טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShachaf Catherine
SubjectTranscriptional Regulation of the Kidney Specific Sodium-
Phosphate Co-Transporter Npt2
DepartmentDepartment of Medicine
Supervisor Professor Karl Skorecki


Abstract

The Npt2 gene encodes the type II sodium-dependent phosphate co-transporter and is expressed specifically in the renal proximal tubule. In order to elucidate the mechanism for cell specific transcription in the proximal tubule, we have isolated and characterized 4,740 bp’s of the 5’-flanking sequence of the rat Npt2 gene. We have demonstrated that this fragment drives reporter gene expression, in the proximal tubule derived opossum kidney (OK) cell line, but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence 10 AP2 sites and a pair of 125 bp inverted repeats flanking 1,325 bp’s of intervening sequence, containing eight of the total ten AP2 sites. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Gelshift analysis, revealed binding to the AP2-site using nuclear extracts from OK cells and AP2 recombinant proteins. We propose that the inverted repeats regulate promoter activity by limiting access to transcription factors that bind to the AP2 sites in the intervening sequence.

In order to confirm the ability of the promoter to drive specific renal expression in vivo, transgenic mice were generated using a construct of the 4.7 kb rat promoter that drives LacZ reporter gene expression. The LacZ reporter gene was found to be expressed specifically in the cortex of the kidney of the transgenic mice specifically in the proximal tubule. The results of these studies confirm that the isolated rat Npt2 promoter is sufficient to confer the proximal tubule specific expression of the Npt2 gene.

The Npt2 promoter can potentially serve as a useful system for gene delivery to the kidney cortex. The Npt2 transgenic mice can serve as a useful model for defining molecular mechanisms of Npt2 gene regulation in response to hormonal and dietary modulation