Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/β-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection

Abstract

Abstract Background We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/β-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. Aim To determine the effects of osmolarity/tonicity changes, Wnt/β-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. Methods Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. β-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). Results Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/β-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, β-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. Conclusions Lcn2-R upregulation and Lcn2 downregulation via Wnt/β-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

Keywords

• Kidney
• hypertonicity
• osmotic stress
• cell death
• proliferation
• lipocalin-2