Localization of NaPi-1, a Na-Pi cotransporter, in rabbit kidney proximal tubules. I. mRNA localization by reverse transcription/polymerase chain reaction.
We have recently isolated from a rabbit cortex cDNA library a cDNA clone (NaPi-1), which, after in vitro transcription (cRNA) and injection into Xenopus laevis oocytes, expresses Na-dependent Pi uptake [Werner A, et al. (1991) Proc Natl Acad Sci USA 88:9608-9612]. The aim of the present work was to study the nephron location of the NaPi-1-related mRNA(s) by combining nephron microdissection procedures, reverse transcription (RT) and amplification of the resultant cDNA by the polymerase chain reaction (PCR). RT-PCR using NaPi-1-specific primers (different combinations) and either total kidney cortex RNA or microdissected proximal tubule segments resulted in two PCR products, both of approximately the expected length (but differing by about 30 base pairs). Restriction-enzyme analysis and nucleotide sequencing confirmed that both PCR products are related to NaPi-1 and that the "longer" PCR product has an insert of 26 base pairs containing an AluI restriction site. Nephron microdissection documents expression of NaPi-1-related mRNA(s) in superficial and deep proximal tubules (S1, S2 and S3 segments) and their absence in glomeruli, thin descending limb and thick ascending limbs of Henle's loop, distal convoluted tubules and cortical and inner medullary collecting ducts. These experiments suggest a "microheterogeneity" of NaPi-1-related mRNA(s) (which is not detected in Northern blot analysis) and proximal tubular expression of NaPi-1.
Custer
M
M
Institute of Physiology, University of Zürich, Switzerland.
Meier
F
F
Schlatter
E
E
Greger
R
R
Garcia-Perez
A
A
Biber
J
J
Murer
H
H
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009399:Nephrons / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014158:Transcription, Genetic; D014982:Xenopus laevis
D002352:Carrier Proteins; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D004996:Ethidium