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Am J Physiol Renal Physiol (April 22, 2003). doi:10.1152/ajprenal.00389.2002
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Submitted on October 31, 2002
Accepted on March 16, 2003

Cloning and localization of KCC4 in the rabbit kidney: Expression in the distal convoluted tubule

Heino Velazquez1* and Teresa C. Silva2

1 Department of Research, Veterans Affairs Medical Center, West Haven, CT, USA; Department of Medicine and Nephrology, Yale University, New Haven, CT, USA
2 Department of Research, Veterans Affairs Medical Center, West Haven, CT, USA

* To whom correspondence should be addressed. E-mail: heino.velazquez{at}yale.edu.

Chloride-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 EST (expressed sequence tag) 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 1106 amino acids. Antibodies were generated to both N-terminal and C-terminal fusion proteins. Northern and Western analysis 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 DCT and CNT 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.




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