1. Primary Vesicoureteral Reflux in Children
Vesicoureteral reflux (VUR) is the most common congenital anomaly of the kidney and urinary tract and is a major risk factor for pyelonephritic scars and chronic kidney disease (CKD) in children. The global incidence of VUR is estimated to be 10% in the general population. Since the 2000s, Feather has published the results of the first genome-wide search for VUR and RN using the GENEHUNTER program. VUR maps to a locus on chromosome 1p13 and 2q37 under autosomal dominant inheritance
[1]. VUR has been described with a prevalence of 27–51% in siblings of patients with VUR, and a 66% rate of VUR has been observed in children whose parents had reflux
[2]. However, it is known that VUR can resolve spontaneously in the first 3 years of life; therefore, the exact prevalence in family members could be underestimated.
2. Neutrophil Gelatinase-Associated Lipocalin
Neutrophil gelatinase-associated lipocalin (NGAL) is a 25 kDa protein belonging to the lipocalin superfamily. Initially found in activated neutrophils, it can be produced in kidney tubular cells in response to various injuries. Urine NGAL (uNGAL) has been proposed to be an early predictor of acute kidney injury (AKI). Both pNGAL and uNGAL have been studied in relation to kidney injury. Nickolas et al. reported the first study of urinary NGAL in adults admitted to emergency departments and demonstrated that uNGAL has a good predictive capability for intrinsic AKI. This research also suggested that NGAL could be useful as a predictive marker for adverse clinical outcomes such as the need for dialysis and admission to the ICU
[3]. Shapiro et al. determined that a level of plasma NGAL above 150 ng/mL at the moment of presentation is predictive of AKI occurrence within 3 days of hospitalization. There is a distinct difference between uNGAL, which is specific to injured epithelial cells of the distal nephron, and plasma NGAL, which results from tubular reabsorption from the injured kidney as well as from organs that ultimately crosstalk with the kidney
[4]. NGAL was tested in multiple renal diseases, starting with AKI, and multiple causes of chronic kidney disease, such as nephrotic syndrome, type 1 diabetes, and urinary tract malformations, including VUR. After the first study by Mishra
[5], Parikh
[6], Krawczeski
[7], and Bennett
[8] also confirmed positive predictive values of uNGAL or pNGAL for AKI after cardiac surgery in pediatric or neonatal contexts. Urinary tract infections are one of the trademarks of subjacent malformations. In this sense, the role of NGAL in pediatric UTIs had to be determined. As a diagnostic tool, Shaikh et al. found that uNGAL was more sensitive and specific for a positive UTI diagnosis than leukocyte esterase
[9]. Moreover, Jagadesan found that uNGAL was more specific than pyuria
[10], while Kim Byunh Kwan
[11] and Ji Hym Moon
[12] found it to be more specific than C-reactive protein for the diagnosis of UTIs. Some studies suggested that NGAL could be used to differentiate between acute pyelonephritis and lower UTI, with plasma NGAL having better results
[13][14][15]. Other causes of CKD include nephrotic syndrome and type 1 diabetes. It has been observed that uNGAL may serve as a differentiation marker between minimal-change nephrotic syndrome and focal and segmental glomerulosclerosis
[16]. Renal survival has been directly associated with proteinuria control in long-term cohort studies in patients with GSFS, so a marker to predict the damage to renal function in glomerular nephropathy can be very useful. uNGAL may also be useful as an early diagnostic marker of diabetic nephropathy in type 1 diabetes patients that is superior to microalbuminuria
[17]. By contrast, some malformations are not prone to UTIs or recurrent UTIs. They do not present with the edema of nephrotic syndrome or symptoms related to diabetes. They may not present as modifications on an ultrasound
[18]. VUR represents a true diagnostic challenge.
3. The Diagnostic Value of Urine Neutrophil Gelatinase-Associated Lipocalin in Children with Primary Vesicoureteral Reflux
Depending on the severity of a kidney injury, increased NGAL production and decreased NGAL reabsorption may occur in patients with renal tubular damage. uNGAL has already proven its validity as a biomarker for the diagnosis of children with acute UTI and for steroid responses in idiopathic nephrotic syndrome or in children with different urologic conditions
[19][20]. Nickavar et al.
[21] recognized the diagnostic role of uNGAL in patients with primary VUR and demonstrated in a prospective case control study of 69 small children (2–3 years old) the accuracy of uNGAL/uCr for VUR diagnosis, demonstrating high specificity, sensitivity, and accuracy. Therefore, imaging exploration (e.g., scintigraphy) might not be compulsory for VUR management in children with low uNGAL. However, there was no correlation between uNGAL and the severity of VUR. In a recent study, Amiri et al.
