Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na+/H+ exchangers that can be distinguished by their differing sens itivities to the amiloride analog N-ethyl-N-isopropylamiloride (EIPA). It h as been shown previously that the basolateral exchanger is encoded by NHE1. In the present study, a combination of reverse transcription-PCR, 5' RACE, and genomic library screening was used to clone the coding region of the p orcine NHE3 gene. There was significant homology between the LLC-PK1 sequen ce and the previously reported rabbit and rat NHE3 genes, with nucleotide a nd deduced amino acid identities of 87 and 85% in rabbit, and 85 and 87% in rat, respectively. To study expression patterns, Northern analysis was car ried out using an NHE3 cDNA to probe poly(A)(+) RNA isolated from LLC-PK1 c ells, and from pig kidney cortex. In all three cases, a major transcript of 6.1 kb was detected along with two minor transcripts of 4.7 and 3.8 kb. II I situ hybridization with two different NHE3 probes gave intense labeling o f the distal convoluted tubule in pig kidney but (unexpectedly) no detectab le labeling of the proximal tubule. These studies suggest that there are ma rked species differences in NHE3 expression in the distal nephron.