Lipocalin 2 Links Aquaporin 2⁺ Progenitor Cell-Derived Distal Renal Segments to Kidney Fibrosis

Loss of Dot1l in Aquaporin 2⁺ progenitor cell (AP)-derived distal renal segments in Dot1l^ffAqp2cre (Dot1l^AC) promotes kidney fibrosis by upregulating endothelin 1 (ET1) through histone deacetylase 2 (HDAC2), defining a Dot1l-HDAC2-ET1 profibrotic pathway. Additionally, Lcn2 was also upregulated in Dot1l^AC mice. Here, we investigated whether and how Lipocalin 2 (Lcn2) contributes to this pathway. We performed in vivo studies using multiple mouse models with targeted deletion of Dot1l, Lcn2, or Edn1 in distal nephron segments or globally. Human CKD RNA-seq data analysis and in vitro studies in IMCD3 cells were conducted to validate the in vivo results. Because Lcn2^-/- mice are available and Lcn2 is barely detectable in IMCD3 cells, Lcn2 down- and upregulation experiments were performed in mice and IMCD3 cells, respectively. Several approaches, including immunofluorescence staining and in situ hybridization, were employed to identify and confirm the Lcn2 profibrotic role. Both Dot1l deletion in AP in Dot1l^AC mice and Dot1l silencing in IMCD3 cells upregulated Lcn2. Global deletion of Lcn2 in Dot1l^AC mice attenuated HDAC2 and ET1 levels and reduced kidney fibrosis, establishing Lcn2 as a downstream effector of Dot1l-deletion-induced kidney fibrosis. Although conditional knockout of Edn1 in Dot1l^AC mice showed Lcn2 expression comparable to that in Dot1l^AC mice, they exhibited reduced kidney fibrosis, suggesting that Lcn2 lies upstream of ET1-mediated injury. Reanalysis of a human chronic kidney disease (CKD) dataset revealed that downregulation of DOT1L was coupled with upregulation of LCN2, HDAC2, and ET1, recapitulating our findings in Dot1l^AC mice. These results were further validated in IMCD3 cells, in which Lcn2 was upregulated by Dot1l silencing, and addition of 293 T cells-secreted or recombinant Lcn2 led to increased HDAC2 and ET1 expression. Loss of Dot1l in AP upregulates Lcn2, which promotes kidney fibrosis by increasing HDAC2 and ET1. Our findings add Lcn2 as a new component to define a more complicated Dot1l-Lcn2-HDAC2-ET1 pro-fibrotic pathway that links AP-derived distal renal segments to kidney fibrosis.