**2. Acute diseases of the kidney and biomarkers**

Biomarkers have been identified for the following acute diseases of the kidney: acute pyelonephritis or upper urinary tract infection (UTI) and acute cystitis/urethritis or lower UTI, as well as acute kidney injury (AKI). UTI ranks third in the hierarchy of common childhood infections in developing countries [7], and remains one of the commonest causes of febrile illness in the pediatric age group [8, 9].

sepsis, as its levels correlate with the severity of sepsis [12]. In comparison with other acute phase reactants such as IL-2, IL-6, IL-8, C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α), PCT has a high sensitivity (85%) and specificity (91%) for discriminating between patients with systemic inflammatory response syndrome (SIRS) and those with sepsis [13]. It is now well established that serum PCT has a good diagnostic accuracy for the diagnosis of acute pyelonephritis than for lower UTI, with reported high sensitivity and specificity values [14–16]. For instance, sensitivity and specificity values of 94.1 and 89.7% respectively, and sensitivity and specificity values of 83.3 and 93.7% respectively, have been documented [15, 16]. PCT also has a good predictive ability for vesico-ureteric reflux (VUR), as high levels of this biomarker are significantly related to high-grade VUR [17–19]. Again, children with VUR reportedly presented with significantly higher median PCT levels than those without VUR; sensitivity and negative predictive values of 94.3 and 95.4% respectively for predicting high-grade VUR (using the biomarker alone), as well as 97.1 and 97.8% respectively (using a combination of the biomarker and ultrasound studies) have been recorded [18]. Finally, PCT can also serve as an early predictor of renal parenchymal damage in children with UTI [20]. The implication is that the prognostic utility of PCT may in future preclude the need for dimercaptosuccinic acid (DMSA) scan, or better still, may serve as an adjunct to this imaging study. Similarly, other biomarkers such as urine 8-hydroxy-2′-deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) are useful in the prediction of renal parenchymal injury in children with UTI [21]. Interestingly, those patients with positive DMSA scan had higher levels of

**Figure 1.** Schematic representation of the list of biomarkers used in the three-fold evaluation of urinary tract infection

Biomarkers of Common Childhood Renal Diseases http://dx.doi.org/10.5772/intechopen.74016 127

(UTI).

## **2.1. Biomarkers of UTI**

Serum procalcitonin (PCT), urine/serum interleukin 6 (IL-6) and 8 (IL-8), and urine neutrophil gelatinase-associated lipocalin (NGAL) are currently the major biomarkers identified in the evaluation of the disease. Other novel biomarkers include urine interleukin 1-beta (IL-1β), urine 8-hydroxy-2-deoxyguanosine (8-oxodG), and urine total anti-oxidant capacity (TAC) (**Figure 1**).

PCT is one of the inflammatory markers recognized as a biomarker of severe bacterial infection [10]. It is essentially a peptide precursor of the hormone- calcitonin - produced by the thyroid parafollicular cells, as well as the neuroendocrine cells of the lungs and intestines. Its serum level in healthy subjects is below the limit of detection (0.01 μg/L) of laboratory assays [11]. Notably, serum PCT level is not elevated significantly with viral or non-infectious inflammatory process, but may rise up to 100 μg/L in systemic inflammatory response from severe bacterial infection. In fact, estimation of PCT can be used as a marker of severe bacterial

need for a paradigm shift to biomarkers. The attraction of these novel biomarkers lies in the non-invasive nature of their use in disease evaluation. However, the wide variation in their

According to the definition by the *National Institute of Health Biomarkers Definitions Working Group*, a biomarker refers to 'a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention' [4, 5]. In other words, it is a measurable indicator of a biological state or condition. Although there are diverse perspectives to the definition of a biomarker, for the purpose of the current topic, a biomarker can be defined as a substance whose detection indicates a particular disease-state, or a change in expression/state of a protein which correlates with the risk/progression of a disease, or with disease susceptibility to a given therapy. In fact, the most actively studied biomarkers presently are the biological macromolecules. For instance, proteins are most easily measured by immunoassays or by mass spectrometry combined with high-resolution separation techniques, while nucleic acids can be amplified by

In the identification of biomarkers of kidney injury, an ideal biomarker should be produced after organ injury in concentrations which directly correlate with the degree of injury; should be easily measured in body fluids; and should serve as a potential tool to monitor therapeutic

In this book chapter, novel biomarkers which essentially meet these requirements with respect

Biomarkers have been identified for the following acute diseases of the kidney: acute pyelonephritis or upper urinary tract infection (UTI) and acute cystitis/urethritis or lower UTI, as well as acute kidney injury (AKI). UTI ranks third in the hierarchy of common childhood infections in developing countries [7], and remains one of the commonest causes of febrile illness

Serum procalcitonin (PCT), urine/serum interleukin 6 (IL-6) and 8 (IL-8), and urine neutrophil gelatinase-associated lipocalin (NGAL) are currently the major biomarkers identified in the evaluation of the disease. Other novel biomarkers include urine interleukin 1-beta (IL-1β), urine 8-hydroxy-2-deoxyguanosine (8-oxodG), and urine total anti-oxidant capacity (TAC) (**Figure 1**). PCT is one of the inflammatory markers recognized as a biomarker of severe bacterial infection [10]. It is essentially a peptide precursor of the hormone- calcitonin - produced by the thyroid parafollicular cells, as well as the neuroendocrine cells of the lungs and intestines. Its serum level in healthy subjects is below the limit of detection (0.01 μg/L) of laboratory assays [11]. Notably, serum PCT level is not elevated significantly with viral or non-infectious inflammatory process, but may rise up to 100 μg/L in systemic inflammatory response from severe bacterial infection. In fact, estimation of PCT can be used as a marker of severe bacterial

sensitivity and specificity is still a major challenge.

126 Biomarker - Indicator of Abnormal Physiological Process

the polymerase chain reaction (PCR) and related techniques.

**2. Acute diseases of the kidney and biomarkers**

in the pediatric age group [8, 9].

**2.1. Biomarkers of UTI**

response, predicated upon a post-injury decrease in its concentration [6].

to evaluating common kidney diseases in children are highlighted and discussed.

**Figure 1.** Schematic representation of the list of biomarkers used in the three-fold evaluation of urinary tract infection (UTI).

sepsis, as its levels correlate with the severity of sepsis [12]. In comparison with other acute phase reactants such as IL-2, IL-6, IL-8, C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α), PCT has a high sensitivity (85%) and specificity (91%) for discriminating between patients with systemic inflammatory response syndrome (SIRS) and those with sepsis [13]. It is now well established that serum PCT has a good diagnostic accuracy for the diagnosis of acute pyelonephritis than for lower UTI, with reported high sensitivity and specificity values [14–16]. For instance, sensitivity and specificity values of 94.1 and 89.7% respectively, and sensitivity and specificity values of 83.3 and 93.7% respectively, have been documented [15, 16]. PCT also has a good predictive ability for vesico-ureteric reflux (VUR), as high levels of this biomarker are significantly related to high-grade VUR [17–19]. Again, children with VUR reportedly presented with significantly higher median PCT levels than those without VUR; sensitivity and negative predictive values of 94.3 and 95.4% respectively for predicting high-grade VUR (using the biomarker alone), as well as 97.1 and 97.8% respectively (using a combination of the biomarker and ultrasound studies) have been recorded [18]. Finally, PCT can also serve as an early predictor of renal parenchymal damage in children with UTI [20]. The implication is that the prognostic utility of PCT may in future preclude the need for dimercaptosuccinic acid (DMSA) scan, or better still, may serve as an adjunct to this imaging study.

Similarly, other biomarkers such as urine 8-hydroxy-2′-deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) are useful in the prediction of renal parenchymal injury in children with UTI [21]. Interestingly, those patients with positive DMSA scan had higher levels of urine 8-oxodG and higher urine TAC than the patients with normal DMSA scan: showing that urine levels of 8-oxodG may directly correlate with renal parenchymal injury.

injury and repair [1]. For example, biomarkers like NGAL, interleukin-18 (IL-18), N-acetylβ-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), accumulate in urine due to an induced tubular epithelial synthesis in different parts of the nephron, or as a consequence of reduced reabsorption of the filtered load in the proximal tubule (NGAL, cystatin C) [34]. Furthermore, production of biomarkers from transmigrated, activated immune cells into the tubular lumen may be contributory (NGAL, IL-18), while increased production of some biomarkers in other tissues (NGAL, IL-18) also occurs, thus raising concerns about their diagnostic value in the disease [35]. Biomarkers of AKI have been classified as follows: *functional markers* (serum cystatin C, urine albumin and NGAL), *up-regulated proteins* (KIM-1, liver-type fatty-acid binding protein [L-FABP], IL-18, β-trace protein and asymmetric dimethyl arginine), *low-molecular weight proteins* (urine cystatin C, NAG, glutathione S-transferase, γ-glutamyltrans peptidase [γGT]) and *enzymes* (alanine amino-peptidase and lactate dehydrogenase) [36]. Other markers which have shown diagnostic ability include hepatocyte growth factor (HGF), vasoactive endothelial growth factor (VEGF), interferon gamma-induced protein 10

Biomarkers of Common Childhood Renal Diseases http://dx.doi.org/10.5772/intechopen.74016 129

(IP-10) and total protein [37]. Some of the novel biomarkers are highlighted as below.

