FP080
THE NONCANONICAL NOTCH LIGAND DLK1 REGULATES RENAL INFLAMMATION Free

INTRODUCTION AND AIMS: The main goal of this work is to study the role of the notch signaling pathway non-canonical ligand DLK1 (epidermal growth factor-like protein Delta-like 1) in experimental renal damage. Notch signaling pathway is highly activated during embryonic development, but it is inhibited in adult tissues. It has been demonstrated that this pathway is overactivated in human renal pathologies. DLK1 is suggested to be a Notch pathway endogenous inhibitor in vitro; however, there are no studies about its role in vivo.

METHODS: Progressive renal damage model of unilateral ureteral obstruction was done in wild-type and dlk1-null mice of SvJ-129 genetic background. These animals were sacrificed after 2, 5, 10 and 14 days of obstruction. Left kidney was obstructed, whilst right kidney was used as control (contralateral).

RESULTS: Non-canonical ligands DLK1 and DLK2 (DLK1 homolog) were analysed. From 5 days of obstruction, both genes increased their expression in WT vs contralateral (dlk1) and in obstructed kidneys of dlk1-null mice vs WT (dlk2). Obstructed kidneys of dlk1-null mice presented an increase in NICD (Notch intracellular domain), fragment of Notch receptor which is translocated to the nucleus and activates the effector genes, hes and hey. Moreover, a significant increase of hes-1 gene expression levels was observed in obstructed kidneys of dlk1-null mice when compared to obstructed WT kidneys at 14 days, and of hey-1 gene expression levels at 5 days. The evaluation of renal damage through PAS tinction revealed a significant increase in inflammatory infiltrate as focal aggregates in obstructed kidneys of transgenic mice at 14 days. Therefore, it was decided to characterised this infiltrate through immunohistochemistry at that time. These aggregates were associated to a significative increase of CD3+, CD4+, F4/80+ infiltrating cells and neutrophils, as well as Th17 lymphocytes. Studying the possible inflammatory mechanism, it was observed that p-IκBα was increased in damaged kidneys of dlk1-null mice when compared to their littermates at 14 days. In addition, a significant increase in ccl-2 gene expression was observed in obstructed kidneys of transgenic mice from 10 days, following 14 days. It is important to remark the increase of the Th17 response in these damaged kidneys of dlk1-null mice when compared to the WT ones, as we demonstrated here by an augment of IL17A renal production and the transcription factors that are implicated in this immune response: RORγt and STAT3.

CONCLUSIONS: The deletion of the non-canonical ligand DLK1 from Notch pathway involves the overactivation of this pathway in a renal experimental damage. This confirms that DLK1 acts as an endogenous antagonist on Notch receptor in pathological processes in the kidney. Notch activation in dlk1 absence is associated in an increase of renal inflammatory infiltrate and in an activation of Th17 immune response, demonstrating the importance of Notch pathway in renal inflammatory processes.