Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus

Abstract

Background: Tubular damage has a role in Diabetic Kidney Disease (DKD). We evaluated the early tubulointerstitial damage biomarkers in type-1 Diabetes Mellitus (T1DM) pediatric participants and studied the correlation with classical DKD parameters.

Methods: Thirty-four T1DM and fifteen healthy participants were enrolled. Clinical and biochemical parameters [Glomerular filtration Rate (GFR), microalbuminuria (MAU), albumin/creatinine ratio (ACR), and glycated hemoglobin A1c (HbA1c)] were evaluated. Neutrophil gelatinase-associated lipocalin (NGAL), Hypoxia-inducible Factor-1α (HIF-1α), and Nuclear Factor of Activated T-cells-5 (NFAT5) levels were studied in the supernatant (S) and the exosome-like extracellular vesicles (E) fraction from urine samples.

Results: In the T1DM, 12% had MAU >20 mg/L, 6% ACR >30 mg/g, and 88% had eGFR >140 ml/min/1.72 m². NGAL in the S (NGAL-S) or E (NGAL-E) fraction was not detectable in the control. The NGAL-E was more frequent (p = 0.040) and higher (p = 0.002) than NGAL-S in T1DM. The T1DM participants with positive NGAL had higher age (p = 0.03), T1DM evolution (p = 0.03), and serum creatinine (p = 0.003) than negative NGAL. The NGAL-E correlated positively with tanner stage (p = 0.0036), the median levels of HbA1c before enrollment (p = 0.045) and was independent of ACR, MAU, and HbA1c at the enrollment. NFAT5 and HIF-1α levels were not detectable in T1DM or control.

Conclusion: Urinary exosome-like extracellular vesicles could be a new source of early detection of tubular injury biomarkers of DKD in T1DM patients.

Keywords: NGAL; children; diabetic kidney disease; type 1 diabetes mellitus; urinary extracellular vesicles.

Figure 1

Extracellular vesicles are present in the urine samples of control and T1DM patients. (A) Representative figure of electron micrograph of isolated exosomes-like extracellular vesicles (scale bar, 100 nm). (B) Flot-1 levels in 100 mg of total protein of extracellular vesicle pellet. The upper panel is a representative picture of the Western blot of flotillin-1 (extracellular vesicle component). The graphic represents the median ± SEM of the levels of Flot-1 in the supernatant (S) and enriched extracellular fractions (E) generated by ultracentrifugation.

Figure 2

The NGAL level in urinary exosome enriched fraction is higher in T1DM than in control participants. Urine samples were processed to purify the extracellular vesicles by ultracentrifugation. The NGAL levels were studied by Western blot in the supernatant and the exosomes fraction (100 ug of total proteins). Upper panel representative picture of the two bands of NGAL (25 and 50 KDa). The lower panel is a graph with the NGAL signal. Arbitrary Unit (Median ± SEM; *p < 0.05).

Figure 3

Correlation analysis between NGAL-E and kidney parameters. (A) NGAL vs ACR; (B) NGAL vs MAU; (C) NGAL vs HbA1c one year before enrollment, and (D) NGAL vs Tanner Stage. The correlation between variables was studied by Spearman test.