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Vasopressin-regulated urea transport in the renal inner medullary collecting duct (IMCD) is mediated by two urea channel proteins, UT-A1 and UT-A3, derived from the same gene (Slc14a2) by alternative splicing. The N[H.sub.2]-terminal 459 amino acids are the same in both proteins. To study UT-A1/3 phosphorylation, we made phosphospecific antibodies to UT-A sequences targeting phospho-serines at positions 84 and 486, sites identified previously by protein mass spectrometry. Both antibodies proved specific, recognizing only the phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat IMCD suspensions or whole inner medullas showed that the V2R-selective vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3) and Ser486 (in UT-A1) by about eighffold. Time course studies in rat IMCD suspensions showed maximum phosphorylation within 1 min of dDAVP exposure, consistent with the time course of vasopressin-stimulated phosphorylation of the vasopressin-sensitive water channel aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. Immuno-electron microscopy studies showed that both phosphorylated forms were present mainly in intracellular compartments in the presence of vasopressin. These studies demonstrate regulated phosphorylation of both UT-A1 and UT-A3 in response to vasopressin in a manner consistent with coordinate regulation of UT-A and aquaporin-2 in the renal IMCD. The findings add to prior evidence for vasopressin-induced phosphorylation of UT-A1, providing evidence that UT-A3 may be regulated by phosphorylation as well. phospho-specific antibody; confocal microscopy; immunofluorescence immunocytochemistry; immuno-electron microscopy doi: 10.1152/ajprenal.00617.2009.

Xie L, Hoffert JD, Chou C, Yu M, Pisitkun T, Knepper MA, AFenton R. Quantitative analysis of aquaporin-2 phosphorylation. Am J Physiol Renal Physiol 298: F1018-F1023, 2010. First published January 20, 2010; doi:10.1152/ajprenal.00580.2009.--The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation. vasopressin; trafficking; quantitative immunoblot analysis; immunogold electron microscopy doi: 10.1152/ajprenal.00580.2009.

We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000-g fraction to the 200,000-g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000-g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000-g fractions. LC-MS/MS analysis showed that markers of 'intracellular vesicles,' chiefly endosomal markers, were present in both the 17,000- and the 200,000-g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000-g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000-g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000-and 200,000-g fractions are highly heterogeneous and cannot be equated with 'plasma membrane' and 'intracellular vesicle' fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane. aquaporin-2; phosphorylation; vasopressin; membrane trafficking

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct. AM J Physiol Renal Physiol 292: F192-F201, 2008. Fist published April 16, 2008: doi:10.1152/ajprenal.00011-2008.--Collecting duct cells swell when exposed to argininge vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated , perfused rate inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomysosin). Whe a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradien and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl contransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs form NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath cased significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry. cell volume; blebbistatin; cytoskeleton; kidney; inner medulla

We recently identified a novel phosphorylation site, serine-261 (pS261), in the COOH-terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). To address whether phosphorylation at this site is regulated by vasopressin, a rabbit polyclonal phospho-specific antibody was generated. Dot blot and immunoblot analysis demonstrated that this antibody specifically recognizes AQP2 phosphorylated at pS261, and that phosphorylation of S256 (pS256), a site already known to be regulated by vasopressin, does not interfere with antibody recognition. Immunohistochemical analysis revealed intense pS261 labeling of inner medullary collecting duct (IMCD) from wild-type mice, while sections from AQP2 knockout animals showed a general absence of labeling. AQP2 pS261 was present in principal cells of all mouse and rat distal tubule segments from the connecting tubule to the terminal IMCD. Co-immunolabeling of collecting duct with phospho-specific and total AQP2 antibodies revealed that pS261 and pS256 have distinct subcellular distributions. Levels of pS256 increased, while the amount of pS261 significantly decreased in freshly isolated rat IMCD samples incubated with 1 nM [deamino-[Cys.sup.1], D-[Arg.sup.8]]vasopressin for 30 min. Similarly, based on immunohistochemical labeling, the amount of pS261 was reduced in all collecting duct segments of Brattleboro rats treated with [deamino-[Cys.sup.1],D-[Arg.sup.8]]vasopressin for 2 h. This study reveals a reciprocal change in S256 and S261 phosphorylation in response to short-term vasopressin exposure, suggesting that these residues may serve distinct roles in regulation of AQP2 subcellular distribution and collecting duct water permeability. IMCD; water transport; serine-256; channel; inner medullary

