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Am J Physiol Renal Physiol 285: F49-F58, 2003. First published April 22, 2003; doi:10.1152/ajprenal.00389.2002
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Cloning and localization of KCC4 in rabbit kidney: expression in distal convoluted tubule

Heino Velázquez and Teresa Silva

Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516

Submitted 31 October 2002 ; accepted in final form 16 March 2003

Cl-dependent K secretion is a feature of renal distal tubules and collecting ducts. Recent cloning and identification of K-Cl cotransporter proteins led us to search for additional novel KCC isoforms expressed in the renal distal nephron. A human expressed sequence tag (EST) with high homology to KCC1 was identified. The rabbit isoform was cloned by homology using degenerate primers and rapid amplification of cDNA ends (RACE). Our isoform is the rabbit homologue of mouse and human KCC4 published previously. The 4.35-kb rabbit KCC4 cDNA encodes a protein of 1,106 amino acids. Antibodies were generated to both NH2-terminal and COOH-terminal fusion proteins. Northern and Western blot analyses showed widespread mRNA and protein expression in many rabbit organs, in renal cortex, outer medulla, and inner medulla but not in skeletal muscle. Immunohistochemical localization of KCC4 showed expression exclusively along the basolateral membrane in many nephron segments. The distal convoluted tubule and connecting tubule exhibited the highest level of KCC4 immunoreactivity, followed by the medullary thick ascending limb. A low level of immunoreactivity was detected in the proximal tubule and collecting ducts. We postulate that KCC4 mediates potassium and chloride exit from the cell and may play an important role in salt absorption by the distal convoluted tubule.

immunolocalization; antibody; potassium chloride cotransport; distal tubule


Address for reprint requests and other correspondence: H. Velázquez, VA Connecticut Healthcare System, Research Office 151, West Haven, CT 06516 (E-mail: heino.velazquez{at}yale.edu).




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