is sustained hypoxia, there are changes in renal blood low, changes in the GFR, and the supply of sodium towards the renal tubules, producing a disequilibrium in between the production and the demand of oxygen nutrients, damaging tubular epithelial cells and leading towards oxidative stress [9,14].
After the renal blood low reduction, the epithelial cells cannot maintain adequate intracellular ATP for the essential metabolic processes. This ATP depletion leads to cellular damage and, if severe enough, can cause cellular death by necrosis or apoptosis. During an ischemic event, all the segments of the nephrons are potentially affected, but the proximal tubular cells are the most commonly damaged [3]. The damaged endothelium in the small renal arterioles of the vasa recta in the ischemic kidney suffers vasoconstriction due to an increase in endothelin-1, angiotensin II, thromboxane A2, prostaglandin H2, leukotriene C4, and D4, as well an increase in the sympathetic nervous system activity [14].
Consequently, these enzymes are secreted by the tubular epithelial cells and they are excreted in the urine as a response to the AKI; among these, N-Acetyl-b-glucosaminidase (NAG), cytoplasmic protein lactate dehydrogenase and glycoprotein 130 (gp130) [15][16][17]. The secreted enzymes are the biomarkers themselves, which are classi ied based on their role in the pathophysiology of AKI [18][19][20][21] (Table 1).

Clinical Manifestations and Complications of AKI
Patients with AKI may present with symptoms speci ic to their underlying condition (e.g heart failure, sepsis, cirrhosis, systemic vasculitis). If there is true involvement of renal function, the dynamic process is as follows: stage 1 (initiation), stage II (oligo-anuria), stage III (polyuria) and stage IV (restitution) [59]. There are numerous complications that can occur if the AKI is left untreated or persists; these include hyperkalemia, uremia, volume overload, metabolic acidosis and progression to chronic kidney disease (CKD). Hyperkalemia, de ined as a potassium above 5, is particularly catastrophic in AKI patients and should be treated in an urgent manner to avoid potential life threatening arrhythmias. Management of hyperkalemia includes calcium gluconate to stabilize the cardiac membrane, IV insulin and dextrose, beta-agonists, potassium binding resins and ultimately RRT for refractory hyperkalemia. Persistent metabolic acidosis (de ined as a serum bicarbonate level less than 24), volume overload (especially in patients with heart failure and or/ increasing oxygen requirements) and uremia (pruritus, neurological manifestations, nausea, vomiting, diarrhea, anorexia, arrhythmias and insomnia) all are indications for urgent RRT [59]. Patients with AKI are at risk of developing CKD, and those with CKD are at risk for progression to ESKD. A Taiwan study investigated long term renal outcomes in hospitalized patients; it showed that the risk of developing CKD stage 3 was double among patients who developed AKI during hospitalization and recovered renal function, compared to those who did not develop AKI [60].

Alpha-1 microglobulin and beta-2 microglobulin:
Both molecules rise in serum and urine due to glomerular or tubular lesions, leading to a considerable reduction in the glomerular iltration rate. Their main advantage is their low cost; despite this, it depends on the urinary pH, decreasing its effectiveness if the pH is less than 5.5 [22]. A retrospective Table 1: Classifi cation and biomarkers of AKI with the highest relevance [18][19][20][21]. Each one of them has diff erent properties as a biomarker (e.g., increasing the sensitivity of the diagnoses, predict prognosis, etc.). On the other hand, it has been proposed that the use of a panel of several biomarkers can improve their individual properties [18].
Enzymes released by tubular damage N-Acetyl-b-glucosaminidase (NAG), Alkaline phosphatase, cytoplasmic protein lactate dehydrogenase, TIMP-2, IGFBP-7. study of 529 patients showed that patients with AKI had higher serum beta-2 microglobulin and urinary beta-2 microglobulin with an area under the curve (AUC) of 0.84 and 0.83, respectively. In the study, 245 patients developed AKI. Patients had a higher median serum and urinary beta 2-microglobulin with each higher AKI stage; however, neither were associated with renal recovery [65].
Biphosphatase Protein (RBP4): RBP4 is an adipocytokine whose main function is to transport retinol to the circulation; its role on the kidney is unknown. Several studies have demonstrated that levels of RBP4 are elevated in diabetes, obesity, cardiovascular diseases and in lammation. It has been described that RBP4 by itself can be pro-in lammatory by increasing the production of other adipocytokines or in lammatory cytokines. It loses sensitivity to identify changes in tubular function when it is below 10%. Similarly, it has been found that it can serve as a biomarker of bladder tissue degradation, as in neoplastic disease [22,23].

