Hematuria is a frequent abnormality in various renal diseases. Isolated hematuria without proteinuria has been considered as a benign condition. Proteinuria rather than hematuria is the main target for treatment and monitoring in renal diseases. However, epidemiological evidences have reported that persistent microscopic hematuria is a risk factor for progression to end-stage renal disease (ESRD) [1-3]. In a study including 1.2 million young Israeli adults, Vivante A. et al. reported that persistent asymptomatic isolated microscopic hematuria significantly increased the risk for developing ESRD (HR 18.5; 95% CI:12.4–27.6) after adjustment for age, sex, body mass index, and blood pressure [1]. In addition, in a large adult cohort enrolled 3,272 participants with moderate chronic kidney disease (CKD), hematuria was found to be a significantly higher risk of CKD progression and death in the first 2 years of follow-up [2]. In a single-center cohort of 112 patients with IgA nephropathy followed for a mean of 14 years, Sevillano et al. reported that the proportion of patients reaching ESRD or a 50% reduction of renal function was significantly greater among patients with persistent hematuria than patients with minimal or negative hematuria, and disappearance of hematuria was associated with a lower decline in renal function [3]. Recently, several studies have suggested that hematuria might be a promoter of tubular injury and that tubular epithelial cells are key injury targets involved in hematuria-related renal damage [4-6]. However, little information has been obtained regarding the association between hematuria and tubular injury in children with hematuria. Neutrophil gelatinase-associated lipocalin (NGAL) is a 25 kDa protein of the lipocalin family [7]. It was originally identified in neutrophils, but is is also expressed in kidney in response to various pathologic states, such as inflammation, infection, ishchemia and neoplastic transformation [7,8] In normal condition, only low levels of NGAL are detectable in urine. However, following renal injury, NGAL is upregulated in the distal part of the nephron and this leads to increased urinary NGAL levels [9-11]. Liver-type fatty acid binding protein (L-FABP) is a protein expressed in the proximal tubule of the kidney [12]. In various stressful events causing renal tubulointerstitial injury such as renal ischemia, hyperglycemia and toxins, renal gene expression for FABP1 (fatty acid binding protein, liver) is upregulated and the urinary excretion of L-FABP is increased [13,14]. A number of studies have demonstrated that urinary NGAL and L-FABP might be noninvasive and suitable markers for renal tubular injury, not only for the early detection of acute kidney injury but also for the progression of chronic kidney disease [7,12,15]. Thus, the objective of this study was to examine the association between hematuria and tubular injury by evaluating two tubular injury markers, urinary NGAL and L-FABP, in children with hematuria.

A total 72 children and adolescents aged 2 to 18 years who had persistent hematuria for more than 6 months and followed up at the Department of Pediatrics, the Catholic University of Korea, Bucheon St. Mary’s Hospital between March 2016 and February 2018 were enrolled. Children who had transient hematuria coincided with such as urinary tract infection or fever, and had other chronic diseases such as cancers, diabetes mellitus and congenital anomalies of kidney and urinary tract (CAKUT) including cystic kidney diseases were excluded. After obtaining informed consent from subjects and their parents, medical histories including family history of renal diseases and anthropometric measurements including height and weight were collected from subjects during their visits to the clinic. Laboratory examinations including white blood cell counts, routine biochemical analysis, and urinalysis with microscopy were also performed. Hematuria was defined when ≥5 red blood cells were detected in a high-power field on microscopic analysis. Proteinuria was defined when a protein score ≥+1 (+1–+4) was obtained in urinalysis and random urine protein to creatinine ratio was ≥0.2 (mg/mg). Hypercalciuria was defined when the calcium to creatinine ratio in spot urine was ≥0.2 (mg/mg). Estimated glomerular filtration rate (eGFR) was calculated using a revision of the Schwartz equation: 0.413×height (in cm)/serum creatinine (mg/dl). The diagnosis of glomerulonephritis (GN) was made based on renal biopsy results except for post-streptococcal glomerulonephritis (PSGN). Renal biopsy was performed for children with hematuria if they had at least one of these criterion -proteinuria, decreased renal function, hypertension, decreased serum C3 level except for PSGN or parental anxiety. Total 30 patients were diagnosed with GN and their distributions were as follows- IgA nephropathy (n=19), IgA vasculitis with nephritis (n=5), Thin basement membrane nephropathy (n=2), mesangial proliferative GN (n=2), PSGN (n=1) and non-specific focal GN (n=1). Urine samples for measuring NGAL and L-FABP were obtained from children upon routine urine collection for urinalysis. Only one sample collection was performed for 46 subjects (46 samples) and more than two sample collections were performed for 26 subjects (66 samples). Therefore, 112 samples were collected. Fifty-four subjects (84 samples) had no proteinuria while 18 subjects (28 samples) had proteinuria. Forty-two subjects (54 samples) had not been diagnosed with GN while 30 subjects (58 samples) had been diagnosed with GN (Fig. 1). Of 26 subjects who offered more than two samples, 9 subjects had initial hematuria only and proteinuria during follow-up, 13 subjects had only hematuria, and 4 subjects had hematuria and proteinuria at every visit. Urine samples were centrifuged at 3000×g for 15 minutes at 4℃ within 30 min of collection and stored at -80℃ until final analyses. Urinary levels of NGAL and L-FABP were measured using ELISA kits for Human Lipocalin-2/NGAL Quantikine ELISA kit and Human L-FABP kits from R&D systems (Minneapolis, MN, USA), respectively, following the manufacturer’s instructions. For detection of NGAL, urine was diluted 1:50. However, no dilution was required for the detection of L-FABP. Urinary levels of NGAL and L-FABP were normalized to urinary creatinine concentration (ng/Cr mg). All samples were run in duplicate. These levels were within the range of the standard curve.

