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Quantitative phosphoproteomics of hypercalcemia induced nephrogenic diabetes insipidus
Author(s) -
Khositseth Sookkasem,
Somparn Poorichaya,
Thippamon Nattakarn,
Uawithya Panapat,
Shen Rong-Fong,
Chen Shu-Hui
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1100.3
Subject(s) - nephrogenic diabetes insipidus , chemistry , phosphopeptide , phosphoproteomics , phosphorylation , aquaporin 2 , medicine , antiporter , parathyroid hormone , endocrinology , protein phosphorylation , biochemistry , diabetes insipidus , calcium , protein kinase a , biology , water channel , mechanical engineering , organic chemistry , membrane , engineering , inlet
Nephrogenic diabetes insipidus (NDI) could result from hypercalcemia (HC). The mechanism of NDI in parathyroid hormone induced HC was studied by using phosphoproteomic technique. Rats were infused with 30 μg/kg/day of PTH or vehicle for 1 day. Inner medulla collecting ducts from HC rats and control (C) were isolated. Tryptic phosphopeptides (immobilized metal affinity chromatography‐enriched) were identified and quantified by mass spectrometry (LTQ‐Orbitrap) using label‐free methodology. Phosphopeptide ratios were calibrated by total protein levels. Sixty nine and 180 phosphorylation sites were increased and decreased in HC, respectively. A down‐regulation of cytoskeletal proteins, nucleotide binding proteins, and transmembrane transporters were revealed. There was a significant decrease in aquaporin‐2(Aqp2) phosphorylation at S256 (by 60%) and S261 (by 50%), and urea transporter (Slc14a2) phosphorylation at S62 and S63 (by 80%) in HC. Immunoblots demonstrated 34% decrease of total Aqp2 (P=0.08), 60% decrease of pS256 (P<0.001), 181% increase of pS261 (P<0.05), and 30% decrease of total Slc14a2 (P<0.05) in the HC as compared to the C. In conclusion, two key proteins in urine concentrating mechanism, Aqp2 and Slc14a2, were found decreased in both phosphorylated forms in HC‐ induced NDI model. Other signaling molecules that might play important roles in this disease process have been identified.