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Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression
H S Tenenhouse1, C Gauthier, J Martel
1Department of Pediatrics, McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Abstract:
While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
Insights
Phosphate reabsorption in the distal tubule involves Na+-dependent cotransport, distinct from the proximal tubule. This study identifies novel Na+-Pi cotransporters in mouse distal convoluted tubule cells.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Physiology
Background:
- Phosphate (Pi) reabsorption is crucial for kidney function.
- Pi transport mechanisms in the distal tubule remain incompletely understood.
Purpose of the Study:
- To investigate Na+-dependent Pi transport and its regulation in immortalized mouse distal convoluted tubule (MDCT) cells.
- To identify the specific Na+-Pi cotransporter genes involved in distal tubule Pi handling.
Main Methods:
- Utilized MDCT cells for in vitro studies of Pi uptake.
- Employed techniques including initial rate kinetics, pH manipulation, substrate depletion, reverse transcriptase polymerase chain reaction (RT-PCR), and ribonuclease protection assay (RPA).
Main Results:
- MDCT cells exhibit Na+-dependent, high-affinity Pi transport inhibited by phosphonoformic acid.
- Pi uptake is stimulated by acidic extracellular pH and unaffected by parathyroid hormone or other signaling molecules.
- MDCT cells express Npt1, Glvr-1, and Ram-1 Na+-Pi cotransporter genes, with Glvr-1 and Ram-1 likely playing significant roles.
Conclusions:
- Na+-Pi cotransport in the distal tubule is regulated differently than in the proximal tubule.
- Multiple Na+-Pi cotransporter gene products, including Glvr-1 and Ram-1, mediate Pi transport in the distal convoluted tubule.