Early dexamethasone-inducible genes in M-1 Cortical Collecting Cells

Precise control of blood pressure and salt balance is ultimately dependent on the regulation of epithelial sodium channels, ENaC, in the distal nephron. Ligand activation of mineralocorticoid and glucocorticoid receptors induces a transcriptional response that leads to an augmentation of ENaC cell surface density and activity, but a complete inventory of the network of genes involved in the response is incomplete. In the present study, we used high-density cDNA microarrays to examine changes in the transcriptional profile of a mouse cortical collecting duct cell line, M-1, in response to a potent glucocorticoid agonist, dexamethasone. Here we identify 64 gene transcripts that are consistently co-regulated with ENaC, increasing within 3 hours of dexamethasone treatment. Within the same early response period, the abundance of 58 diiferent gene transcripts significantly decreased. A subset of up-regulated genes (12) was selected for further validation and the majority (10) of these were confirmed as dexamethone-regulated transcripts by real time RT-PCR. The products of these genes are known to function in intracellular trafficking operations (Numb, tetraspanin CD63, and Vps41), signal transduction, or to operate as modifiers of the cytoskeleton (gelsolin, tektin 2 and transgelin). These studies have identified new group of candidate genes that potentially regulate sodium transport in the collecting duct. Keywords: Gene identification in a cultured kidney cell model Experimental design. Cells were then treated with 100 nM hydrosoluble dexamethasone or steroid-free medium for 3 hours at 37°C with 5% CO2. RNA samples from dexamethasone-treated and control cells were extracted with Trizol reagent, and DNA-decontaminated using DNA-free kit, commercially available (Ambion, Austin, TX). Yield and concentration was quantified by spectrophotometry. RNA quantity and integrity was confirmed by denaturing formaldehyde agarose gel electrophoresis and ethidium bromide staining. These samples were used for microarray labeling. Microarrays, RNA labeling, and hybridization. High quality spotted cDNA on glass slide microarrays were purchased from the Microarray Centre of the University Health Network (Toronto, Canada). These arrays contained PCR-generated cDNA inserts of all 15,264 sequence-verified mouse ESTs, from the National Institute of Aging collection. Duplicate 100-mm diameter cDNA spots are represented on GAPS II slides (Corning, Inc.). Spot cDNA identity is tracked by composed identifiers including clone identifier, description, and Unigene, and catalogued as “M15Kv1”. Briefly, 5 µg total RNA was reverse-transcribed using oligo-dT flanked at 5’ end with capture sequences specific to fluorescent Cy3- or Cy5-coupled dendrimer complexes. The resulting cDNAs from the control and test samples were mixed and hybridized with the mouse cDNA arrays in humidified hybridization chambers at 60°C for 18 hours. Following hybridization, microarrays were washed three times as described above. Quantitation. Slides were read using a laser confocal scanner, the GSI Lumonics ScanArray 5000 imaging system, at 10-µm resolution set up for excitation/emission at 543/570 and 649/670 nm, and for acquisition of Cy3 and Cy5-labeled spots respectively. Scanned image data were saved in 16-bit tiff files and quantitated with Imagene 4.0 (BioDiscovery, Marina del Rey, CA). The mean intensities of fluorescent spots were measured.