**4. Novel diagnostic and predictive biomarkers of AKI in children**

#### **4.1. Biomarkers used in sickle cell disease**

New biomarkers are promising for the early detection of renal function loss in patients with SCD (**Table 3**), including cystatin C, plasma neutrophil gelatinase-associated lipocalin (NGAL), serum liver fatty acid-binding protein (L-FABP), serum kidney injury marker 1 (KIM-1), serum interleukin 18 (IL-18), soluble FMS-like tyrosine kinase-1 (sFLT-1) and N-acetyl-b-d glucosaminidase (NAG) [106].

#### *4.1.1. Serum cystatin C*

Cystatin C (CysC) or cystatin 3, a protein encoded by the CST3 gene, is mainly used as a biomarker of kidney function. Recently, it has been studied for its role in predicting new-onset or deteriorating cardiovascular disease (CVD). CysC is a nonglycosylated low molecular weight (13 kDa) basic protein that inhibits cysteine proteases and it has been demonstrated its closely correlation to glomerular filtration rate (GFR) in children [107]. CysC crosses the glomerular membrane, and it is reabsorbed and metabolized in the renal tubules and not returned to the bloodstream. It is not secreted by the tubules, even in cases of reduced GFR, and is not affected by muscle mass, protein intake, metabolic factors, drugs and inflammatory stimuli. It has also been reported that serum CysC correlates with the level of albuminuria [108, 109] and its levels well perform as marker of renal function to detect cardiovascular outcome both in Sickle Cell Nephropathy: Current Understanding of the Presentation, Diagnostic and… http://dx.doi.org/10.5772/intechopen.76588 165


**Table 3.** Novel urinary biomarkers.

**4. Novel diagnostic and predictive biomarkers of AKI in children**

New biomarkers are promising for the early detection of renal function loss in patients with SCD (**Table 3**), including cystatin C, plasma neutrophil gelatinase-associated lipocalin (NGAL), serum liver fatty acid-binding protein (L-FABP), serum kidney injury marker 1 (KIM-1), serum interleukin 18 (IL-18), soluble FMS-like tyrosine kinase-1 (sFLT-1) and N-acetyl-b-d

Cystatin C (CysC) or cystatin 3, a protein encoded by the CST3 gene, is mainly used as a biomarker of kidney function. Recently, it has been studied for its role in predicting new-onset or deteriorating cardiovascular disease (CVD). CysC is a nonglycosylated low molecular weight (13 kDa) basic protein that inhibits cysteine proteases and it has been demonstrated its closely correlation to glomerular filtration rate (GFR) in children [107]. CysC crosses the glomerular membrane, and it is reabsorbed and metabolized in the renal tubules and not returned to the bloodstream. It is not secreted by the tubules, even in cases of reduced GFR, and is not affected by muscle mass, protein intake, metabolic factors, drugs and inflammatory stimuli. It has also been reported that serum CysC correlates with the level of albuminuria [108, 109] and its levels well perform as marker of renal function to detect cardiovascular outcome both in

**4.1. Biomarkers used in sickle cell disease**

164 Hematology - Latest Research and Clinical Advances

**Table 2.** (A) GFR categories in CKD; (B) ACR categories (KDIGO 2012) [105].

glucosaminidase (NAG) [106].

