Suppression subtractive hybridization analysis of low-protein diet- and vitamin D-induced gene expression from rat...

Protein restriction and hypercalcemia result in a urinary concentrating defect in rats and humans. Previous tubular perfusion studies show that there is an increased active urea transport activity in the initial inner medullary (IM) collecting duct in low-protein diet (LPD) and vitamin D (Vit D) animal models. To investigate the possible mechanisms that cause the urinary concentrating defect and to clone the new active urea transporter, we employed a modified two-tester suppression subtractive hybridization (ttSSH) approach and examined gene expression induced by LPD and Vit D in kidney IM base. Approximately 600 clones from the subtracted library were randomly selected; 150 clones were further confirmed to be the true positive genes by slot blot hybridization with subtracted probes from LPD and Vit D and sent for DNA sequencing. We identified 10 channel/transporter genes that were upregulated in IM base in LPD and Vit D animal models; 8 were confirmed by real-time PCR. These genes include aquaporin 2 (AQP2), two-pore calcium channel protein 2, brain-specific organic cation transporter, Na(+)- and H(+)-coupled glutamine transporter, and solute carrier family 25. Nine genes are totally new, and twelve are uncharacterized hypothetical proteins. Among them, four genes were shown to be new transmembrane proteins as judged by Kyte-Doolittle hydrophobic plot analysis. ttSSH provides a useful method to identify new genes from two conditioned populations.