N-acetyl Glucosaminidase (NAG):
N-acetyl Glucosaminidase (NAG) is a lysosomal enzyme produced by the proximal and distal tubular cells; it's detected 12 hours after injury [15]. It rises after exposure to several toxic substances such as lead and cadmium, solvents, contrast media, aminoglycosides, nephrotoxic cancer medications, as well as glomerular diseases, including diabetic nephropathy [16].

Alkaline
Phosphatase and Gamma-Glumatil Transferase: Alkaline Phosphatase (AP) and Gamma-Glumatil Transferase (GGT) are enzymes in the brush border villi of the proximal tubular cells. They are excreted in the urine when there is signi icant damage to the microvilli in the brush border membrane [15].
Alkaline phosphatase is an endogenous metalloenzyme found in the serum and multiple organs, including kidneys, liver, bone and intestines. Phase 2 studies in humans have shown ef icacy in sepsis-induced AKI with AP [17].
Gamma-Glutamyl Transferase (GGT) is an enzyme located in the cell membrane; it is found in the proximal renal tubules, liver, pancreas and intestines. Urinary GGT is an indicator of tubular damage as it can show changes in renal function before identifying these via conventional methods [24].
Glutathione-S-Transferase: Glutathione-S-Transferase is a soluble cytoplasmic enzyme with two subunits: α y π, expressed in the proximal and distal convoluted tubule, respectively. Epithelial tubular cell damage leads to the accumulation of this enzyme, becoming detectable in urine. It is worth mentioning there are four subtypes; however, α y π are the two most studied ones in AKI [15].

Proteins produced in AKI
NGAL: Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a pro-in lammatory marker of the innate immune system, a useful tool to differentiate between pre-renal and intrinsic AKI. NGAL expression is regulated in the irst few hours after ischemic injury in animal models and has been identi ied as an early biomarker to diagnose AKI. It is a glycoprotein expressed on many cells, including hepatocytes, tubular and endothelial cells [16,17,25].
NGAL is the most studied biomarker in AKI. It was discovered initially in the granules of neutrophils [1]. It is reabsorbed almost totally in the proximal tubule and elevated levels can indicate proximal tubular damage. Production of NGAL can be increased up to 1000 times in Henle's loop and the distal tubule when AKI is present [2]. To summarize, its expression is increased as soon as renal cells detect stress and/or damage [6]. It is also a potential tool to distinguish in between pre-renal azotemia and ATN, as it just elevates mildly in volume depletion, along with L-FABP, with no histologic damage [63].

KIM-1:
Biomarkers such as the kidney injury molecule-1 are fundamental for the kidney's response to proximal tubular damage [2]. KIM-1 is a 38.7 kDa transmembrane type 1 glycoprotein with a similar domain as extracellular immunoglobulin, similar to mucin. It is expressed in low levels in the kidney and other organs, but its expression is accentuated in pre-renal kidney injury and after reperfusion [6].
It is found in higher concentrations in people with AKI in comparison to people with CKD without acute injury. A metaanalysis of 11 clinical trials showed that it has a diagnostic sensitivity and speci icity of 74% and 86% in AKI [25,28]. Sabisetti, et al. demonstrated signi icantly elevated plasma KIM-1 levels in serum in patients with AKI compared to patients without AKI, with an AUC of 0.74% (95% CI 0.48-0.91, p < 0.01). In 2002, Han et al showed that KIM-1 was speci ic for ATN; an increase of 1 unit in normalized KIM-1 was associated with an odd's ratio (OR) of 12.4 (95% CI, 1.2-119) for the presence of ATN [61]. This makes it a useful tool in differentiating between pre-renal azotemia and intrinsic injury.

IL-18:
Also known as a factor that enhances interferongamma, IL-18 is a 24kDa cytokine that belongs to the superfamily of IL-1. It is initially synthesized as a signal-free inactive precursor and remains in the intracellular space until it is excised by caspase-1. Posteriorly, it gets secreted by monocytes and macrophages [6]. It is an in lammatory mediator produced in response to ischemia of several organs, including the heart, brain, and kidneys [25]. In the kidney, it is produced and excreted in the proximal tubule secondary to ischemia or toxic damage [28].
IL-18 plays a vital role in tubular apoptosis, with high urinary concentrations in the urine of patients with ischemic AKI [29]. It serves as an early diagnostic marker given that it can predict mortality in several clinical settings, including ICU and after cardiovascular surgery [2]. Elevated levels of IL-18 in the urine was shown by Parikh, et al. in 2011 to be an independent predictor of dialysis or death, with an OR of 6.8 [62].
Cysteine 61: Cysteine 61 (Cyr61) is a cysteine-rich secretory protein found in human renal tissue. It promotes cellular proliferation, adhesion, chemotaxis, embryo development and neovascularization. Previous studies have detected low levels of Cyr61 expression in the healthy adult kidney, but increased expression in ischemic renal tissue. It is also elevated in in lammatory states, suggesting it may be a potential biomarker for AKI [30,31]. Xu, et al. found that Cyr 61 could protect tubular epithelial cells from apoptosis; however, its capability to detect AKI and when it is detected is still unknown [32].