*4.1.1. Serum cystatin C*

population-based studies and in patients with non-ST-elevation acute coronary syndrome [110]. Therefore CysC is an ideal biomarker for study the renal function in SCD patients which is ongoing hemolysis and inflammation secondary to the sickling phenomenon and in which creatinine clearance is generally increased and serum creatinine is low. Tantawy et al. [111] have found significantly higher serum CysC in patients with SCD compared to healthy controls. In particular, patients of their court of study with nephropathy had higher cystatin levels than those without, and a significant positive correlation was found with ACR. The authors have demonstrated also that patients with SCD treated with hydroxyurea had lower CysC levels than untreated patients, possibly due to the role of hydroxyurea in decreasing inflammation [111]. In agreement with Tantawy's results, Alvarez et al. [108] examined the value of serum CysC as a marker for GFR in small cohort of 20 children with SCD with and without albuminuria, compared to serum creatinine and creatinine clearance. The mean GFR derived from serum cystatin was significantly different among these subgroups, becoming abnormal in the proteinuric cohort (63 mL/min per 1.73 m2 ), compared to 94 mL/min per 1.73 m2 for the microalbuminuric and 103 mL/min per 1.73 m2 for the normal subgroups. Serum creatinine or creatinine clearance did not change significantly with the level of albuminuria. The authors concluded that serum CysC was higher than serum creatinine in SCD, and this probably relates to the fact that serum CysC is not secreted by the kidney, as creatinine. Moreover other studies have demonstrated the utility of CysC in patients with SCD. Asnani and Reid [109] have proved in 98 adults with SCD that CysC levels were significantly correlated with measured GFR, hemoglobin, serum creatinine, urinary albumin-creatinine ratio (UACR) and systolic blood pressure. In addition to urine, screening for albuminuria may help in the diagnosis of early renal impairment in the patients with SCD before a significant rise in serum creatinine is observed. Receiver-operating characteristic curve (ROC) analysis has revealed that the cut-off value of CysC at 580 ng/mL could differentiate patients having SCD with and without nephropathy with 87.8% sensitivity and 84.6% specificity. Further prospective studies are needed to validate this threshold. On the other hand, Cho et al. [112] have evaluated the significance of serum cystatin C levels in pediatric patients with chronic kidney disease diagnosed by renal biopsy and showed normal serum creatinine levels. The authors have found that 95% of the patients showed only slightly increased cystatin C levels from the upper normal limit of the reference range and suggested that mildly increased cystatin C without increased creatinine might not have clinical significance.

the other hand, the authors have found significant positive correlations between serum levels of sFLT-1 and microalbuminuria, LDH and indirect bilirubin. Meanwhile, there were no significant correlations between serum levels of sFLT-1 and creatinine, eGFR, serum ferritin and erythrocyte sedimentation rate (ESR). This association between sFLT-1 levels and microalbuminuria combined with the association of sFLT-1 with soluble vascular cell adhesion molecule (VCAM) in prior studies [124], suggests that sFLT-1 may contribute to the pathogenesis of

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Serum liver-type fatty acid-binding protein (L-FABP) is an anti-oxidant, renoprotective molecule induced in the proximal tubule early after experimental AKI. It has been reported in children undergoing cardiopulmonary bypass (CPB), the increase in urinary L-FABP maybe occur within 4 h of initiating CPB [125]. However, there are conflicting data about L-FABP, such as a prospective multi-center study of 311 children undergoing cardiac surgery did not show similar increase in L-FABP [106]. In SCD patients, Sundaram et al. [57] reported that urine L-FABP level was highest in the youngest group (6–12 years old) even with little evidence of renal injury. In fact urine L-FABP levels are reduced with increasing albuminuria.

Preclinical studies have identified the kidney injury molecule-I (KIM-1) gene to be induced in the proximal tubule cells of ischemic rat kidneys [114]. KIM-1 protein regulates phagocytosis of damaged cells and thereby limits injury. An extracellular domain of KIM-1 can be detected by enzyme-linked immunosorbent assays and is useful as a urinary biomarker in patients with AKI. In fact, in a study conducted by Han WK et al., it was shown that in 40 children undergoing CPB, urinary KIM-1 levels were markedly increased in those who have developed AKI [126]. On the other hand, a prospective multi-center study of 311 children undergoing cardiac surgery confirmed the delay in upregulation of urinary KIM-1 in AKI patients and showed that KIM-1 was not significantly associated with AKI after adjusting for other injury biomarkers [127]. This data is in contrast with the results published by Sundaram et al. [57]. Indeed when KIM-1 levels, detected in all SCA urine samples, were compared within the different albuminuria groups, they were detected at lowest levels in patients with normal albuminuria, significantly increased in patients with moderately increased albuminuria and further increased in the severely increased albuminuria group, suggesting this may be another biomarker of relevance in sickle nephropathy that needs to be confirmed in longitudinal studies.

Interleukin-18 (IL-18) represents a proinflammatory cytokine that might worsen the degree of AKI. Animal studies have shown that IL-18 is induced in the proximal tubule and detectable in the urine following ischemic AKI. Numerous pediatric studies have proved that urine IL-18 obtained 6- to 12-h post-CPB moderately predicts AKI [128–131]. Cerqueira et al. [131] performed a cross-sectional study composed of 45 SCA patients. They founded that IL-18 levels were correlated closely with markers of hemolysis, endothelial dysfunction and others cytokines levels. These findings suggest probable influences of IL-18 in the pathophysiology

albuminuria in SCD by promoting endothelial dysfunction.

