Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry.
We have recently identified a rat kidney cortex Na-dependent transport system for phosphate (P(i)) by expression cloning (NaP(i)-2) (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979, 1993). In this study we have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to establish the sites of expression of the NaP(i)-2-related mRNA and protein. RT-PCR was performed with single microdissected nephron segments. From these experiments we conclude that NaP(i)-2 mRNA is predominantly expressed in the proximal tubules of superficial and deep nephrons. No NaP(i)-2 mRNA was detected in the thick ascending limb of Henle's loop; however, faint NaP(i)-2 related PCR products were also observed in collecting ducts. Expression of the NaP(i)-2 protein was examined with the use of polyclonal antibodies raised against synthetic NaP(i)-2-derived peptides. Strong specific anti-NaP(i)-2 antiserum-mediated immunofluorescence was found in the convoluted part of proximal tubules and gradually decreased along the straight part. Immunofluorescence indicated that the NaP(i)-2 protein is present in the brush border of proximal tubular cells. In addition, NaP(i)-2-specific immunofluorescence was also observed in subapical vesicles. The described distribution of the NaP(i)-2 protein is in agreement with previously described nephron sites of P(i) reabsorption in the rat kidney and therefore suggests that the NaP(i)-2 transport system represents an Na-P(i) cotransporter involved in proximal tubular apical transport of phosphate.
Custer
M
M
Institute of Anatomy, University of Zürich-Irchel, Switzerland.
Lötscher
M
M
Biber
J
J
Murer
H
H
Kaissling
B
B
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis*; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009399:Nephrons / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D012313:RNA