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SALT AUGMENTS TH17 CELL RESPONSES IN ANCA ASSOCIATED VASCULITIS PATIENTS AND IN MODELS OF INFLAMMATORY KIDNEY DISEASE INTRODUCTION: Recent evidence has demonstrated a detrimental effect of salt on innate and adaptive immunity. A raised extracellular concentration of sodium can cause naïve T cells to differentiate into pathogenic Th17 cells, and alter macrophage phenotype. The kidney is the main salt transporting organ and Th17 cells are implicated in both glomerular and interstitial inflammatory disease. The effect of extracellular sodium concentrations on T cell activation states in renal inflammatory disease is unknown. AIMS: To determine the effect of increased extracellular sodium concentration on Th17 cell activation in patients with ANCA associated glomerular disease; to determine if a high-salt diet worsens renal inflammation in an experimental model of glomerulonephritis; and to identify intracellular signalling molecules involved in T cell activation in patients with autoimmune interstitial disease. METHODS: Peripheral blood mononuclear cells (PBMCs) from 10 patients with ANCA associated vasculitis (AAV)(7 PR3- and 3-MPO-ANCA) and 7 healthy controls were isolated and cultured with CD3/CD28 stimulating antibodies and Th17 polarising cytokines (IL1,IL6,IL21,IL23, TGFβ), in the presence or absence of additional salt (20 and 40mmol NaCl). Th17 cell frequencies were analysed by FACS. IL17-YFP tracker mice were subjected to nephrotoxic nephritis (NTN). One group was fed normal chow (n=4) and the other group high salt (6%) chow (n=5). The percentage of YFP positive cells infiltrating the kidney was analysed using FACS. The inflammatory infiltrate in renal biopsy tissue from patients with Primary Sjögren’s Syndrome associated tubulointerstitial nephritis (pSS TIN) was isolated via laser microdissection. Fragments were digested into peptides and analysed by liquid chromatography electrospray tandem mass spectroscopy. Proteins were compared to patients with drug induced TIN. RESULTS: The mean percentage of CD4 +IL17-expressing cells in patients with AAV under standard conditions was 27.6 +/- 10.7%, as compared to 36.1+/- 12.7% with 20mmol NaCl, and 46.6 +/- 15.0% with 40mmol NaCl (p<0.01); no differences found between MPO- and PR3-ANCA patients. In healthy controls there was a more variable effect of salt, with a general upward trend, but overall no significant difference in CD4+IL17 frequencies after addition of salt (figure A Patients; B Controls). The median number of CD3+YFP positive cells infiltrating the kidney in normal salt fed animals was 0.37% (0.08-0.75) compared with 0.80% (0.6-1.1) in those on high salt diet (p=0.06) (figure C). Many of these cells also expressed IFN-g in both normal and high salt groups, suggesting a switch of cytokine phenotype. Impact of salt on other disease biomarkers including renal function is currently being analysed. Candidate proteins present in pSS but not drug induced TIN infiltrate were serine/threonine kinases or phosphatases; WNK kinase-1, SPAK, 14-3-3 protein and protein phosphatase 1. C B 1.5 1.0 0.5 Sa h Hi g ls ma N or lt 0.0 al t % CD3+YFP cells in kidney A CONCLUSION: CD4+Th17 cell responses are significantly augmented by additional salt in patients with ANCA associated glomerular disease, while a high-salt diet leads to greater Th17 cell renal infiltration in experimental glomerulonephritis. This salt effect may be mediated by Serine/threonine kinases and phosphatases. !!