Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 ± 3.4 vs. 4.1 ± 0.3 mmHg, and reduced plasma sodium concentrations (142.2 ± 1.6 vs. 149.1 ± 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 ± 53%, n=12) relative to sham-operated controls (100 ± 13%, n=14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective.Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 ± 28% of sham levels, n=6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF

Long-term regulation of proximal tubule acid–base transporter abundance by angiotensin II

In the proximal tubule, angiotensin II (Ang-II) regulates HCO−3 reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na+/HCO−3 cotransport and Na+/H+ exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na+/HCO−3 cotransporter (NBC1) or the apical membrane type 3Na+/H+ exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59±9% of control; P<0.05) and Ang-II infusion increased abundance (130±7% of control; P<0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52±7% of control; P<0.05) and BBM (32±7% of control; P<0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P<0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption

Proteomic analysis of long-term vasopressin action in the inner medullary collecting duct of the Brattleboro rat.

Item does not contain fulltextVasopressin 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. Immunocytochemistry 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)

Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins.

Item does not contain fulltextIn 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 fraction 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

Single-tubule RNA-Seq uncovers signaling mechanisms that defend against hyponatremia in SIADH.

Item does not contain fulltextIn the syndrome of inappropriate antidiuretic hormone secretion (SIADH), hyponatremia is limited by onset of vasopressin-escape caused by loss of the water channel aquaporin-2 in the renal collecting duct despite high circulating vasopressin. Here, we use the methods of systems biology in a well-established rat model of SIADH to identify signaling pathways activated at the onset of vasopressin-escape. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical collecting ducts of vasopressin-treated rats at 1, 2, and 4 days after initiation of oral water loading in comparison to time-control rats without water loading. The time-dependent mRNA abundance changes were mapped to gene sets associated with curated canonical signaling pathways and revealed evidence of perturbation of transforming growth factor beta signaling and epithelial-to-mesenchymal transition on Day 1 of water loading simultaneous with the initial fall in Aqp2 gene expression. On Day 2 of water loading, transcriptomic changes mapped to Notch signaling and the transition from G0 into the cell cycle but arrest at the G2/M stage. There was no evidence of cell proliferation or altered principal or intercalated cell numbers. Exposure of vasopressin-treated cultured mpkCCD cells to transforming growth factor beta resulted in a virtually complete loss of aquaporin-2. Thus, there is a partial epithelial-to-mesenchymal transition during vasopressin escape with a subsequent shift from quiescence into the cell cycle with eventual arrest and loss of aquaporin-2.1 januari 201