Basolateral transport of the fluorescent organic cation tetramethylammonium by insect renal tubules

Organic cations (OCs) include a diverse class of endogenous metabolites and xenobiotics whose excretion is essential for animal homeostasis. The vast majority of insects the Malpighian tubules (MTs), hindgut and midgut serve as the primary epithelial organs responsible for OC excretion. Historically, the use of radiolabeled OC substrates and electrophysiological approaches have provided foundational insights into OC transport. These techniques are limited by their need for technical expertise and their relative low throughput. We used the fluorescent organic cation probe [2-(4-nitro-2,1,3-benzoxadiazol-7-yl) aminoethyl] trimethylammonium) (NBD-TMA) to investigate OC uptake and transport in isolated MTs and midgut of Drosophila melanogaster. Using a fluorescent plate-reader, we quantified accumulation and transport of NBD-TMA in the MTs of Drosophila melanogaster. NBD-TMA uptake was electrogenic, competitively inhibited, carrier mediated, saturable, which is consistent with epithelial transport of OCs. When compared to previous radiotracer and microelectrode-based approaches, this fluorescence-based strategy provides enhanced sensitivity, higher experimental throughput, and minimizes the requirements for specialized training and safety. This study demonstrates that NBD-TMA is a powerful tool for studying OC transport physiology in insect renal epithelia and further highlights the utility of fluorescence-based assays for mechanistic and comparative analyses of epithelial detoxification systems.