Browsing by Author "Rocco, Jocelyn"
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Item Emerging new role of NFAT5 in inducible nitric oxide synthase in responseto hypoxia in mouse embryonic fibroblast cells(2019) Serman, Yair; Fuentealba, Rodrigo A; Pasten, Consuelo; Rocco, Jocelyn; Ko, Ben; Carrión, Flavio; Irarrázabal, CarlosWe previously described the protective role of the nuclear factor of activated T cells 5 (NFAT5) during hypoxia. Alternatively, inducible nitric oxide synthase (iNOS) is also induced by hypoxia. Some evidence indicates that NFAT5 is essential for the expression of iNOS in Toll-like receptor-stimulated macrophages and that iNOS inhibition increases NFAT5 expression in renal ischemia-reperfusion. Here we studied potential NFAT5 target genes stimulated by hypoxia in mouse embryonic fibroblast (MEF) cells. We used three types of MEF cells associated with NFAT5 gene: NFAT5 wild type (MEF-NFAT5+/+), NFAT5 knockout (MEF-NFAT5-/-), and NFAT5 dominant-negative (MEF-NFAT5Δ/Δ) cells. MEF cells were exposed to 21% or 1% O2 in a time course curve of 48 h. We found that, in MEF-NFAT5+/+ cells exposed to 1% O2, NFAT5 was upregulated and translocated into the nuclei, and its transactivation domain activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5-/- or MEF-NFAT5Δ/Δ cells, the basal levels of iNOS and AQP-1 expression were strongly downregulated, but not for UTA-1. The upregulation of AQP-1, UTA-1, and iNOS by hypoxia was blocked in both NFAT5-mutated cells. The iNOS induction by hypoxia was recovered in MEF-NFAT5-/- MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5Δ/Δ cells, confirming the dominant-negative effect of MEF-NFAT5Δ/Δ cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information about the signaling pathway of NFAT5 during responses to oxygen depletion in mammalian cells and suggests that the expression of iNOS induced by hypoxia is dependent on NFAT5.Item Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus(2021) Ugarte, Francisca; Santapau, Daniela; Gallardo, Vivian; Garfias, Carolina; Yizmeyián, Anahí; Villanueva, Soledad; Sepúlveda, Carolina; Rocco, Jocelyn; Pasten, Consuelo; Urquidi, Cinthya; Cavada, Gabriel; San Martin, Pamela; Cano, Francisco; Irarrázabal, Carlos E.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-1a (HIF-1a), 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-1a 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.