Abstract P241: Salt Directly Alters Molecular Trafficking Of Uromodulin In Renal Thick Ascending Limb Cells

Abstract

Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending epithelial (TAL) cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. We have previously demonstrated that salt induces a reduction in urinary UMOD excretion in normotensive Wistar-Kyoto (WKY) rats after 3 weeks, which was exacerbated in a chronic hypertensive rat model with increased accumulation of UMOD in the endoplasmic reticulum (ER). We further showed that these effects occur at the protein trafficking level. Our objective was to understand whether UMOD trafficking is influenced by acute salt exposure. Further, we aimed to dissect the molecular pathways through an untargeted proteomics approach. To study acute effects, WKYs were subjected to 1% NaCl on an intermittent basis over 3 weeks in an on-off-on pattern (control n=4, salt n=6). In an ex vivo experiment, TAL tubules isolated from WKY were incubated with salt (+300mOsm) (control/salt n=3). Protein extractions were labelled with isobaric tandem mass tags and analysed by mass spectrometry. Acute salt-loading significantly lowered UMOD excretion after one week (9-fold, p<0.05), which returned to control levels when halted, and decreased again when resumed a week later (4-fold, p<0.05). Together, this confirmed that salt directly influences UMOD trafficking even after a brief exposure. Proteomic analyses of ex vivo tubule incubated with salt revealed 45 differentially expressed proteins (p<0.05), of which 15 were upregulated (>1.2 fold) in the salt group. Gene ontology analysis results showed that these differentially expressed proteins were enriched in the mitochondria/electron transport chain (NDUFS6, FDX1, NDUFV2, TXNRD2), oxidation-reduction processes (PP1R1A, SUOX, CYB5R1), and protein transport (EXOC6, MACF1, GLG1, SORT1, BNIP1). In summary we demonstrate that UMOD excretion responds to changes in exposure to salt in vivo and in vitro and that altered UMOD trafficking may be due to changes in mitochondrial function and oxidative stress. The exact role of the identified proteins in UMOD trafficking warrants further study.