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Immunohistochemical Demonstration of Avian Multi‐drug Resistance Protein 4 (Mrp4) in Renal Proximal Tubule Epithelium

Birds are uricotelic and most of the urate is excreted by active tubular secretion into the urine. The mechanism of urate transport across the apical membrane of the intact proximal tubule epithelium during secretion is uncertain. Our previous studies of urate transport have implicated Mrp4 as a dominant pathway for urate secretion across the apical membrane. Short hairpin RNA interference (shRNAi) effectively knocked down expression of Mrp4 mRNA in chick renal proximal tubule epithelial cell primary cultures (PTCs) and reduced transepithelial urate secretion approximately 80%. Here, we report the production of a rabbit custom polyclonal antibody designed against chicken Mrp4 (Genscript). Immunoblots of isolated brush border membrane samples from chicken kidney and allyl alcohol‐stimulated mouse liver samples confirmed the labeling of Mrp4 by the antibody (~ 150 kDa). Antigenicity was not detected in isolated basolateral membrane vesicles of chick kidney. Fluorescence immunohistochemistry and confocal microscopy of intact renal proximal tubules and PTCs showed localization of Mrp4 to the apical membrane of the cells and confirmed the lack of Mrp4 in the basolateral membranes. In PTCs where shRNAi was introduced to knockdown expression of Mrp4 mRNA, immunohistochemical fluorescence microscopy confirmed a substantial reduction of Mrp4 protein in the apical membrane microvilli at seven days post‐transfection. Supported by NSF.