Response of IMCD3 cells to hypertonic challenges as analyzed by electron microscopy.

This work defines the ultrastructural responses of immortalized cells from the inner medullary collecting duct cells (IMCD3 cells) to hypertonic challenges. The cultured cells were either acutely exposed to hypertonic medium (550 mOsm/kgH₂O) for 24-72 h or gradually adapted to 600 or 900 mOsm/kgH₂O media with sodium chloride. After short (24 h) hypertonic challenges, there was an expansion of the Golgi apparatus with distinct expression of the γ subunit of Na,K-ATPase.

Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli.

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na(+)/H(+) exchanger isoform 3 (NHE3), Na(+)/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl.

Expression and trafficking of the gamma subunit of Na,K-ATPase in hypertonically challenged IMCD3 cells.

The gamma subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of gamma to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the gamma subunit and 58K Golgi protein in the cytoplasm, but no co-localization of alpha1 and Golgi protein.

Effects of dietary salt on renal Na+ transporter subcellular distribution, abundance, and phosphorylation status.

During high-salt (HS) diet the kidney increases urinary Na+ and volume excretion to match intake. We recently reported that HS provokes a redistribution of distal convoluted tubule Na+-Cl- cotransporter (NCC) from apical to subapical vesicles and decreases NCC abundance. This study aimed to test the hypothesis that the other renal Na+ transporters' abundance and or subcellular distribution is decreased by HS diet.

A metabonomic and proteomic analysis of changes in IMCD3 cells chronically adapted to hypertonicity.

Background: The genomic response to adaptation of IMCD3 cells to hypertonicity results in both upregulation and downregulation of a variety of genes.

Method: The present study was undertaken to assess the metabonomic and proteomic response of IMCD3 cells that have been chronically adapted to hypertonicity (600 and 900 mosm/kg H(2)O) as compared to cells under isotonic conditions.

Results: Adaptation of IMCD3 cells to hypertonic conditions resulted in a change of a wide range of organic osmolytes, including sorbitol (+8,291%), betaine (+1,099%), myo-inositol (+669%), taurine (+113%) and glycerophosphorylcholine (+61%).

ANG II provokes acute trafficking of distal tubule Na+-Cl(-) cotransporter to apical membrane.

The distal convoluted tubule (DCT) Na+-Cl(-) cotransporter (NCC), the target of thiazide diuretics, is responsible for the reabsorption of 5-10% of filtered NaCl. The aim of this study was to test the hypothesis that acute infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril (at 12 microg/min) for 20 min provokes trafficking of NCC from apical plasma membranes (APM) to subapical cytoplasmic vesicles (SCV), which is reversed by acute ANG II infusion (ANG II at 20 ng.kg(-1).

Expression of the calcium-binding protein S100A4 is markedly up-regulated by osmotic stress and is involved in the renal osmoadaptive response.

Proteomic analysis of Inner Medullary Collecting Duct (IMCD3) cells adapted to increasing levels of tonicity (300, 600, and 900 mosmol/kg H(2)O) by two-dimensional difference gel electrophoresis and mass spectrometry revealed several proteins as yet unknown to be up-regulated in response to hypertonic stress. Of these proteins, one of the most robustly up-regulated (22-fold) was S100A4. The identity of the protein was verified by high pressure liquid chromatography-mass spectrometry.

Expression of Vitreoscilla haemoglobin in hybrid aspen (Populus tremula x tremuloides).

We describe the first ever expression of Vitreoscilla haemoglobin (VHb) in an economically important boreal woody plant hybrid aspen (Populus tremula x tremuloides). VHb has mainly been expressed in biotechnologically important unicellular organisms of both prokaryotic and eukaryotic origin. VHb expression, in this study, was analysed under different greenhouse cultivation conditions and under elevated UV-B illumination.

Locations, abundances, and possible functions of FXYD ion transport regulators in rat renal medulla.

The gamma-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of gamma and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting.

Interaction with the Na,K-ATPase and tissue distribution of FXYD5 (related to ion channel).

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y.

The gamma-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity.

Hypertonicity mediated by chloride upregulates the expression of the gamma-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428-6433, 2003). The purpose of this study was to examine the cellular locations and the time course of gamma-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts.

Immunocytochemical localization of Na,K-ATPase gamma subunit and CHIF in inner medulla of rat kidney.

The gamma subunit of Na,K-ATPase and CHIF both belong to the FXYD single-membrane-spanning protein family and have been suggested to have regulatory functions in kidney tubules. CHIF is known to be present in the collecting duct, and gamma has been demonstrated in several segments of the rat kidney tubule, but never clearly in the inner medullary collecting duct (IMCD). Here, we demonstrate the cellular and subcellular localization of the gamma subunit and CHIF in the IMCD in inner medulla by using Western blotting, laser-scanning confocal immunofluorescence, and immunoelectron microscopy.