Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis.
The renal carbonic anhydrases, CA II (cytosolic) and CA IV (membrane bound), are believed to facilitate renal acid secretion. We have recently shown that renal cortical sodium dodecyl sulfate (SDS)-resistant hydratase (presumably CA IV) activity was stimulated 241% during chronic metabolic acidosis (CMA). In the present study, we examined the expression and regulation of CA IV mRNA in kidneys from control and acidotic rabbits. To obtain a CA IV probe, we reverse transcribed rabbit kidney total RNA and amplified a approximately 780-base pair (bp) DNA product using primers derived from the human CA IV sequence. Using this product, we screened one-half of a kidney cortex cDNA library and sequenced a 1,194-bp cDNA, which contained the entire open-reading frame of rabbit CA IV. The cDNA was 78% identical to human and 71% to rat CA IV. The deduced amino acid sequence projected an active zinc binding site and two glycosylation sites. Northern analysis yielded a single transcript of approximately 1,600 bp in size expressed more abundantly in cortex and inner medulla than in outer medulla. CA IV mRNA was also expressed abundantly in lung but not in liver or spleen. The high abundance of CA IV mRNA in inner medulla was localized by in situ hybridization to medullary collecting duct cells. Rabbits exposed to CMA showed significant upregulation of CA IV mRNA expression in kidney cortex and outer medulla. Despite a numerical increase, excessive variability precluded statistical significance in the inner medulla. Thus CA IV mRNA was expressed abundantly in kidney and stimulated by CMA, similar to what has been previously observed for SDS-resistant hydratase (presumed CA IV) activity. It is likely that the regulation of CA IV mRNA and activity is relevant to the kidney's adaptation to CMA.
Winkler
C A
CA
Department of Pediatrics, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Kittelberger
A M
AM
Schwartz
G J
GJ
D000138:Acidosis / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007672:Kidney Cortex / Q000201:enzymology; D007679:Kidney Medulla / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000201:enzymology; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases