Intrarenal distribution of the colonic H,K-ATPase mRNA in rabbit
G Fejes-Tóth1, A Náray-Fejes-Tóth, H Velázquez
1Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001, USA. Geza.Fejes-Toth@dartmouth.edu
Background:
Evidence suggests that the colonic H,K-ATPase isoform is expressed in the kidney and that a mRNA species highly homologous to the rat and guinea pig HKalpha2 is expressed in the cortical collecting duct (CCD) of the rabbit. The goals of this study were to determine if this mRNA is the rabbit homologue of HKalpha2 or a novel isoform and to determine intrarenal distribution of the HKalpha2 mRNA in rabbit.
Methods:
5'-RACE and Dye Deoxy Terminator chemistry were used to determine the full-length sequence of the rabbit HKalpha2 mRNA. The intrarenal distribution of HKalpha2 mRNA was determined in microdissected nephron segments, connecting tubule (CNT), and CCD cells isolated by immunodissection, as well as in the three cell types of the CCD. Principal cells and alpha- and beta-intercalated cells were isolated by fluorescence-activated cell sorting. HKalpha2 mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) or single-nephron RT-PCR (SN-RTPCR).
Results:
The full-length sequence of the rabbit kidney HKalpha2 mRNA was determined. This transcript is identical to the one expressed in rabbit distal colon. In microdissected nephron segments, strong HKalpha2 amplicons were present in the CNT, CCD, and outer medullary collecting duct (OMCD), whereas no signal was detected in the proximal tubule, distal convoluted tubule, think ascending limb, and inner medullary collecting duct. Roughly comparable levels of HKalpha2 mRNA were present in all three CCD cell types, and the highest levels were observed in a subpopulation most likely corresponding to CNT cells.
Conclusions:
These results suggest that the HKalpha2 mRNA is expressed in rabbit collecting duct is identical in size and sequence to the one expressed in rabbit distal colon. HKalpha2 mRNA in the rabbit kidney is selectively expressed in the CNT, CCD, and OMCD, and all three collecting duct subtypes express its mRNA.
Insights
Rabbit kidney HKalpha2 mRNA, identical to the colon isoform, is found in the collecting duct. This study identifies its specific locations within the kidney nephron segments.
Area of Science:
- Nephrology
- Molecular Biology
- Renal Physiology
Background:
- The H,K-ATPase isoform is suggested to be expressed in the kidney.
- A highly homologous mRNA species to rat and guinea pig HKalpha2 is found in rabbit cortical collecting duct (CCD).
Purpose of the Study:
- Determine if the rabbit kidney mRNA is a homologue of HKalpha2 or a novel isoform.
- Map the intrarenal distribution of HKalpha2 mRNA in rabbits.
Main Methods:
- Utilized 5'-RACE and Dye Deoxy Terminator chemistry for full-length mRNA sequencing.
- Employed microdissection and immunodissection to isolate nephron segments and collecting duct cells.
- Quantified mRNA levels using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and single-nephron RT-PCR (SN-RT-PCR).
Main Results:
- Full-length rabbit kidney HKalpha2 mRNA sequence determined; identical to rabbit distal colon transcript.
- Strong HKalpha2 mRNA signals detected in connecting tubule (CNT), CCD, and outer medullary collecting duct (OMCD).
- No signal in proximal tubule, distal convoluted tubule, or inner medullary collecting duct; highest levels in CNT cells.
Conclusions:
- Rabbit kidney HKalpha2 mRNA is identical in sequence and size to the colon isoform.
- HKalpha2 mRNA is selectively expressed in rabbit CNT, CCD, and OMCD.
- All three collecting duct subtypes express HKalpha2 mRNA.
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