Data are presented as mean±standard deviation for normally distributed values and median and interquartile range for nonparametric values. Categorical variables are expressed as frequencies and proportions. Differences between two groups were analyzed using unpaired Student’s t-test (parametric distributions) or Mann–Whitney U test (nonparametric distributions). Associations between urinary NGAL or L-FABP levels and other clinical parameters were analyzed using Spearman’s correlation coefficient r. All statistical analyses were performed using SPSS Statistics for Windows (version 20.0; IBM Corp., Armonk, NY, USA). P values <0.05 were considered significant.

Hematuria may be considered as the first sign of many glomerular diseases. Recently, several studies have suggested that hematuria is the forgotten CKD factor and that tubular injury promoted by hematuria might be associated with the development or progression to CKD [4,5]. The present study evaluated the association of hematuria with tubular injury through two tubular injury markers, uNGAL and uL-FABP.

NGAL is a 25 kDa protein of the lipocalin family [7]. L-FABP is a protein expressed in the proximal tubule of the kidney. It is involved in fatty acid metabolism [12]. In patients with acute kidney injury, increased levels of urinary NGAL and L-FABP can detect renal injury more sensitively than the conventional diagnostic criterion (i.e., increase of serum creatinine and/or decrease of urine output [10,16,17]. Several studies have reported that urinary L-FABP levels may reflect renal injury in early stages of nephropathy in patients with diabetes, even in normoalbuminuric state [14,18]. Smith et al. have shown that urinary NGAL in addition to conventional established cardiovascular and renal risk factors may improve the prediction of disease progression in patients with non-proteinuric stages 3 and 4 CKD [11]. Under the assumption that patients with glomerulonephritis or decreased eGFR or proteinuria in children with hematuria, compared patients without GN nor proteinuria or higher eGFR may have more tubular injury, we compared urinary NGAL and L-FABP levels based on these parameters. However, we did not find any difference. The present study was also limited by the small number of subjects in a single center. It did not contain healthy controls. Furthermore, some data were cross-sectional. However, we found that urinary levels were positively associated with proteinuria and that the development of proteinuria for patients with hematuria caused increases in levels of uNGAL/Cr and L-FABP/Cr. These results may suggest that monitoring uNGAL and L-FABP levels in addition to conventional risk factors such as proteinuria and eGFR may improve the prediction of renal injury in patients with hematuria. To confirm the role of uNGAL and uL-FABP as a marker for tubular injury in children with hematuria, further studies including longitudinal observations in a larger population and investigations of other tubular injury markers such as beta-2-microglobulin or N-acetyl-β-D-glucosaminidase (NAG) are needed.

Hematuria without proteinuria is regarded as a benign condition. Thus, many nephrologists only monitor patients’ serum creatinine and proteinuria without performing renal biopsy. However, recent studies have reported that isolated hematuria is associated with the progression to ESRD [1,5]. Therefore, there is a continuing need for identifying more specific markers of renal injury besides relying on proteinuria or eGFR in patients with hematuria. Toward this goal, results of the present study suggest that monitoring urinary NGAL and L-FABP levels may help identify renal injury as an adjuvant method to standard risk factors in children with hematuria in a non-invasive manner. Further studies are warranted to investigate the role of these markers in the development or progression of renal injury in order to elucidate their values as prognostic and therapeutic targets for children with hematuria.