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. The frequency of active caspase-3-positive cells was unchanged as was also the measured activity of caspase-3. Immunoelectron microscopy showed that active caspase-3 in the positive cells was localized in cytoplasmic bodies 0.5-1 µm in diameter but not in other structures. Apoptotic bodies with the nuclei were only rarely observed following acute hypertonicity for 24 to 72 h. Following prolonged hypertonic challenges, some cells showed condensation of the chromatin but still few apoptotic bodies. Gradual hypertonicity to 900 mOsm/kgH2O led to a decrease of microvilli, dilated cisternae of the endoplasmic reticulum (ER), increased abundance of free ribosomes and longitudinal mitochondrial cristae. Virus particles were present inside and outside the cells in all experimental conditions and appeared unrelated to the apoptotic process. The results suggest that cultured IMCD3 cells are resistant to short hypertonic challenge or gradual adaptation to moderate hypertonicity and only rarely exhibit more ultrastructural apoptotic changes than control cells. The presence of caspase-3-containing bodies is a novel finding, and we suggest that they arise from the ER and are involved in the apoptotic signaling system.

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. Male Sprague-Dawley rats were infused with a dose of captopril (12 microg/min for 20 min) that increased PT flow rate approximately 20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng x kg(-1) x min(-1) for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H(+)-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.

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. Exposure of the challenged cells to BFA prevented the subsequent incorporation of gamma into the basolateral plasma membrane. The gamma subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of gamma in the basolateral membrane. The alpha1 subunit was only marginally influenced by BFA. The results demonstrate that the gamma subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the gamma and alpha subunits do not traffic together to the plasma membrane, and that the gamma and alpha subunit have different turnover rates during these experimental conditions.

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. Six-week-old Sprague-Dawley rats were fed a normal (NS) 0.4% NaCl diet or a HS 4% NaCl diet for 3 wk or overnight. Kidneys excised from anesthetized rats were fractionated on density gradients or analyzed by microscopy; transporters and associated regulators were detected with specific antibodies. Three-week HS doubled Na+/H+ exchanger (NHE)3 phosphorylation at serine 552 and provoked a redistribution of NHE3, dipeptidyl peptidase IV (DPPIV), myosin VI, Na+-Pi cotransporter (NaPi)-2, ANG II type 2 receptor (AT2R), aminopeptidase N (APN), Na+-K+-2Cl- cotransporter (NKCC2), epithelial Na+ channel (ENaC) beta-subunit, and Na+-K+-ATPase (NKA) alpha1- and beta1-subunits from low-density plasma membrane-enriched fractions to higher-density intracellular membrane-enriched fractions. NHE3, myosin VI, and AT2R retraction to the base of the microvilli (MV) during HS was evident by confocal microscopy. HS did not change abundance of NHE3, NKCC, or NKA alpha1- or beta1-subunits but increased ENaC-beta in high-density intracellular enriched membranes. Responses to HS were fully apparent after just 18 h. We propose that retraction of NHE3 to the base of the MV, driven by myosin VI and NHE3 phosphorylation and accompanied by redistribution of the NHE3 regulator DPPIV, contributes to a decrease in proximal tubule Na+ reabsorption during HS and that redistribution of transporters out of low-density plasma membrane-enriched fractions in the thick ascending limb of the loop of Henle and distal nephron may also contribute to the homeostatic natriuretic response to 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%). Evaluation of the polyol pathway for sorbitol production revealed a reduction in sorbitol dehydrogenase and an increase in aldose reductase mRNA in adapted cells. Proteome analysis revealed increased expression of six glycolytic proteins, including malic enzyme and pyruvate carboxylase, indicating the activation of the pyruvate shunt and changes in glucose metabolism. This study showed that the observed reduction in cell replication could possibly reflect a redirection of cellular energy from cell growth and replication to maintenance of intracellular ion levels in chronically adapted cells. CONCLUSION: The combined metabonomic and proteomic analysis was shown to be a very helpful tool for the analysis of the effects caused by chronic adaptation to hypertonicity. It made it possible to better evaluate the importance of certain changes that occur in the process of adaptation.