Structural and functional tubular proteins
F-actin and Na+/H+ exchanger isoforms: Actin plays a fundamental role in maintaining structural and cellular functions, acting as a barrier between adhesion/union complexes and the cellular matrix [33].
The integrity of the cytoskeleton is essential for proximal tubular cells, given that the ampli ication of the apical membrane through the microvilli is decisive for healthy cellular function. Loss of actin in the cytoskeleton increases interstitial permeability and an interstitial leak of urine through the tubular basement membrane, causing bidirectional sodium transport between the apical and basolateral portions of the cell. Increased microvascular permeability is seen in AKI, increasing the amount of iltered sodium towards the distal tubule. This mechanism reduces GFR [33].

YKL-40:
YKL-40 is a 40 kDa chitinase-like glycoprotein implied in in lammation, cellular protection and repair. It is synthesized by renal macrophages and contributes to tissue remodeling and scarring by limiting cellular apoptosis; it promotes cellular repair after ischemic renal injury, becoming a good option when it comes to predicting AKI recovery [34][35][36][37].

GFR biomarkers
Cystatin C: Cystatin C is a low molecular weight protein produced in the nucleated cells. It is freely iltered by the glomeruli and almost completely reabsorbed in the proximal tubule. Its constant production and renal elimination make it an excellent biomarker of glomerular iltration. However, Cystatin C does not estimate GFR in real-time. Serum Cystatin C can be a useful biomarker in AKI prediction; its urinary excretion indicates tubular damage, and it has a moderate diagnostic utility [28,38]. Given this, it has been studied for its use in several clinical settings, especially in patients with a high risk of developing AKI [15,25]. Its concentrations are elevated in acute and chronic kidney disease, and contrary to creatinine, it does not depend on height, weight, age, sex, nutritional status, and in lammatory processes [16].
In 2017, Deng et al. carried out an observational, multicentric and prospective trial in which Cystatin C was found to be useful when associated with NAG for the early detection and prognosis of AKI, as well as a predictor of mortality [39,40]. Patients with Cystatin C levels below 0.8 mg/L have less chance of developing AKI after kidney injury, while patients with levels superior to 2.04 mg/L have a more signi icant chance of developing AKI [38]. Another metaanalysis of 19 studies in 11 countries involving 3,336 patients showed that across all settings, the diagnostic OR of serum cystatin C of predicting AKI was 23.5 (95% CI, 14.2-38.9), with sensitivity and speci icity of 0.84 and 0.82, respectively [65].

The best evidence-based biomarker in AKI
The list of biomarkers of kidney function and damage has surged in light of the evidence with an uncertain assertive value, with its clinical utility questioned in the context of AKI [41]. Thus, serum creatinine and urine output are very useful tools in the diagnosis of AKI. However, serum creatinine is a late marker of kidney injury [15].
A signi icant reduction in GFR can be challenging to appreciate given the initial changes in sCr in the acute phase, considering that creatinine excretion decreases during a steady drop in GFR, making the serum creatinine rise with time. Urine output is a result of those mentioned above, conceived as an additional severity marker [39]. Consequently, kidney function changes are usually evaluated by monitoring the solutes eliminated by glomerular iltration, such as creatinine [3].
Serum and urinary creatinine act as functional biomarkers, but its utility is limited, given that 400 to 500 milliliters of urine have to be excreted daily to eliminate nitrogen waste products [8]. Serum creatinine and urine output are nor sensitive or speci ic for AKI detection. Nonetheless, these are the mainstay markers for diagnosis. That is why a decrease in serum creatinine, though not de ining AKI, should prompt clinicians to suspect AKI; it can also be useful when the baseline creatinine is unknown [42].
The increase in serum creatinine and the decrease in urine output are not enough for AKI diagnosis, and their sensitivity and speci icity are too low to establish a diagnosis. It's also worth to mention that a healthy renal functional reserve can mitigate the rise in serum creatinine [6]. Despite its prognostic value, a rise in serum creatinine typically occurs 24 to 48 hours after the event. Similarly, its levels are affected by independent factors such as muscle mass, sex, diet, and some medications [2]. Even so, the diagnosis and strati ication of AKI are made by obtaining serum creatinine levels [43].
However, due to pathophysiological reasons, one can suppose that early intervention is a crucial factor for treatment success, justifying the search for new biomarkers that not only show a decline in kidney function but also permits earlier detection of structural damage [8] (Table 2).

