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

Christine A. Shugrue, Nicholas Obermüller, Sebastian Bachmann, Carolyn W. Slayman, Robert F. Reilly

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1649-1657
Number of pages9
JournalJournal of the American Society of Nephrology
Volume10
Issue number8
StatePublished - Aug 1999

Fingerprint

LLC-PK1 Cells
Molecular Cloning
Swine
Kidney
Rabbits
Kidney Cortex
Sodium-Hydrogen Antiporter
Genomic Library
Amiloride
Nephrons
Genes
Reverse Transcription
In Situ Hybridization
Nucleotides
Complementary DNA
Clone Cells
Amino Acids
Cell Line
Polymerase Chain Reaction
Messenger RNA

ASJC Scopus subject areas

  • Nephrology

Cite this

Shugrue, C. A., Obermüller, N., Bachmann, S., Slayman, C. W., & Reilly, R. F. (1999). Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney. Journal of the American Society of Nephrology, 10(8), 1649-1657.

Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney. / Shugrue, Christine A.; Obermüller, Nicholas; Bachmann, Sebastian; Slayman, Carolyn W.; Reilly, Robert F.

In: Journal of the American Society of Nephrology, Vol. 10, No. 8, 08.1999, p. 1649-1657.

Research output: Contribution to journalArticle

Shugrue, CA, Obermüller, N, Bachmann, S, Slayman, CW & Reilly, RF 1999, 'Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney', Journal of the American Society of Nephrology, vol. 10, no. 8, pp. 1649-1657.
Shugrue, Christine A. ; Obermüller, Nicholas ; Bachmann, Sebastian ; Slayman, Carolyn W. ; Reilly, Robert F. / Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney. In: Journal of the American Society of Nephrology. 1999 ; Vol. 10, No. 8. pp. 1649-1657.
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