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).min(-1) along with 12 microg/min captopril) for 20 min in male Sprague-Dawley rats (250-350 g). By immuno-electron microscopy using an anti-NCC (D. Ellison) 71.5 +/- SD 4.9% of the NCC gold labeling was associated with the APM in control, sham operated, and infused rats, while captopril infusion reduced NCC in APM to 54.9 +/- 6.9% (P < 0.001) and markedly increased immunogold labeling of SCV. Subsequent infusion of ANG II with captopril restored NCC immunogold labeling of APM to 72.4 +/- 4.2%, that is, 20% of the total NCC trafficked between APM and SCV. Likewise, on density gradients of cortex, captopril provoked redistribution of 27.3% of total NCC from low-density APM-enriched membranes to higher-density membranes and ANG II+captopril restored 20.3% of the NCC to APM-enriched fractions. Redistribution occurred independent of a change in NCC total abundance. In conclusion, this study demonstrates that ACE inhibition provokes acute trafficking of NCC out of the plasma membrane, which likely decreases DCT Na+ reabsorption, while ANG II provokes rapid trafficking of NCC from stores in subapical vesicles to the plasma membrane, which likely increases DCT Na+ reabsorption.

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. Western blot analysis confirmed increased expression with increased tonicity, both acute and chronic. S100A4 protein expression was further confirmed by immunocytochemical analysis. Cells grown in isotonic conditions showed complete absence of immunostaining, whereas chronically adapted IMCD3 cells had uniform cytoplasmic localization. The protein is also regulated in vivo as in mouse kidney tissues S100A4 expression was many -fold greater in the papilla as compared with the cortex and increased further in the papilla upon 36 h of thirsting. Increased expression of S100A4 was also observed in the medulla and papilla, but not the cortex of a human kidney. Data from Affymetrix gene chip analysis and quantitative PCR also revealed increased S100A4 message in IMCD3 cells adapted to hypertonicity. The initial expression of message increased at 8-10 h following exposure to acute sublethal hypertonic stress (550 mosmol/kg H(2)O). Protein and message half-life in IMCD3 cells were 85.5 and 6.8 h, respectively. Increasing medium tonicity with NaCl, sucrose, mannitol, and choline chloride stimulated S100A4 expression, whereas urea did not. Silencing of S100A4 expression using a stable siRNA vector (pSM2; Open Biosystems) resulted in a 48-h delay in adaptation of IMCD3 cells under sublethal osmotic stress, suggesting S100A4 is involved in the osmoadaptive response. In summary, we describe the heretofore unrecognized up-regulation of a small calcium-binding protein, both in vitro and in vivo, whose absence profoundly delays osmoadaptation and slows cellular growth under hypertonic conditions.

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. The results showed that antibodies against the gamma-subunit COOH terminus and splice variant gamma(a), but not splice variant gamma(b), labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the gamma-subunit COOH terminus, splice variant gamma(a), and CHIF. Splice variant gamma(b) was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase alpha(1)-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of gamma and CHIF in the renal medulla and show a new gamma variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the gamma-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion.

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., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

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. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase gamma-subunit or the gamma(b) splice variant. Cells grown in 300 mosmol/kgH(2)O showed no immunoreactivity for the gamma-subunit, whereas cells adapted to 600 or 900 mosmol/kgH(2)O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH(2)O with sodium chloride or choline chloride showed incorporation of gamma into plasma membrane 12 h after osmotic challenge and distinct membrane staining in approximately 40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH(2)O by addition of sodium acetate did not result in expression of the gamma-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase gamma-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the gamma-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.

Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus.

