*Purpose: Neutrophil gelatinase-associated lipocalin (NGAL) is a potential biomarker for early detection of acute kidney injury (AKI) in human, but the mechanistic role for NGAL in AKI has not been well studied.

*Methods: CD4 cells were isolated from kidneys and spleens of healthy C57BL6 wild-type male mice and after renal ischemia-reperfusion injury (IRI). RNA-sequencing was performed using Illumina NextSeq 500. Trranscriptional status of lipocalin (Lcn2) gene was measured using Quantstudio 12K flex real-time PCR (Life Technologies). The amount of NGAL protein was measured using enzyme-linked immunosorbent assay (ELISA) kit (BioLegend). Data are presented as the mean±SEM, and Statistical significance of difference was defined as a P value ≤0.05.

*Results: While conducting discovery studies in CD4 T cells using next generation RNA sequencing in a mouse model of ischemic AKI, we found that the Lcn2 gene, which encodes NGAL, had a ~60 fold increase following IRI in renal CD4 T lymphocytes among 52,633 genes screened. Quantitative real-time (qRT)-PCR confirmed the Lcn2 upregulation in renal CD4 T lymphocytes during AKI. This was verified at the protein level using enzyme-linked immunosorbent assay (ELISA). Increased NGAL was found in renal CD4 T lymphocytes after AKI (244±24 ng/mg vs. 8±5 ng/mg in normal, p < 0.001) and similar results were observed in spleen CD4 T cells (261±44 ng/mg after IRI vs. 42±19 ng/mg in normal, p < 0.01). To evaluate the pathophysiologic role of CD4 T cell NGAL in AKI, we transferred ~10⁷ splenic CD4 cells isolated from NGAL KO mice or WT B6 mice into CD4 KO mice or WT B6 mice, and induced ischemic AKI 72hrs after the adoptive transfer. PBS injected CD4 KO mice or WT B6 mice also underwent surgery as additional controls. After 24 hours of reperfusion, the NGAL KO CD4 T cell-transferred group had significantly higher serum creatinine than WT CD4 T cells-transferred group or PBS-injected group in WT recipients (NGAL KO CD4 T cell-transferred group, 1.47±0.17 mg/dL; WT CD4 T cell-transferred group, 0.93±0.17 mg/dL; PBS-injected group, 0.73±0.17 mg/dL; p = 0.02) (n=7-11 per each group) as well as in CD4 KO recipients (NGAL KO CD4 T cell-transferred group, 1.80±0.26 mg/dL; WT CD4 T cell-transferred group, 1.06±0.21 mg/dL; PBS-injected group, 0.97±0.25 mg/dL; p = 0.04) (n=9-10 per each group). We then performed in vitro simulated hypoxia experiment using mineral oil covering cells, and 7.5 minutes of hypoxia of renal CD4 T cells from WT mice induced an increase of Lcn2 gene expression by 1.9 fold which was accompanied by an increase of Il-10 gene expression by 1.8 fold.

*Conclusions: We conclude that NGAL, previously thought to be a biomarker for AKI and derived from renal epithelial cells, is highly expressed post-ischemic renal CD4 T lymphocytes. This T cell derived NGAL directly mediates the outcome of experimental AKI. Further studies to investigate the underlying mechanisms and human relevance are needed.

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