Abstract P2039: Nitric Oxide Inhibition Enhances the Protein Expression of TNF-α Receptor Type 1 in Cultured M-1 Cells With Characteristics of Renal Collecting Duct Epithelium

Abstract

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.