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University of Rochester School of Medicine, Strong Children's Research Center, Rochester, New York 14642
Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1-2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney's adaptation to chronic metabolic acidosis.
reverse transcription-polymerase chain reaction; microdissected tubule segments; ribonucleic acid; Southern analysis; malate dehydrogenase
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