[22] also found higher levels of uNGAL in VUR patients vs. control and higher but not significant levels of uNGAL in the severe compared to mild/moderate forms of VUR as well as in bilateral vs. unilateral involvement. However, when adjusting for uCr, significant differences in the uNGAL/uCr ratio between study groups (patients and controls) and between the patient subgroups, according to the severity of the disease, have been found. No significant differences were found for uNGAL and the uNGAL/uCr ratios between the patients with and without renal scars. This means that uNGAL could not predict RN and could only predict the severity of VUR. In a similar sample (71 children aged 1–5 years), Eskandarifar et al.
[23] confirmed significant differences between the uNGAL levels and the uNGAL/uCr ratios in the VUR group compared with a healthy group. Furthermore, contrary to Nickavar
[21] and Amiri
[22], they detected a significant correlation between uNGAL and the VUR grade. Thus, these authors, referring specifically to the diagnostic role of NGAL in VUR (with no attempt to describe the predictive value for renal scars), reinforced that VCUGs should not be routinely performed in small children after a first febrile UTI, as long as the NGAL/Cr ratio can help, at least in the preliminary stages of VUR management. As there are disagreements regarding the relation to the severity of VUR, the role of uNGAL in predicting the severity of reflux is not well established.
4. Neutrophil Gelatinase-Associated Lipocalin as a Predictive Tool for Renal Scarring and Reflux Nephropathy
Four original studies aimed specifically to assess the predictive value of uNGAL for the development of RN after they had already demonstrated the significant relationship between increased uNGAL and VUR
[24][25][26][27]. Apart from this, the correlation with the severity of VUR as well as a comparison with other biomarkers such as kidney injury molecule-1 (KIM 1) and liver-type fatty-acid-binding protein (L-FABP) was also sought. The Parmaksız et al. study
[28] had the most complex study design. They studied uNGAL in a sample of 123 children with primary VUR. They split the sample into four groups: group A—VUR + renal scars, group B—VUR without renal scars, group C—renal scars and resolved VUR, group D—resolved VUR + no renal scars, and group E—healthy children. The uNGAL/uCr ratio was significantly higher in the groups with scarring than in those without it, demonstrating that uNGAL corelates with scarring and not with VUR (contrary to Amiri
[22]). Moreover, the uNGAL levels in patients with renal scars and VUR (group A) were significantly higher than in the group with VUR without renal scars (group B), the group with fully resolved VUR without renal scars (group D), and the control group, demonstrating that renal scars were corelated with increased uNGAL levels. A particularity of this research was that it also demonstrated a correlation with renal scars: the uNGAL/uCr ratio significantly increased with the severity of renal scars. Similar outcomes were found in Ichino’ study
[20] aiming to assess the predictive value of uNGAL for renal scar development in children with primary VUR. Their results demonstrated a significant relationship between the presence of renal scars and high uNGAL values. However, no significant relationship between uNGAL and the severity of VUR has been proven. Thus, contrary to the results reported by Amiri
[22] and Eskandarifar
[23], in the studies by Ichino
[20], Nickawar
[21], and Parmaksız
[25], the uNGAL levels did not appear to reflect the severity of VUR. Parmaksız’s results
[28] were consistent with Naik’s conclusions: the uNGAL values were higher in children with renal scars than in those without renal scars
[26]. Unlike uNGAL, uKIM-1 and uKIM-1/Cr were not able to predict renal scar formation. Anand et al.
[24] underlined the importance of NGAL in predicting the functional deterioration associated with VUR. They studied uNGAL (among three other biomarkers: trefoil family factors (TFF 1 and 3) and microalbuminuria), in a sample of 50 children with congenital anomalies of the kidney and urinary tract (of which 18 had primary VUR). Their main outcome was a progressive decline in renal function (a decrease in GFR from ≥60 to <60 mL/min/1.73 m
2 and/or new-onset kidney scars or the growth of previous scars on DMSA scans. uNGAL proved to be an accurate biomarker for the progression of chronic kidney disease: the median concentrations of NGAL were significantly higher in children with the progressive deterioration of kidney function. In the newer study by Eskandarifar, the average urinary NGAL level was found to be 524.05 ± 166.65 ng/dL in the group of patients with renal scars, while in the group without renal scars, it was 125.77 ± 61.06 ng/dL (
p < 0.05). The authors showed that urinary NGAL levels had a positive predictive value of 100% and a negative predictive value of 81.25% for predicting the presence of renal scars
[27]. In the study by Eskandarifar, the patients with renal scars had significantly higher levels of NGAL in their urine compared to those without renal scars, similar to the study by Parmaksız
[23][25]. In a mouse model, Han et al. reported that there was a significant increase in NGAL levels two weeks after pyelonephritis that did not definitively return to basal levels after 4–6 weeks. The initial increase in NGAL levels was the result of an acute inflammatory response, but the persistent levels after six weeks indicated that the origin of the high NGAL levels was related to tubular dysfunction
[18].
