Membrane Of Collecting Duct Cells Research Articles

Absence of E-Cadherin and β-Catenin in the Basal Plasma Membrane of Collecting Duct Cells During NDI Development and Recovery.

Lithium (Li) induces severe polyuria and polydipsia in up to 40% of patients undergoing Li treatment. In rats, Li treatment induces a reversible cellular remodeling of the collecting duct (CD), decreasing the fraction of principal-to-intercalated cells. To investigate the potential role of adherens junction proteins, we performed immunohistochemistry on kidney cross-sections from rats treated with Li as well as rats undergoing recovery on a normal diet following 4 weeks of Li-treatment. We performed immunoelectron microscopy on cryosections to determine the ultrastructural localizations. Immunohistochemistry showed that E-cadherin and β-catenin were present in both the lateral and basal plasma membrane domains of CD cells. Immunoelectron microscopy confirmed that β-catenin was localized both to the lateral and the basal plasma membrane. The basal localization of both proteins was absent from a fraction of mainly principal cells after 10 and 15 days of Li-treatment. After 4 weeks of Li-treatment few to no cells were absent of E-cadherin and β-catenin at the basal plasma membrane. After 12 and 19 days of recovery some cells exhibited an absence of basal localization of both proteins. Thus, the observed localizational changes of E-cadherin and β-catenin appear before the cellular remodeling during both development and recovery from Li-NDI.

Abstract 408: Hyperglycemia Increases the Prorenin Receptor in the Plasma Membrane of Collecting Duct Cells in Rats with Type 1 Diabetes Mellitus

In type 1 diabetes mellitus (T1DM) there is increased prorenin secretion by the principal cells of the collecting duct. Binding of prorenin to prorenin receptor (PRR) on intercalated cells increases its catalytic activity, increases local angiotensin (Ang) II formation, and stimulates intracellular MAPK signaling responsible for inflammation and tissue fibrosis. Thus, changes in the amount of membrane bound PRR may be a key factor in stimulating these pathways. However, it has not been established that activation of PRR in the collecting duct contributes to increased intrarenal Ang II and tubulointerstitial inflammation via stimulation of inflammatory pathways including transforming growth factor-beta (TGF-β). This study tested the hypothesis that hyperglycemia increases the PRR abundance at the plasma membrane (PM) in the collecting duct cells, thus allowing greater capability to be activated by locally produced prorenin. Streptozotocin (STZ; 60 mg/kg; ip single dose) was used to induce T1DM in Sprague-Dawley rats (N=10) and compared to control rats (N=8). After 7-days induction, STZ-rats showed plasma glucose levels of 428±13 vs. 138±9 mg/dL and insulin of 0.05±0.02 vs. 2.4±0.6 ng/mL, compared to control. Although PRR transcript in the renal medulla were not different between groups; PRR localized predominantly on the apical aspects of collecting duct cells in STZ-induced rats; while in controls it was primarily found intracellularlly. These changes were accompanied by greater levels of active renin and Ang II in the urine and increased TGF-β mRNA levels in the renal medulla of STZ-rats (Renin: 186± 34 vs. 6± 3 ng Ang I/mL/h; P<0.01; Ang II: 884± 147 vs. 42± 14 fmol/h; P<0.05; TGF-β: 1.22 ± 0.06 vs. 0.97 ± 0.03 mRNA ratio; P<0.01). To further assess if hyperglycemia induced in vitro PRR trafficking alterations, collecting duct M-1 cells were treated with normal glucose (NG; 1mM glucose + 1 mM mannitol) and high glucose (HG; 4mM) for 5, 60, and 360 min. PRR protein levels were higher in the PM fractions in cells treated with HG, compared to cells treated with NG. Thus, hyperglycemia increases PRR abundance in the PM of the collecting duct and stimulates TGF-β synthesis in the renal medulla which may underlie the development of tubulointerstitial inflammation.

Relative CO2/NH3 Permeabilities of Human RhAG, RhBG and RhCG

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (DpHS) caused by exposing the same oocyte to 5 % CO₂/33 mM HCO₃⁻ (an increase) or 0.5 mM NH₃/NH₄⁺ (a decrease). Subtracting the respective values for day-matched, H₂O-injected control oocytes yielded channel-specific values (*). (ΔpH*(S))(CO₂) and (-ΔpH*(S))(NH₃) were each significantly >0 for all channels, indicating that RhBG and RhCG--like RhAG--can carry CO₂ and NH₃. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH₃ transport. However, surface biotinylation experiments indicate the mutants RhBG(D178N) and RhCG(D177N) have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG--like RhAG--have significant CO₂ permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH₃ permeability. However, as evidenced by (ΔpH*(S))(CO₂)/ (-ΔpH*(S))(NH₃) values, we could not distinguish among the CO₂/ NH₃ permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH₃ but also CO₂ across the membranes of CD cells.