The Protective Role of Tubular Epithelial Cells Produced Fms-like Tyrosine Kinase Receptor 3 Ligand in Acute Kidney Injury

Introduction: Dendritic cells (DC) accumulate in the kidneys of patients with acute kidney injury (AKI), but the Fms-like tyrosine kinase receptor 3 ligand (Flt3L)-dendritic cell axis is poorly defined in kidneys with AKI. Meanwhile it has not been reported whether Flt3L is produced by kidney cells under ischemic-reperfusion injury (IR) induced microenvironment.

Methods: (1) Blood specimens and urine were collected from two patient cohorts: a) patients with pre-renal AKI and healthy controls for analysis of serum Flt3L. b) patients with acute myocardial infarction (AMI) developed with or without AKI on admission for analysis of serum and urine Flt3L. (2) Wild type mice and T cell-deficient mice ( Tcra^-/- mice) were applied. IR induced AKI mice and hypoxia-reoxygenation stimulated HK-2 cell line (human tubular epithelial cell line) were constructed. The expression of Flt3L in kidney, tubular epithelial cells, and T cells were measured by RT-PCR, ELISA, immunohistochemistry, immunofluorescence staining, RNAscope, and flow cytometry. (3) Wild type mice and type I conventional dendritic cell (cDC1)-deficient mice ( Irf8^-/- mice) were treated with 15 μg rFlt3L or 10mg/kg gilteritinib (Flt3 inhibitor). They were subjected to IR. Serum and kidney tissues were collected. Kidney function, tubular injury, and DC accumulation were quantified.

Results: Initially, among patients with pre-renal AKI, we observed an increased level of serum Flt3L within two days after admission. Another patient cohort revealed that this increase was specific for AMI patients with AKI rather than without AKI or healthy controls. Furthermore, among these AMI patients, serum Flt3L levels correlated with Creatinine increase (R ² = 0.4532, p = 0.0002) and BUN increase (R ² = 0.3417, p = 0.0017) within 48 hours after admission. Similarly, urine Flt3L levels among these AMI patients also steadily elevated with Creatinine increase (R ² = 0.6395, p < 0.0001) and BUN increase (R ² = 0.5115, p = < 0.0001) within 48 hours after admission. Flt3L was also enhanced in wild type mice with IR-AKI but not affected in T cell-deficient mice ( Tcra^-/-) with IR-AKI. Among kidney tissue, Flt3L-positive cells scattered within tubules in outer stripe of the outer medulla (OSOM) and mRNA and protein increased within 24 hours after IR-AKD. In vitro, FLT3L mRNA was markedly induced in HK-2 cells during hypoxia and FLT3L protein remained enhanced during reoxygenation. Phenotypically, the numbers of renal cDC1 and CD64 ⁺DC were significantly decreased in gilteritinib-treated IR-AKI mice, which were associated with more severe tubular injury. With reduced kidney cDC1, Irf8^-/- mice also showed worsen kidney injury and aggravated functional failure upon IR-AKI. Therapeutic administration of rFlt3L significantly increased kidney cDC1 and CD64 ⁺DC upon IR-AKI in wild type but not Irf8^-/- mice. This was associated with significantly reduced tubular injury of wild type mice .In addition, pre-treatment of wild type mice with rFlt3L increased kidney cDC1 and protected the mice from severe IR-AKI.

Conclusion: Flt3L is upregulated in human and mice and produced by tubular epithelial cells during IR-AKI. It fosters the accumulation of kidney cDC1 and CD64 ⁺DC, thereby limiting the severity of kidney injury in mice. The current study implies the possibility of DC-based immunotherapy for treatment of AKD.