5. Neutrophil Gelatinase-Associated Lipocalin —Predictive Tool for Chronic Kidney Disease Progression in Children
Chronic kidney disease (CKD) involves irreversible structural and/or functional damage to the kidneys, with evolutionary potential and an evolution longer than 3 months. It is usually accompanied by albuminuria and histopathological and imaging changes characteristic of its etiological type
[29]. A drop in the glomerular filtration rate below 60 mL/min/1.73 m
2 is considered to be a sign of advanced CKD
[30]. Due to the ever-increasing number of patients with CKD, the disease is called “the epidemic of the 21st century”. The incidence of CKD in children under age 16 is 1.5–3 per 1,000,000
[31]. The early stages of CKD are asymptomatic most of the time, so the diagnosis can be delayed in many cases. It is known, however, that the early diagnosis of CKD allows the implementation of an effective strategy to slow down the progression of the disease
[32]. Currently, the traditional markers used for the diagnosis of CKD, i.e., creatinine, urea, GFR, albuminuria, and proteinuria, do not have a high sensitivity
[33]. GFR reflects the total number of functional nephrons. Although measuring GFR using inulin clearance is still the gold standard, the method is far too laborious and is not used in clinical practice. The Schwartz formula, which was developed in 1976, uses serum creatinine (mg/dL, measured using the Jaffe test), height (cm), and the coefficient k, which is proportional to muscle mass and depends on age and sex. However, the error of GFR calculated from serum creatinine has been shown to be approximately ±20% and ±30–40% in children
[34]. In children under 2 years of age, GFR can also be underestimated due to the immaturity of the urinary tract. The measurement of endogenous creatinine also has its limitations. The creatinine level depends, among others, on muscle mass and diet, which can generate significant variation in children, especially in children under 3 years old. Although albuminuria and proteinuria are important parameters for the assessment of the CKD status in adults, they are not always applicable in pediatric practice. In accordance with the 2012 KDIGO guideline
[35], the range of values for albuminuria in children is the same as in adults, but these criteria are not useful in children under 2 years of age due to renal immaturity and protein reabsorption in the proximal convoluted tubule compared to adults
[36]. In addition, CKD in children is often due to congenital abnormalities associated with the tubular loss of albumin; therefore, albuminuria is a less sensitive renal marker in this category of patients
[37]. Due to the mentioned limitations, there is a continuous search for new, early, sensitive, and specific markers of kidney damage, whose introduction into daily clinical practice would lead to the early establishment of the diagnosis of CKD. New markers with particular potential in the diagnosis of CKD include uromodulin, KIM-1, fibroblast growth factor 23 (FGF23), urinary N-acetyl-β-D-glucosaminidase (NAG), NGAL, glomerular biomarker soluble urokinase-type plasminogen activator receptor (suPAR), and urinary retinol-binding protein 4 (RBP4). Serum creatinine, blood urea nitrogen, creatinine clearance, urinalysis, and radiological findings have traditionally been the markers used to indicate renal damage in VUR. However, these markers lack the sensitivity or specificity to accurately diagnose kidney damage and scarring. On the other hand, a high level of urinary NGAL has been associated with kidney damage and an increased risk of kidney scarring, making it a potentially useful diagnostic marker
[27]. Bolignano et al. evaluated the utility of NGAL as an independent marker of CKD progression. The serum and urinary levels of NGAL were inversely proportional to the decrease in GFR
[36]. Because NGAL measurements were more reliable in patients with stage-1–3 CKD than in patients in stages 4 or 5, NGAL was considered to be a useful marker for the diagnosis of early CKD. In the CRISIS study, Alderson et al. also found NGAL to be a marker of CKD
[38]. In addition, there appears to be a correlation between NGAL and serum creatinine levels. Mori et al.
[39] postulated in the “Forest Fire Theory” that increasing the concentration of NGAL is not only a consequence of decreased renal clearance in CKD patients but also the result of increased production of this protein in damaged tubular epithelial cells. According to this theory, the NGAL concentration is assumed to reflect the activity of ongoing processes of kidney damage during CKD. While the increase in the serum creatinine concentration is the result of the loss of a number of functional nephrons, the increase in NGAL reflects the rate of progression of CKD
[40].