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 (NaPi-a) (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 NaPi-2-related mRNA and protein. RT-PCR was performed with single microdissected nephron segments. From these experiments we conclude that NaPi-2 mRNA is predominantly expressed in the proximal tubules of superficial and deep nephrons. No NaPi-2 mRNA was detected in the thick ascending limb of Henle's loop; however, faint NaPi-2 related PCR products were also observed in collecting ducts. Expression of the NaPi-2 protein was examined with the use of polyclonal antibodies raised against synthetic NaPi-2-derived peptides. Strong specific anti-NaPi-2 antiserum-mediated immunofluorescence was found in the convoluted part of proximal tubules and gradually decreased along the straight part. Immunofluorescence indicated that the NaPi-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 NaPi-2 protein is in agreement with previously described nephron sites of P-i reabsorption in the rat kidney and therefore suggests that the NaPi-2 transport system represents an Na-P-i cotransporter involved in proximal tubular apical transport of phosphate.