The interstitium of the renal medulla is the only tissue in mammals that normally undergoes large changes in osmolality. The changes are part of the renal mechanism for producing a concentrated or dilute urine. When the medulla is hypertonic, the cells of the medulla balance the high extracellular concentration of NaCl by accumulating high concentrations of small organic solutes such as betaine, sorbitol, myo-inositol, and glycerophosphorylcholine, which in contrast to high concentrations of electrolytes, do not perturb cell function. This proposal is designed to gain understanding of the mechanism of the cellular response to hypertonicity that results in the accumulation of betaine. Studies will use kidney-derived cells in culture that accumulate betaine and some other non-perturbing solutes by specifically increasing their rate of uptake when the culture medium is made hypertonic. We have recently cloned the cDNA for the renal betaine transporter. The sequence of the open reading frame of the cDNA indicates that the transporter is a member of the brain GABA/noradrenaline transporter gene family. We will prepare antibodies to the transporter to quantify the amount of transporter protein to determine whether hypertonic cells increase betaine uptake by making more transporters, as suggested by the results of kinetic experiments. Focusing on the mechanism by which hypertonicity increases the accumulation of betaine, we have performed preliminary experiments that indicate that hypertonicity raises the cell level of mRNA for the betaine transporter, at least in part by increasing the rate of transcription of the gene for the transporter. We will also determine whether hypertonicity affects the rate of degradation of transporter mRNA. If further experiments establish that the rate of synthesis of transporter mRNA is elevated in hypertonic cells, the mechanism by which hypertonicity regulates transcription will be studied by cloning the gene for the transporter and the adjacent 5' and 3' regions to search for DNA sequences that serve a regulatory function. Also, if mRNA stability is regulated by hypertonicity, the sequences responsible will be identified. Abnormal medullary osmolyte metabolism has been implicated in diseases of the renal medulla, particularly interstitial nephritis due to chronic ingestion of analgesics.
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