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were ~95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis. difference gel electrophoresis; mass spectrometry; phosphoenolpyruvate carboxykinase; glutaminase; mRNA stabilization

LC--M[S.sup.n] has become a popular option for high-throughput quantitative proteomics, thanks to the availability of stable-isotope labeling reagents. However, the vast quantity of data generated from LC--M[S.sup.n] continues to make the postacquisition quantification analyses challenging, especially in experiments involving multiple samples per experimental condition. To facilitate data analysis, we developed a computer program, QUIL, for automated protein quantification. QUIL accounts for the dynamic nature of spectral background and subtracts this background accordingly during ion chromatogram reconstruction. For elution profile identification, QUIL minimizes the inclusion of coeluted neighbor peaks, yet tolerates imperfect peak shapes. Outlier-resistant methods have been implemented for better protein ratio estimation. The utility of QUIL was validated by quantitative analyses of a standard protein as well as complex protein mixtures, which were labeled with cICAT or [sup.18]O and analyzed using LCQ, LTQ, or FT-ICR instruments. For samples that no prior knowledge of relative protein quantities was available, Western blotting was performed for confirmation. For the standard protein, the coefficient of variation (CV) of peptide ratio estimation was 6%. For complex mixtures, the median CV for protein ratio calculations was less than 10%. Computed protein abundance ratios exhibited a relatively high degree of correlation with those obtained from Western blot analyses. Compared with a widely used commercial software tool, QUIL showed improvement in ion chromatogram construction and peak integration and significantly reduced relative errors in abundance ratio assessment.

Vasopressin regulates water and solute transport in the renal collecting duct. In addition to short-term regulation of aquaporin-2 trafficking, vasopressin also has long-term effects to regulate the abundances of aquaporins-2 and -3 and [beta]- and [gamma]-subunits of the epithelial sodium channel in collecting duct principal cells. To investigate further the direct and indirect long-term regulatory actions of vasopressin in the inner medullary collecting duct (IMCD), we used a proteomic approach [difference gel electrophoresis (DIGE) coupled with MALDI-TOF identification of differentially expressed protein spots]. DDAVP or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCD cells were purified from the inner medullas for proteomic analysis. Forty-three proteins were found to be regulated in response to vasopressin infusion, including 18 that were increased in abundance, 22 that were decreased, and 3 that were shifted in the gel, presumably because of posttranslational modification. Immunocyto-chemistry confirmed collecting duct expression of several of the proteins that were identified. Immunoblot analysis of nine of the proteins confirmed the changes seen by the DIGE method. Of these nine proteins, six were increased in response to DDAVP infusion: nitric oxide synthase-2 (NOS2), GRP78, heat shock protein-70, annexin II, glutaminase, and cathepsin D. The remaining three were decreased in response to DDAVP: aldehyde reductase I, adenylyl cyclase VI, and carbonic anhydrase II. The findings point to a role for vasopressin in the coordinate regulation of several determinants of nitric oxide levels (NOS2, arginase II, NADPH oxidase) and of proteins potentially involved in vasopressin escape (adenylyl cyclase VI and G protein-coupled receptor kinase 4). vasopressin: mass spectrometry; collecting duct

In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with matrix-assisted laser desorption-ionization-time of flight mass spectrometry. Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly purified IMCD cell traction in which aquaporin-2 was enriched 10-fold. When DIGE was initially applied to rat inner medullas fractionated into IMCD cells (labeled with Cy3) and non-IMCD cells (labeled with Cy5), we identified 50 highly abundant proteins expressed in the IMCD cells. These proteins, identifiable without subcellular fractionation, included chiefly enzymes, structural proteins, and signaling intermediates. An additional 35 proteins were found predominantly in the non-IMCD cell types. Proteins that were highly enriched in the IMCD fraction included cytokeratin 8, cytokeratin 18, transglutaminase II, aminopeptidase B, T-plastin, heat shock protein (HSP) 27, HSP70, and lactate dehydrogenase A. Semiquantitative immunoblotting and immunohistochemistry confirmed relative expression levels and distribution of selected proteins. An additional 40 IMCD proteins were identified in separate experiments aimed at further enrichment of proteins through optimization of sample loading. These studies document the applicability of a standardized approach to purification of IMCD cells for proteomic analysis of IMCD proteins and demonstrate the feasibility of large-scale identification of proteins in the native IMCD cell. inner medullary collecting duct; kidney; two-dimensional electrophoresis; difference gel electrophoresis; proteomics