LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

Sachs, Aaron N.; Pisitkun, Trairak; Hoffert, Jason D.; Yu, Minshu; Knepper, Mark A.

doi:10.1152/ajprenal.90510.2008

Cited by 34 publications

(19 citation statements)

References 22 publications

(30 reference statements)

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“…Procedures were as described previously (37). Briefly, kidney cortices were excised and homogenized in buffer containing 250 mM sucrose, 10 mM triethanolamine, protease inhibitors (Complete; Roche Diagnostics), and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1; Sigma) (pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

Mutig

Saritas

Uchida

et al. 2010

American Journal of Physiology-Renal Physiology

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Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V 2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na ϩ -Cl Ϫ cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH 2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V 2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11␤-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V 2 receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT. antidiuretic hormone; distal convoluted tubule; phosphorylation; sodium-chloride cotransporter trafficking ANTIDIURETIC HORMONE [arginine vasopressin (AVP)] serves to control extracellular fluid homeostasis. The principal effect of AVP is found in the collecting duct where AVP increases water reabsorption. The prerequisite to ensure this function is the creation of a hypertonic interstitium via action of the thick ascending limb (TAL), which is also promoted by AVP. These epithelial effects of AVP are mediated by type 2 AVP receptors (V 2 R) (2, 29), whereas type 1a receptor signaling rather serves to limit the antidiuretic effects of AVP (31). Mapping receptorspecific probes to the tubular segments has revealed subtypeselective distribution along TAL, macula densa, distal convolutions, and collecting ducts (3, 28). In TAL, strong V 2 R expression has been observed, which agreed with increased abundance and phosphorylation of the furosemide-sensitive NaϪ cotransporter (NKCC2) and enhanced NaCl reabsorption in TAL in response to AVP (8, 13, 28). V 2 R signaling also activates epithelial Na ϩ channel (ENaC)-dependent Na ϩ reabsorption in collecting ducts (16); AVP-induced antinatriuresis in this segment has therefore been considered as a causal element in the context of salt-sensitive hype...

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Section: Methodsmentioning

confidence: 99%

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

Mutig

Saritas

Uchida

et al. 2010

American Journal of Physiology-Renal Physiology

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“…Primary antibodies for AQP2 included K5007 detecting the COOH terminus (29) and N-20 detecting the NH 2 terminus (SC-9880, Santa Cruz Biotechnology). Phospho-specific antibodies against AQP2 were generated in our laboratory (29,30).…”

Section: Methodsmentioning

confidence: 99%

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

Miller

Uawithya

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

116

139

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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.aquaporin 2 ͉ kidney ͉ microarrays ͉ transcription ͉ vasopressin R enal water excretion is tightly regulated chiefly through effects of vasopressin on the molecular water channel, aquaporin-2 (AQP2) (1). AQP2 gene expression in the kidney is restricted to collecting duct principal cells and connecting tubule cells (2, 3). Aside from control of trafficking of AQP2-containing vesicles (1), AQP2 is regulated through changes in the total abundance of the AQP2 protein in collecting duct cells. Vasopressin increases the renal abundance of the AQP2 protein (4) via changes in AQP2 mRNA levels (5), in part by transcriptional regulation. Studies in transgenic mice in which 14-15 kb of the 5Ј-flanking region of the AQP2 gene was coupled to reporters established that cell-specific expression of the AQP2 gene in the collecting duct is dependent on cis-elements in this region (6, 7). Altered AQP2 protein abundance in the renal collecting duct is largely responsible for water balance abnormalities associated with diverse clinical states including lithium-induced diabetes insipidus, congestive heart failure, and the syndrome of inappropriate antidiuresis (1). Understanding the roles of AQP2 in these clinical states hinges largely on understanding the mechanism of cell-specific expression of the AQP2 gene.Sequencing of the 5Ј-flanking region of the AQP2 gene revealed several putative cis-binding element (BE) motifs including a c...