First, KIM-1 is a transmembrane tubular glycoprotein which is upregulated approximately 50 to 100-fold in the kidney, and is secreted into the urine after proximal tubular injury. KIM-1 is highly expressed in renal tubules, and is typically seen in areas of fibrosis and inflammation. The results of a meta-analysis indicate that urine KIM-1 represents a promising biomarker for early detection of AKI, with a good predictive value, especially in cardiac surgery patients [38]. Furthermore, in comparison to other biomarkers used as indicators of drug toxicity, KIM-1 significantly performed better than serum creatinine and blood urea nitrogen at detecting renal tubular injury in murine models; this makes it a useful marker for determining drug toxicity [39].

Second, NAG (another biomarker of kidney injury) is a large (>130 kDa) lysosomal enzyme which is located in several human cells including the renal tubule [34]. Its size hinders glomerular filtration, and elevated urine levels are thus presumed to emanate from the tubules, indicating tubular injury. In fact, during active renal disease, urine NAG activity is increased

Third, cystatin C is a low-molecular-weight protein produced by all nucleated cells in the body at a constant rate, freely filtered by the glomeruli but completely reabsorbed and catabolized by the renal tubule [41]. Thus, its elevated urine levels are also seen in a tubulopathy because of the reduced re-absorptive capacity of the proximal tubules: making it a non-specific biomarker of AKI [42]. However, urine cystatin C could correctly predict the need for dialysis in intensive care unit (ICU) patients with established AKI [43]; could be an early predictor of AKI in children and in pediatric RIFLE classification, as well as a predictor of reduced estimated GFR (eGFR) after cardiac surgery [44]; while serum cystatin C could be used alone or in combination with serum creatinine and eGFR for early and accurate diagnosis of AKI in patients at emergency settings [45]. Finally, NGAL (which plays a role in the evaluation of UTI as previously mentioned) is a universal iron-transporter protein expressed in the tubular epithelium of the distal nephron and released into the blood and urine following tubular injury. This biomarker was first identified as a 25 kDa protein in the secondary granules of human neutrophils which is released into the bloodstream in response to bacterial infection. Interestingly, its elevated level in the urine may be diagnostic of AKI using the Acute Kidney Injury Network (AKIN) criteria: although

[40]. This feature makes it a potential and sensitive biomarker of AKI.

Urine NGAL is another biomarker for the diagnostic evaluation of UTI. NGAL (also called lipocalin-2) is a protein involved in innate immunity as it is involved in the sequestration of iron which ultimately blocks bacterial growth [22]. It is expressed mainly in neutrophils and in low concentrations in the kidney, prostrate, as well as respiratory and gastrointestinal epithelia [23]. NGAL is currently employed as a biomarker of renal injury [24], given that it is secreted in high concentrations into the blood and urine within 2 hours of AKI [25]. In addition, the biomarker is easily excreted and detected in the urine because it is protease-resistant. Thus, urine NGAL appears to be a specific and sensitive biomarker for the diagnosis of UTI and for the prediction of renal parenchymal injury in the disease [26, 27].

Finally, some interleukins also play a role in disease diagnosis and in differentiating upper from lower UTI. Generally, the infection stimulates both local and systemic cytokine responses [28, 29]. For instance, *Escherichia coli* is known to activate a cytokine response in the uroepithelial cell lines to produce IL-6 and IL-8, and in peripheral blood monocytes to produce interleukin 1-alpha (IL-1α), IL-1β, IL-8, IL-6, and TNF-α [28]. Adherence of this bacterium on the mucosal sites of the urinary tract triggers inflammation, including a mucosal cytokine-response which results in the secretion of IL-6 and IL-8 by activated uroepithelial cells [29, 30]. Thus, since IL-6 and IL-8 are expressed early after a UTI episode and are secreted by uroepithelial cells, these cytokines have clearly fulfilled some of the five characteristics of an ideal biomarker outlined in the introductory section. Reports indeed suggest that urine IL-6 and IL-8 are proven biomarkers for UTI in children [31]. The serum and urine levels of IL-6 were more sensitive and specific for upper UTI than levels of IL-8 [32]. Sensitivity and specificity values for serum IL-6 were noted to be 88 and 83% respectively, while for urine IL-6, sensitivity and specificity values were 86 and 81%, respectively [32]. Furthermore, serum IL-6 was able to differentiate upper from lower UTI with a sensitivity of 88% and specificity of 74%, while serum IL-1β was known to have a sensitivity of 97% and a specificity of 59% for detecting upper UTI [14].

#### **2.2. Biomarkers of AKI**

Serum creatinine represents a poor traditional biomarker for AKI due to some limitations. First, its levels are only altered when renal function diminishes by 50% [33]. Second, there are well-known confounders to serum creatinine levels such as muscle size, chronologic age, gender, drugs and state of hydration [34]. Third, a sudden reduction in renal function may not be shown by a rise in serum creatinine until after 24–48 hours. Finally, it provides little information about the underlying cause and nature of kidney injury, and is less accurate for patients with small muscle mass and unusual diets [1]. These draw-backs have resulted in a paradigm shift to novel biomarkers.

Generally, there are two main types of AKI biomarkers: *biomarkers of kidney function* and *biomarkers of kidney injury*. Irrespective of the type of kidney injury and the clinical scenario, an inflammatory response appears to play a significant role in the pathogenesis of AKI [34]. The major triggers of AKI (ischemia, nephrotoxins, and bacterial endotoxins) stimulate the release of inflammatory mediators from renal endothelial and tubular cells. As AKI progresses, a number of causative factors result in the accumulation of biomarkers in plasma and urine, and possibly indicate different pathophysiologic events during the process of kidney injury and repair [1]. For example, biomarkers like NGAL, interleukin-18 (IL-18), N-acetylβ-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), accumulate in urine due to an induced tubular epithelial synthesis in different parts of the nephron, or as a consequence of reduced reabsorption of the filtered load in the proximal tubule (NGAL, cystatin C) [34]. Furthermore, production of biomarkers from transmigrated, activated immune cells into the tubular lumen may be contributory (NGAL, IL-18), while increased production of some biomarkers in other tissues (NGAL, IL-18) also occurs, thus raising concerns about their diagnostic value in the disease [35]. Biomarkers of AKI have been classified as follows: *functional markers* (serum cystatin C, urine albumin and NGAL), *up-regulated proteins* (KIM-1, liver-type fatty-acid binding protein [L-FABP], IL-18, β-trace protein and asymmetric dimethyl arginine), *low-molecular weight proteins* (urine cystatin C, NAG, glutathione S-transferase, γ-glutamyltrans peptidase [γGT]) and *enzymes* (alanine amino-peptidase and lactate dehydrogenase) [36]. Other markers which have shown diagnostic ability include hepatocyte growth factor (HGF), vasoactive endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10) and total protein [37]. Some of the novel biomarkers are highlighted as below.

urine 8-oxodG and higher urine TAC than the patients with normal DMSA scan: showing that

Urine NGAL is another biomarker for the diagnostic evaluation of UTI. NGAL (also called lipocalin-2) is a protein involved in innate immunity as it is involved in the sequestration of iron which ultimately blocks bacterial growth [22]. It is expressed mainly in neutrophils and in low concentrations in the kidney, prostrate, as well as respiratory and gastrointestinal epithelia [23]. NGAL is currently employed as a biomarker of renal injury [24], given that it is secreted in high concentrations into the blood and urine within 2 hours of AKI [25]. In addition, the biomarker is easily excreted and detected in the urine because it is protease-resistant. Thus, urine NGAL appears to be a specific and sensitive biomarker for the diagnosis of UTI

Finally, some interleukins also play a role in disease diagnosis and in differentiating upper from lower UTI. Generally, the infection stimulates both local and systemic cytokine responses [28, 29]. For instance, *Escherichia coli* is known to activate a cytokine response in the uroepithelial cell lines to produce IL-6 and IL-8, and in peripheral blood monocytes to produce interleukin 1-alpha (IL-1α), IL-1β, IL-8, IL-6, and TNF-α [28]. Adherence of this bacterium on the mucosal sites of the urinary tract triggers inflammation, including a mucosal cytokine-response which results in the secretion of IL-6 and IL-8 by activated uroepithelial cells [29, 30]. Thus, since IL-6 and IL-8 are expressed early after a UTI episode and are secreted by uroepithelial cells, these cytokines have clearly fulfilled some of the five characteristics of an ideal biomarker outlined in the introductory section. Reports indeed suggest that urine IL-6 and IL-8 are proven biomarkers for UTI in children [31]. The serum and urine levels of IL-6 were more sensitive and specific for upper UTI than levels of IL-8 [32]. Sensitivity and specificity values for serum IL-6 were noted to be 88 and 83% respectively, while for urine IL-6, sensitivity and specificity values were 86 and 81%, respectively [32]. Furthermore, serum IL-6 was able to differentiate upper from lower UTI with a sensitivity of 88% and specificity of 74%, while serum IL-1β was known

urine levels of 8-oxodG may directly correlate with renal parenchymal injury.