Nephrocheck
AKI is very common in ICU patients, associated with increased short and long term complications; additionally, it is associated with increased morbidity and mortality.
In September 2014, the FDA allowed the commercialization of Nephrocheck, a test made to determine if certain critical patients were at risk of developing AKI [45].
In this sophisticated setting, the idea of introducing a biomarker in clinical practice that is synchronized with renal damage arose, since traditional criteria based on serum creatinine and urine output are not timely in the identi ication of patients at risk for AKI. This, because clinical manifestations occur when the residual iltration rate cannot compensate [46]. Taking the aforementioned into account, it is necessary to introduce a biomarker capable of identifying high-risk patients to implement preventative strategies even before sCr rises or urine output decreases [46,47].
From that standpoint, in recent years, several studies focused on discovering new biomarkers with the goal of early AKI detection. These include NGAL, IL-18, L-FABP, KIM-1, TIMP-2, and IGFBP-7. Among these, IGFBP-7 and TIMP-2 have shown the most signi icant utility and have been proposed as a predictive tool in the early detection of AKI in critical care settings [48][49][50].
TIMP-2 and IGFBP-7 are expressed and secreted in the kidneys as well as other tissues. Some authors postulated that these proteins halt the G1 phase of the cell cycle in response to different factors (for example, oxidative stress, toxins, ischemia, sepsis, and in lammation); their signaling pathways also have an autocrine and paracrine pattern [51,52]. Hence, markers of cellular cycle detention like TIMP-2 and IGFBP-7 act as a warning sign that tubular cells have been a subject of stress and have to shut down their function to preserve energy.
Katani, et al. validated IGFBP-7 and TIMP-2 through a prospective, multicenter clinical trial compared to other biomarkers in AKI. The authors selected a cohort of critical care patients without evidence of AKI on presentation. The primary endpoint was moderate to severe AKI based on the KDIGO guidelines (KDIGO stage 2-3) within the irst 12 hours, which occurred in 14% in the patients.  x According to this trial,  x  were notably superior to other AKI biomarkers (p < 0.002), including NGAL and KIM-1, nor of which made an AUC of > 0.27 [53].
Evidence shows that these biomarkers (TIMP-2 and IGFBP-7), identi ied as Nephrocheck, have a high predictive capacity of identifying patients at risk for AKI. Sapphire investigators and others demonstrated that Nephrocheck predicts stage 2-3 AKI twelve hours before the rise in serum creatinine [53][54][55][56]. McCullough et al. evaluate serial Nephrocheck measurements in a retrospective post hoc analysis in the Sapphire ICU cohort and suggest that these can be useful in detecting stage 2-3 AKI in the irst seven days of the ICU course [55]. The authors concluded that serial urine Nephrocheck tests  x [IGFBP-7] at 0, 12, and 24 hours, and even 72 hours are prognostic for AKI development. Three consecutive negative values [< 0.3 (ng/mL) 2/ 1000] are associated with a low incidence (13%) of stage 2-3 AKI in the irst seven days. On the contrary, positive results [> 2.0 (ng/mL) 2 /1000) predict a higher incidence (up to 94.4%) of stage 2-3 AKI. Other authors previously suggested that serial measurements of these AKI biomarkers could further characterize patients' risk during their ICU stay [56].
Other studies suggest that these new biomarkers add diagnostic and prognostic certainty, allowing for a better evaluation of patients at risk for AKI. Although they do not differentiate between the type of injury (ischemic, toxins, in lammatory) or the speci ic site of damage in the nephron, its use today seems to be justi ied [57,58].
With the routine use of these biomarkers, we can use the data to develop algorithms via arti icial intelligence to guide our practice. We shall see these AKI biomarkers utilized to evaluate AKI risk and diagnosis in the upcoming years.

Conclusion
Nowadays, some biomarkers play an essential role in detecting structural and functional abnormalities in the kidneys. There has not been an established speci ic biomarker that predicts AKI to its fullest, why it is necessary to introduce clinical biomarkers synchronized with acute kidney injury, like Nephrocheck. With the routine use of these biomarkers, we will have an enormous quantity of useful data to design algorithms for our clinical practice. We will notice an increase in the use of these biomarkers in the upcoming years. Early detection and intervention in AKI decrease the damage caused, reducing the chance of complications and mortality.