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Molecular Cloning of NHE3 from LLC-PK₁ Cells and Localization in Pig Kidney

CHRISTINE A. SHUGRUE^*, NICHOLAS OBERMÜLLER^||, SEBASTIAN BACHMANN^§, CAROLYN W. SLAYMAN^, and ROBERT F. REILLY^¶,#

^* Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
^§ Department of Anatomy, Charité, Humboldt University, Berlin, Germany
^|| Medical Research Center, Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany
^¶ The University of Colorado Health Sciences Center, Colorado
^# Department of Internal Medicine, Denver Veterans Administration, Denver, Colorado

Correspondence to Dr. Robert F. Reilly, Section of Nephrology, 111C, Department of Internal Medicine, Denver Veterans Administration, 1055 Clermont Street, Denver, CO 80220. Phone: 303-399-8020, extension 3101; Fax: 303-393-4611; E-mail: Robert.Reilly{at}uchsc.edu

Abstract

Abstract. LLC-PK₁ cells, an established line from pig kidney, express basolateral and apical Na⁺/H⁺ exchangers that can be distinguished by their differing sensitivities to the amiloride analog N-ethyl-N-isopropylamiloride (EIPA). It has 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 porcine NHE3 gene. There was significant homology between the LLC-PK₁ sequence and the previously reported rabbit and rat NHE3 genes, with nucleotide and deduced amino acid identities of 87 and 85% in rabbit, and 85 and 87% in rat, respectively. To study expression patterns, Northern analysis was carried out using an NHE3 cDNA to probe poly(A)⁺ RNA isolated from LLC-PK₁ cells, 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. In situ hybridization with two different NHE3 probes gave intense labeling of the distal convoluted tubule in pig kidney but (unexpectedly) no detectable labeling of the proximal tubule. These studies suggest that there are marked species differences in NHE3 expression in the distal nephron.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673