*4.1.4. Serum liver-type fatty acid-binding protein*

*4.1.5. Kidney injury molecule-I*

*4.1.6. Interleukin-18*

#### *4.1.2. Urine neutrophil gelatinase-associated lipocalin*

As it has been showed in several preclinical gene expression analyses performed in AKI murine and human models, neutrophil gelatinase-associated lipocalin (NGAL) gene has been revealed the to be one of the most upregulated genes in the kidney soon after an ischemic or a nephrotoxic insult [113, 114]. NGAL is filtered across the glomerulus, is reabsorbed in proximal tubules and its urinary concentration increases early during ischemic insults [115, 116]. The NGAL protein is also highly induced in regenerating and recovering kidney tubule cells. NGAL binds iron; chelation of toxic iron is an important mechanism that protects the kidney tubules from worsening injury. Thus, the biological role of NGAL in AKI is one of enhanced tubule cell proliferation and recovery [117]. Measurement of urinary NGAL (uNGAL) has been demonstrated to be an early, non-invasive marker of AKI due to a variety of etiologies, such as cardiac surgery [118], intravenous contrast administration [119], critical care settings [120] and kidney transplantation [121]. NAGL has an enormous dynamic range, responds in a dosedependent fashion to injury, responds within 3 h of injury, and responds to a wide range of injuries, easy to measure due to the recent availability of clinical platforms including a new NGAL dipstick. Thus, uNGAL values may then be used to initiate AKI patient care algorithms earlier than serum creatinine alone. Although multiple investigations have demonstrated that uNGAL is a promising AKI biomarkers, a study by Sundaram et al. [57] has not showed any relationship with albuminuria in patients with SCD. This study has also showed that uNGAL levels were significantly subnormal (<50 ng/mL) in most patients with SCA and the overall uNGAL in most patients were well below levels usually seen in patients with acute or chronic renal injury. The authors have explained the results obtained based on the fact that proximal tubular function is supra-normal in SCA, and it is likely that any filtered NGAL may be reabsorbed much more efficiently, resulting in subnormal urinary NGAL levels in SN.

#### *4.1.3. Soluble FMS-like tyrosine kinase-1*

Soluble FMS-like tyrosine kinase-1 (sFLT-1) is a member of the vascular endothelial growth factor receptor family (VEGFR) and has an antiangiogenic effect. Soluble FLT-1 is increased in SCD due to its over-expression by vascular endothelial cells, vascular smooth muscles, activated blood monocytes and proximal tubular cells of the renal epithelia [122]. A recent study by Youssry et al. [123] investigated the relationship between serum levels of sFLT-1 and other conventional biomarkers of renal damage. The serum level of sFLT-1 in SCD patients was significantly higher than controls and its median level showed no significant difference when comparing patients with SS and Sβ genotypes, hydroxyurea therapy and iron chelation. On the other hand, the authors have found significant positive correlations between serum levels of sFLT-1 and microalbuminuria, LDH and indirect bilirubin. Meanwhile, there were no significant correlations between serum levels of sFLT-1 and creatinine, eGFR, serum ferritin and erythrocyte sedimentation rate (ESR). This association between sFLT-1 levels and microalbuminuria combined with the association of sFLT-1 with soluble vascular cell adhesion molecule (VCAM) in prior studies [124], suggests that sFLT-1 may contribute to the pathogenesis of albuminuria in SCD by promoting endothelial dysfunction.

#### *4.1.4. Serum liver-type fatty acid-binding protein*

Serum liver-type fatty acid-binding protein (L-FABP) is an anti-oxidant, renoprotective molecule induced in the proximal tubule early after experimental AKI. It has been reported in children undergoing cardiopulmonary bypass (CPB), the increase in urinary L-FABP maybe occur within 4 h of initiating CPB [125]. However, there are conflicting data about L-FABP, such as a prospective multi-center study of 311 children undergoing cardiac surgery did not show similar increase in L-FABP [106]. In SCD patients, Sundaram et al. [57] reported that urine L-FABP level was highest in the youngest group (6–12 years old) even with little evidence of renal injury. In fact urine L-FABP levels are reduced with increasing albuminuria.