BACKGROUND/AIM: In a rat model, lithium treatment is associated with polyuria and severe downregulation of aquaporin-2 (AQP2) protein in the inner medulla (IM) or in the whole kidney. However, it is not known (1) to what extent this downregulation occurs at the mRNA level; (2) whether the main sodium transporter of the nephron, Na,K-ATPase, is regulated in parallel at the mRNA level, and (3) whether lithium treatment induces zonal or segmental differences in AQP2 and Na,K-ATPase mRNA levels. METHOD: We examined the changes in mRNA expression levels for AQP2 and Na,K-ATPase in kidney cortex, inner stripe of the outer medulla (ISOM), and IM of rats treated with lithium orally using semiquantitative Northern blot analyses and in situ hybridization at the light and electron microscopic levels. RESULTS: The AQP2 mRNA levels decreased significantly (p < 0.01) in lithium-treated rats to 37 +/- 4% in the cortex, to 17 +/- 4% in the ISOM, and to 23 +/- 5% in the IM, while the Na,K-ATPase mRNA levels were not altered in the cortex, but were significantly (p < 0.05) altered in the ISOM (144 +/- 15% after 10 days, but 68 +/- 4% after 4 weeks) and in the IM (63 +/- 8% after 10 days, but normalized after 4 weeks). In situ hybridization showed reduced levels of AQP2 mRNA in all zones of the kidney, but the Na,K-ATPase mRNA expressions were slightly decreased only in IM collecting ducts. At the ultrastructural level, principal cells in the IM collecting ducts showed slight hypertrophy, but no cell damage after 4 weeks of lithium treatment. The results demonstrate substantial downregulation of AQP2 at the mRNA level throughout the collecting duct in experimental lithium-induced nephrogenic dabetes insipidus and moderately decreased Na,K-ATPase mRNA levels in the ISOM and in the IM. CONCLUSION: The results suggest that decreased mRNA expressions of AQP2 and Na,K-ATPase contribute to the development of lithium-induced nephrogenic diabetes insipidus.

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. In the initial quarter of the IMCD (next to the outer medulla), antibodies against the C-terminal of gamma as well as splice variant gammaa labeled the basolateral surface of intercalated cells (ICs), while principal cells (PCs) remained unlabeled. In the middle segment of the IMCD, all PCs exhibited distinct basolateral staining for the gammaC-terminal as well as gammaa and CHIF. Immunoelectron microscopy showed that the gammaC-terminal and CHIF were associated with the inner leaflet of the basolateral plasma membrane in the labeled cells. Immunoblotting demonstrated the presence of both the gammaC-terminal and gammaa in inner medullary tissue. However, splice variant gammab was not detected in inner medulla by immunocytochemistry or immunoblotting. The present observations demonstrate that the Na,K-ATPase gamma subunit and CHIF are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of the Na,K-ATPase activity in the IMCD.

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. Microscope analyses of leaves grown under optimized conditions revealed significant differences both in cell structure and size when the transgenic VHb lines were compared with the control lines. VHb lines displayed a higher relative volume of mitochondria and a significantly enhanced accumulation of starch in chloroplasts, all of which pointed towards changes in cellular energy production. Under elevated UV-B illumination, the differences between VHb lines became evident. Some specific VHb lines had elevated levels of total flavonoids, individual quercetin, kaempferol- and myricetin-derivatives relative to controls and other transgenic lines. This observation may reflect the availability of extra energy resources for secondary metabolite production and possibly an enhanced protection ability of these transgenic lines against UV-B illumination. Thus, all these findings point to changes in the energy metabolism of VHb lines. In the cultivation conditions tested this observation did not, however, result in a general improvement of elongation growth.

Genes encoding chitinase-antifreeze proteins are regulated by cold and expressed by all cell types in winter rye shoots.

One group of antifreeze proteins (AFPs) is composed of two chitinases that accumulate in the apoplast of winter rye leaves during cold acclimation. In this study, the 28- and 35-kDa chitinase-AFPs were localized in nonacclimated and cold-acclimated rye leaves by immunoelectron microscopy with an antiserum produced against the purified winter rye 35-kDa chitinase-AFP. In cold-acclimated winter rye leaves, labelled chitinase-AFPs were abundant in the walls of epidermal, parenchymal sheath and mesophyll cells and xylem vessels, while less label was present in walls of vascular parenchyma cells. In contrast, chitinase labelling was essentially absent in the nonacclimated cells except in xylem vessels. As shown by RNA blotting, the transcripts of chitinase-AFPs accumulated to a high level in rye leaves during cold acclimation, to a lesser extent in crowns and were not detectable in roots. mRNA transcripts of the 28-kDa chitinase-AFP were localized in rye leaves by in situ hybridization. The chitinase-AFP transcripts were found in the same cell types as the protein itself. We conclude that all metabolically active cell types in cold-acclimated winter rye leaves and crowns are able to synthesize chitinase-AFPs and secrete them into adjacent cell walls, where they may interact with ice to delay its propagation through the plant and modify its growth.