Abstract
BackgroundWe 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.AimTo 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.MethodsNormosmolarity/-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).ResultsHyperosmotic/-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.ConclusionsLcn2-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.
Highlights
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
Statistical analysis shows means ± SEM of 4 experiments and compares the two osmotic conditions by unpaired t-test. a.u. = arbitrary units. c Immunoblotting of plasma membranes (PM) of mIMCD3 cells grown for 72 h in norm- or hyperosmotic media
We hypothesized that Lcn2-R expression is linked to hyperosmolarity. Quantitative PCR (qPCR) of mIMCD3 cells exposed to 600 mosmol/l showed increased Lcn2-R mRNA expression at 24 and 48 h exposure, compared to normosmotic conditions (Fig. 1a)
Summary
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 cells of the inner medullary collecting duct (IMCD) are key elements of the nephron in the process of urinary concentration and dilution [1] For these processes to be operative, interstitial hyperosmolarity/-tonicity needs to be established by accumulation of high interstitial levels of NaCl and urea [2, 3]. Cells can counteract high osmolality stress by initiating survival mechanisms that activate the transcription factor TonEBP/OREBP/NFAT5 (reviewed in [4]). These survival mechanisms include accumulation of organic osmolytes and increased expression of heat shock proteins through numerous pathways, resulting in osmotolerance. The unique hypertonic environment in the renal medulla induces a nephron segment-specific gene expression pattern [5]
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