Sulfate (S042-) plays an essential role during growth, development, bone/cartilage formation and cellular metabolism. NaSi-1 is a Na+-S042- cotransporter expressed on the apical membrane of the renal proximal tubule and suggested to play an important role in maintaining S042- homeostasis. To gain a better understanding of the physiological role(s) of the human renal Na+-S042- cotransporter, this study presents the isolation and characterization of the human NaSi-1 cDNA (hNaSi-1), gene (NASI) and promoter region. The hNaSi-1 cDNA encodes a protein of 595 amino acids (66.05 kDa) with 13 putative transmembrane domains. hNaSi-1 mRNA expression was exclusive to the human kidney. Expression of hNaSi-1 protein in Xenopus oocytes demonstrated a high affinity Na+-S042- cotransporter (Km for S042- ~0.3 mM) which was inhibited by selenate, thiosulfate, molybdate, tungstate, citrate and succinate. Antisense inhibition experiments suggest hNaSi-1 to represent the major Na+-S042- cotransporter in the human kidney. NASI was localized on human chromosome 7, mapped to 7q3l-7q32, near the S042- transporter genes, CLD and SUT-1. The NASI gene contains 15 exons, spanning over 85-kb in length. Transcription initiation occurs from a single site, 26-bp downstream to a TATA box-like sequence. The promoter is ~63% A+T rich and contains a number of well characterized cis-acting elements, including sequences resembling responsive elements for hormones (T3RE and VDRE) and xenobiotics (XRE). Hormone and xenobiotic responsiveness of the NASI gene was assessed by fusing its promoter region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal OK, LLC-PKl or MDCK cells. Treatment with T3 and l,25-(OH)2D3 had no significant effect on the luciferase activity of the chimeric gene in OK and LLC-PKl renal cell lines; however, treatment with the xenobiotic 3-methyl cholanthrene significantly increased the luciferase activity of the chimeric gene in MDCK cells. By site directed mutagenesis, the XRE was identified at position -2052 in the NASI promoter to be responsible for this activity. This study represents the first characterization of xenobiotic regulation of the NASI gene in a renal cell line. It also provides the framework for a more detailed analysis of NASI gene expression through the characterization of NASI promoter function.
In addition to NaSi-1, this study has focussed on sat-1, a S042- anion transporter expressed on the basolateral membranes of renal proximal tubules, also believed to play a role in the maintenance of SO42- homeostasis. In order to provide insights into the molecular mechanisms underlying tissue-specific and hormonal regulation of sat-1 and its role in S042- homeostasis, the mouse sat-1 cDNA (msat-1), gene (Sat1) and promoter region were isolated and characterized, msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains, which induces SO42- (also oxalate and chloride) transport in Xenopus oocytes, msat-1 mRNA was expressed in kidney, liver, caecum, calvaria, brain, heart and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Satl gene spans ~6-kb and consists of 4 exons. 5'-RACE analysis revealed a single transcription initiation site 358-bp upstream from the ATG start codon. Neither a consensus TATA nor a CAAT box was identified in the proximity to the transcription start site. The promoter region was found to be -52% G+C rich, contains a number of well characterized cis-acting elements (including sequences resembling hormone responsive elements T3RE and VDRE) and can drive basal transcriptional activity in OK cells. This study demonstrated that T3 but not l,25-(OH)2D stimulated the transcriptional activity of the Sat1 promoter in transiently transfected OK cells. Site-directed mutagenesis identified an imperfect T3RE at position -454 in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the genomic structure, function and hormonal regulation of the Sat1 gene encoding a SO42- /chloride/oxalate anion transporter. This information allows further investigation into Sat1 gene expression and provides the tools for assessing the role of Sat1 in the maintenance of S042- homeostasis through the generation and analysis of Sat1-deficient mice.