128 Biomarker - Indicator of Abnormal Physiological Process

and for the prediction of renal parenchymal injury in the disease [26, 27].

to have a sensitivity of 97% and a specificity of 59% for detecting upper UTI [14].

Serum creatinine represents a poor traditional biomarker for AKI due to some limitations. First, its levels are only altered when renal function diminishes by 50% [33]. Second, there are well-known confounders to serum creatinine levels such as muscle size, chronologic age, gender, drugs and state of hydration [34]. Third, a sudden reduction in renal function may not be shown by a rise in serum creatinine until after 24–48 hours. Finally, it provides little information about the underlying cause and nature of kidney injury, and is less accurate for patients with small muscle mass and unusual diets [1]. These draw-backs have resulted in a paradigm shift to novel biomarkers.

Generally, there are two main types of AKI biomarkers: *biomarkers of kidney function* and *biomarkers of kidney injury*. Irrespective of the type of kidney injury and the clinical scenario, an inflammatory response appears to play a significant role in the pathogenesis of AKI [34]. The major triggers of AKI (ischemia, nephrotoxins, and bacterial endotoxins) stimulate the release of inflammatory mediators from renal endothelial and tubular cells. As AKI progresses, a number of causative factors result in the accumulation of biomarkers in plasma and urine, and possibly indicate different pathophysiologic events during the process of kidney

**2.2. Biomarkers of AKI**

First, KIM-1 is a transmembrane tubular glycoprotein which is upregulated approximately 50 to 100-fold in the kidney, and is secreted into the urine after proximal tubular injury. KIM-1 is highly expressed in renal tubules, and is typically seen in areas of fibrosis and inflammation. The results of a meta-analysis indicate that urine KIM-1 represents a promising biomarker for early detection of AKI, with a good predictive value, especially in cardiac surgery patients [38]. Furthermore, in comparison to other biomarkers used as indicators of drug toxicity, KIM-1 significantly performed better than serum creatinine and blood urea nitrogen at detecting renal tubular injury in murine models; this makes it a useful marker for determining drug toxicity [39].

Second, NAG (another biomarker of kidney injury) is a large (>130 kDa) lysosomal enzyme which is located in several human cells including the renal tubule [34]. Its size hinders glomerular filtration, and elevated urine levels are thus presumed to emanate from the tubules, indicating tubular injury. In fact, during active renal disease, urine NAG activity is increased [40]. This feature makes it a potential and sensitive biomarker of AKI.

Third, cystatin C is a low-molecular-weight protein produced by all nucleated cells in the body at a constant rate, freely filtered by the glomeruli but completely reabsorbed and catabolized by the renal tubule [41]. Thus, its elevated urine levels are also seen in a tubulopathy because of the reduced re-absorptive capacity of the proximal tubules: making it a non-specific biomarker of AKI [42]. However, urine cystatin C could correctly predict the need for dialysis in intensive care unit (ICU) patients with established AKI [43]; could be an early predictor of AKI in children and in pediatric RIFLE classification, as well as a predictor of reduced estimated GFR (eGFR) after cardiac surgery [44]; while serum cystatin C could be used alone or in combination with serum creatinine and eGFR for early and accurate diagnosis of AKI in patients at emergency settings [45].

Finally, NGAL (which plays a role in the evaluation of UTI as previously mentioned) is a universal iron-transporter protein expressed in the tubular epithelium of the distal nephron and released into the blood and urine following tubular injury. This biomarker was first identified as a 25 kDa protein in the secondary granules of human neutrophils which is released into the bloodstream in response to bacterial infection. Interestingly, its elevated level in the urine may be diagnostic of AKI using the Acute Kidney Injury Network (AKIN) criteria: although with a moderate predictive value [46, 47]. In addition, its combination with another urine biomarker such as L-FABP resulted in early detection of AKI after cardiac surgery in a sample of adult patients before a rise in serum creatinine was noted [48]. Similarly, in murine models of ischemic and toxic AKI, NGAL was identified as one of the most speedily-induced proteins; its level was elevated by several folds in both serum and urine within hours of the insult [49].
