Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells.

Quantitative mass spectrometry was used to identify hormone-dependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCl across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin). mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by "proline-directed" motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins. Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the Na(+):K(+):2Cl(-) cotransporter NKCC2, at Ser552 of the Na(+):H(+) exchanger NHE3, and at Ser552 of beta-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved.

Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5'-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at -513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and -224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles.