Abstract
The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50–100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400–500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.
Highlights
The chief site of urine concentration is in the collecting duct (CD) system [1]
The CD can be separated into the cortical CD (CCD), outer medullary CD (OMCD) and inner medullary CD (IMCD)
We have previously shown that increased extracellular osmolarity augments abundance of the LCN2 receptor SLC22A17 in a mouse IMCD cell line [20]
Summary
The chief site of urine concentration is in the collecting duct (CD) system [1]. There, the major effector in the regulation of renal water excretion is the antidiuretic hormone arginine vasopressin (AVP), which binds to the AVP type-2 receptor (V2R) and signals through cAMP [2]. This, in turn, triggers the trafficking of intracellular storage vesicles expressing AQP2 to the luminal plasma membrane, within a range of 10–30 min (short-term regulation), and increases Aqp gene transcription, via the increased activity of cAMP-responsive element binding protein (CREB) and AP-1 [4,5,6], over a time period ranging from hours to days (long-term regulation). Na+ reabsorption in the thick ascending limb results in a renal cortico–papillary osmotic gradient. This gradient exposes renal cells to substantial osmotic stress by causing numerous perturbations (reviewed in [9]). Cells can respond to high osmotic stress by activating adaptive mechanisms through various pathways that activate the transcription factor NFAT5 ( known as tonicity-named responsive enhancer binding protein (TonEBP or OREBP)), culminating in the accumulation of organic osmolytes and increased expression of heat shock proteins (reviewed in [9]). Activation of the NF-κB transcriptional factor by pro-inflammatory signals reduces Aqp gene transcription (reviewed in [3,10])
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