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“…6B), the amount of protein loaded (12 g for PM, 45 g for ICM), and the total amount of protein recovered in PM versus ICM (90% in PM, 10% in ICM)]. The difference between these two estimates may be a result of incomplete separation of PM from ICM or incomplete recovery of NCC in low-density ICM (43). The direct counting of silver grains in PM versus subapical pools is likely to be more accurate.…”

Section: Subcellular Localization Of Phosphorylated Nccmentioning

confidence: 99%

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

Lee

Maunsbach

Riquier‐Brison

et al. 2013

American Journal of Physiology-Cell Physiology

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Lee DH, Maunsbach AB, Riquier-Brison AD, Nguyen MT, Fenton RA, Bachmann S, Yu AS, McDonough AA. Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties. Am J Physiol Cell Physiol 304: C147-C163, 2013. First published October 31, 2012; doi:10.1152/ajpcell.00287.2012.-The renal distal tubule Na-Cl cotransporter (NCC) reabsorbs Ͻ10% of the filtered Na ϩ but is a key control point for blood pressure regulation by angiotensin II (ANG II), angiotensin-converting enzyme inhibitors (ACEI), and thiazide diuretics. This study aimed to determine whether NCC phosphorylation (NCCp) was regulated by acute (20 -30 min) treatment with the ACEI captopril (12 g/min ϫ 20 min) or by a sub-pressor dose of ANG II (20 ng·kg Ϫ1 ·min Ϫ1 ) in Inactin-anesthetized rats. By immuno-EM, NCCp was detected exclusively in or adjacent to apical plama membranes (APM) in controls and after ACEI or ANG II treatment, while NCC total was detected in both APM and subapical cytoplasmic vesicles (SCV) in all conditions. In renal homogenates, neither ACEI nor ANG II treatment altered NCCp abundance, assayed by immunoblot. However, by density gradient fractionation we identified a pool of low-density APM in which NCCp decreased 50% in response to captopril and was restored during ANG II infusion, and another pool of higher-density APM that responded reciprocally, indicative of regulated redistribution between two APM pools. In both pools, NCCp was preferentially localized to Tritonsoluble membranes. Blue Native gel electrophoresis established that APM NCCp localized to ϳ700 kDa complexes (containing ␥-adducin) while unphosphorylated NCC in intracellular membranes primarily localized to ϳ400 kDa complexes: there was no evidence for native monomeric or dimeric NCC or NCCp. In summary, this study demonstrates that phosphorylated NCC, localized to multimeric complexes in the APM, redistributes in a regulated manner within the APM in response to ACEI and ANG II. blood pressure; trafficking; captopril; ␥-adducin; subcellular fractionation; immuno-EM THE RENAL DISTAL CONVOLUTED TUBULE (DCT) is a key control point for extracellular volume and blood pressure regulation. The Na-Cl cotransporter (NCC), expressed in the apical membrane of the DCT, is the target of thiazide diuretics prescribed widely to reduce extracellular fluid volume in edema and to lower blood pressure (1,20,32,38). In addition, inactivating mutations of either NCC or the NCC regulator SPAK (STE20/ SPS1-related proline/alanine-rich kinase) provoke salt wasting and lower blood pressure (37,46,60). In contrast, mutations in WNK ("with no lysine") kinases, which directly or indirectly activate NCC, provoke familial hyperkalemic hypertension (12,14,58). Taken together, these findings suggest that conditions that increase NCC activity elevate blood pressure (which drives pressure natriuresis to match salt output to input) while conditions that decrease NCC activity lower blood pressure (by increasing salt excretion). ...

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LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

Abstract: Sachs AN, Pisitkun T, Hoffert JD, Yu M-J, Knepper MA. LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat.

Cited by 34 publications

References 22 publications

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

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

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

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LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

Abstract: Sachs AN, Pisitkun T, Hoffert JD, Yu M-J, Knepper MA. LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat.

Cited by 34 publications

References 22 publications

Short-term stimulation of the thiazide-sensitive Na+-Cl−cotransporter by vasopressin involves phosphorylation and membrane translocation

Short-term stimulation of the thiazide-sensitive Na+-Cl−cotransporter by vasopressin involves phosphorylation and membrane translocation

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

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties

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Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation