H(+)V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways

2000 ◽  
Vol 203 (1) ◽  
pp. 137-145 ◽  
Author(s):  
D. Brown ◽  
S. Breton
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Vas Deferens ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
A Cells ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Many vertebrate transporting epithelia contain characteristic ‘mitochondria-rich’ cells that express high levels of a vacuolar proton-pumping ATPase (H(+)V-ATPase) on their plasma membrane and on intracellular vesicles. In the kidney cortex, A-cells and B-cells are involved in proton secretion and bicarbonate secretion, respectively, in the distal nephron and collecting duct. A-cells have an H(+)V-ATPase on their apical plasma membrane and on intracellular vesicles, whereas the cellular location of the H(+)V-ATPase can be apical, basolateral, bipolar or diffuse in B-cells. The rat epididymis and vas deferens also contain a distinct population of H(+)V-ATPase-rich epithelial cells. These cells are involved in generating a low luminal pH, which is involved in sperm maturation and in maintaining sperm in an immotile state during their passage through the epididymis and vas deferens. In both kidney and reproductive tract, H(+)V-ATPase-rich cells have a high rate of apical membrane recycling. H(+)V-ATPase molecules are transported between the cell surface and the cytoplasm in vesicles that have a well-defined ‘coat’ structure formed of the peripheral V(1) subunits of the H(+)V-ATPase. In addition, we propose that B-type intercalated cells have a transcytotic pathway that enables them to shuttle H(+)V-ATPase molecules from apical to basolateral plasma membrane domains. This hypothesis is supported by data showing that A-cells and B-cells have different intracellular trafficking pathways for LGP120, a lysosomal glycoprotein. LGP120 was found both on the basolateral plasma membrane and in lysosomes in B-cells, whereas no LGP120 was detectable in the plasma membrane of A-cells. We propose that the ‘polarity reversal’ of the H(+)V-ATPase in B-intercalated cells is mediated by a physiologically regulated transcytotic pathway that may be similar to that existing in some other cell types.

scholarly journals Immunoelectron Microscopic Localization of the Electrogenic Na/HCO3 Cotransporter in Rat and Ambystoma Kidney

2000 ◽  
Vol 11 (12) ◽  
pp. 2179-2189
Author(s):  
ARVID B. MAUNSBACH ◽  
HENRIK VORUM ◽  
TAE-HWAN KWON ◽  
SØREN NIELSEN ◽  
BRIAN SIMONSEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Rat Kidney Cortex ◽  
C Terminus ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Abstract. Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

Activation of acid-secreting intercalated cells in rabbit collecting duct with ammonium chloride loading

1994 ◽  
Vol 266 (4) ◽  
pp. F633-F645 ◽  
Author(s):  
J. W. Verlander ◽  
K. M. Madsen ◽  
J. K. Cannon ◽  
C. C. Tisher
Keyword(s):  
Plasma Membrane ◽  
Morphometric Analysis ◽  
Collecting Duct ◽  
Band 3 ◽  
Apical Plasma Membrane ◽  
Multivesicular Bodies ◽  
Intercalated Cells ◽  
Cytoplasmic Vesicles ◽  
Basolateral Plasma Membrane ◽  
Acid Loading

In normal rabbit, immunolabeling of intercalated cells in the outer medullary collecting duct (OMCD) demonstrates band 3-like protein in the basolateral plasma membrane (15) and H(+)-adenosinetriphosphatase (H(+)-ATPase) in the apical plasma membrane and cytoplasmic vesicles (30). However, in type A intercalated cells in the cortical collecting duct (CCD), band 3-like protein is located primarily in multivesicular bodies and cytoplasmic vesicles (15), whereas H(+)-ATPase is present in cytoplasmic vesicles only in most intercalated cells (30). In this study, we observed the effect of chronic acid loading on immunolocalization of these transporters in the collecting duct. Adult New Zealand White rabbits received either normal tap water (controls) or 75 mM NH4Cl for 12 days plus eight daily gavages of 2-6 meq NH4Cl/kg body wt. At time of death, mean urine pH of acid-loaded animals was 5.96 (SD = 0.69), vs. 8.47 (SD = 0.07) in controls. Kidneys were fixed by in vivo perfusion and processed for light and electron microscopic immunoperoxidase localization of band 3-like protein and immunogold localization of H(+)-ATPase. In controls, band 3-like protein was largely confined to multivesicular bodies in the majority of positive-staining intercalated cells in the CCD and to the basolateral plasma membrane of intercalated cells in the OMCD. In acid-loaded rabbits, band 3 protein-positive intercalated cells in the inner CCD and the in the outer stripe of the OMCD (OMCDo) were strikingly stellate in form. Basolateral plasma membrane label was intensified, while the number of labeled multivesicular bodies was diminished. Morphometric analysis demonstrated an increase in the amount of basolateral plasma membrane in these intercalated cells. In control rabbits, H(+)-ATPase immunoreactivity in intercalated cells in the CCD was located predominantly over cytoplasmic vesicles. A minority of intercalated cells exhibited basolateral plasma membrane label, and only an occasional cell displayed apical plasma membrane label. In acid-loaded rabbits, H(+)-ATPase immunoreactivity was enhanced along the apical plasma membrane of intercalated cells in the inner CCD, and morphometric analysis demonstrated increased apical plasma membrane in band 3-positive intercalated cells in this segment. These results suggest that rabbits respond to acid loading via enhancement of both electrogenic proton secretion and Cl-/HCO3- exchange in intercalated cells in the inner CCD and the OMCDo.

scholarly journals Intercalated Cell Subtypes in Connecting Tubule and Cortical Collecting Duct of Rat and Mouse

1999 ◽  
Vol 10 (1) ◽  
pp. 1-12 ◽  
Author(s):  
JIN KIM ◽  
YOUNG-HEE KIM ◽  
JUNG-HO CHA ◽  
C. CRAIG TISHER ◽  
KIRSTEN M. MADSEN
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Collecting Duct ◽  
Type A ◽  
Band 3 ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Type B ◽  
Intercalated Cell ◽  
Basolateral Plasma Membrane

Abstract. At least two populations of intercalated cells, type A and type B, exist in the connecting tubule (CNT), initial collecting tubule (ICT), and cortical collecting duct (CCD). Type A intercalated cells secrete protons via an apical H+ - ATPase and reabsorb bicarbonate by a band 3-like Cl-/HCO3- exchanger, AE1, located in the basolateral plasma membrane. Type B intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger that is distinct from AE1 and remains to be identified. They express H+ -ATPase in the basolateral plasma membrane and in vesicles throughout the cytoplasm. A third type of intercalated cell with apical H+ -ATPase, but no AE1, has been described in the CNT and CCD of both rat and mouse. The prevalence of the third cell type is not known. The aim of this study was to characterize and quantify intercalated cell subtypes, including the newly described third non A-non B cell, in the CNT, ICT, and CCD of the rat and mouse. A triple immunolabeling procedure was developed in which antibodies to H+ -ATPase and band 3 protein were used to identify subpopulations of intercalated cells, and segment-specific antibodies were used to identify distal tubule and collecting duct segments. In both rat and mouse, intercalated cells constituted approximately 40% of the cells in the CNT, ICT, and CCD. Type A, type B, and non A-non B intercalated cells were observed in all of the three segments, with type A cells being the most prevalent in both species. In the mouse, however, non A-non B cells constituted more than half of the intercalated cells in the CNT, 39% in the ICT, and 22% in the CCD, compared with 14, 7, and 5%, respectively, in the rat. In contrast, type B intercalated cells accounted for only 8 to 16% of the intercalated cells in the three segments in the mouse compared with 26 to 39% in the rat. It is concluded that striking differences exist in the prevalence and distribution of the different types of intercalated cells in the CNT, ICT, and CCD of rat and mouse. In the rat, the non A-non B cells are fairly rare, whereas in the mouse, they constitute a major fraction of the intercalated cells, primarily at the expense of the type B intercalated cells.

scholarly journals Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

1996 ◽  
Vol 7 (12) ◽  
pp. 2533-2542 ◽  
Author(s):  
S M Ginns ◽  
M A Knepper ◽  
C A Ecelbarger ◽  
J Terris ◽  
X He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Specific Affinity ◽  
Basolateral Plasma Membrane ◽  
Medullary Collecting Duct ◽  
Antipeptide Antibody

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

scholarly journals Ultrastructural localization of H+ATPase in rabbit cortical collecting duct.

1994 ◽  
Vol 4 (8) ◽  
pp. 1546-1557 ◽  
Author(s):  
J W Verlander ◽  
K M Madsen ◽  
D K Stone ◽  
C C Tisher
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rabbit Kidney ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Thin Sections ◽  
Cytoplasmic Vesicles ◽  
Basolateral Plasma Membrane

In contrast to results obtained in the rat kidney, studies of H+ATPase localization in the rabbit kidney have failed to demonstrate basolateral plasma membrane H+ATPase immunoreactivity in intercalated cells in the cortical collecting duct (CCD). Previous studies have relied on light microscopic immunofluorescence techniques, which have limited resolution. Therefore, the immunogold procedure was used to localize H+ATPase in rabbit collecting ducts at the ultrastructural level. Rabbit kidneys were preserved in vivo with periodate-lysine-paraformaldehyde or glutaraldehyde solutions, and samples of cortex were embedded in Lowicryl K4M. Thin sections were labeled for H+ATPase by the immunogold procedure with a rabbit polyclonal antibody against the 70-kd subunit of bovine brain H+ATPase. Three patterns of localization of H+ATPase were observed. The majority of intercalated cells in the CCD exhibited label over cytoplasmic vesicles only. In these cells, no label was associated with either the apical or basolateral plasma membranes. In a second group of cells, label for H+ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout the cell. Rarely, intercalated cells with H+ATPase label along the apical plasma membrane and over the apical cytoplasmic vesicles were observed in the CCD. In the initial collecting tubule and connecting segment, intercalated cells with either pronounced apical or basolateral plasma membrane label prevailed, whereas few cells exhibited label restricted to the cytoplasmic vesicles. In summary, in the rabbit CCD, three patterns of H+ATPase distribution exist in intercalated cells, two of which conform to published models of type A and type B intercalated cells.

Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney

2002 ◽  
Vol 283 (4) ◽  
pp. F744-F754 ◽  
Author(s):  
Young-Hee Kim ◽  
Tae-Hwan Kwon ◽  
Sebastian Frische ◽  
Jin Kim ◽  
C. Craig Tisher ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Mouse Kidney ◽  
Laser Scanning Microscopy ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Type B ◽  
Connecting Tubule ◽  
Intracellular Vesicles

Recent studies have demonstrated that a novel anion exchanger, pendrin, is expressed in the apical domain of type B intercalated cells in the mammalian collecting duct. The purpose of this study was 1) to determine the expression and distribution of pendrin along the collecting duct and connecting tubule of mouse and rat kidney and establish whether pendrin is expressed in the non-A-non-B intercalated cells and 2) to determine the intracellular localization of pendrin in the different populations of intercalated cells by immunoelectron microscopy. A peptide-derived affinity-purified antibody was generated that specifically recognized pendrin in immunoblots of rat and mouse kidney. Immunohistochemistry and confocal laser scanning microscopy demonstrated the presence of pendrin in apical domains of all type B intercalated cells in mouse and rat connecting tubule and collecting duct. In addition, strong pendrin immunostaining was observed in non-A-non-B intercalated cells. There was no labeling of type A intercalated cells. Immunoelectron microscopy demonstrated that pendrin was located in the apical plasma membrane and intracellular vesicles of both type B intercalated cells and non-A-non-B cells; the latter was identified by the presence of H+-ATPase in the apical plasma membrane. The results of this study demonstrate that both pendrin and H+-ATPase are expressed in the apical plasma membrane of non-A-non-B intercalated cells, suggesting that these cells are capable of both HCO[Formula: see text] and proton secretion. Furthermore, the presence of pendrin in both the apical plasma membrane and the apical intracellular vesicles of type B and non-A-non-B intercalated cells suggests that HCO[Formula: see text] secretion may be regulated by trafficking of pendrin between the two membrane compartments.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

scholarly journals Maintained ENaC trafficking in aldosterone-infused rats during mineralocorticoid and glucocorticoid receptor blockade

2007 ◽  
Vol 292 (1) ◽  
pp. F382-F394 ◽  
Author(s):  
Jakob Nielsen ◽  
Tae-Hwan Kwon ◽  
Jørgen Frøkiær ◽  
Mark A. Knepper ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Glucocorticoid Receptor ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Protein Abundance ◽  
Cortical Collecting Duct ◽  
Plasma Aldosterone Concentration ◽  
Mediated Effects ◽  
Plasma Membrane Domain

Aldosterone induces redistribution of epithelial sodium channel (ENaC) to the apical plasma membrane from intracellular vesicles in renal connecting tubule (CNT) and cortical collecting duct (CCD). The role of the classical mineralocorticoid receptor (MR) in ENaC trafficking is still debated. We examined whether the MR antagonist spironolactone affects ENaC regulation in the kidney cortex of aldosterone-infused rats. Aldosterone infusion for 7 days resulted in a plasma aldosterone concentration in the high physiological range (3 to 4 nM). Aldosterone infusion decreased plasma K+ concentration compared with untreated control rats. Cotreatment with spironolactone completely blocked the aldosterone-induced decrease in plasma K+. Immunoblotting and immunohistochemistry showed increased protein abundance of Na-K-ATPase α1-subunit and NCC in the kidney cortex, in response to aldosterone infusion that was blocked by spironolactone. In contrast, aldosterone-induced redistribution of ENaC subunits from the cytoplasm to the apical plasma membrane domain in CNT and CCD was unaffected by spironolactone. Immunoblotting of αENaC showed increased protein abundance in aldosterone-infused rats that was not blocked by spironolactone treatment. To exclude possible glucocorticoid receptor (GR)-mediated effects of aldosterone, we treated aldosterone-infused rats with both spironolactone and the GR antagonist RU486. Combined MR and GR blockade prevented neither ENaC trafficking nor the upregulation of αENaC protein abundance in aldosterone-infused rats. We provide new evidence for ENaC trafficking occurring independent of MR and GR activation in aldosterone-infused rats.

Membrane recycling and epithelial cell function

1989 ◽  
Vol 256 (1) ◽  
pp. F1-F12 ◽  
Author(s):  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Function ◽  
Collecting Duct ◽  
Water Channel ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Membrane Recycling ◽  
Membrane Composition

The plasma membrane composition of virtually all eucaryotic cells is established, maintained, and modified by the process of membrane recycling. Specific plasma membrane components are inserted by exocytosis of transport vesicles, and are removed by endocytosis of segments of the membrane in which particular proteins are concentrated. In the kidney collecting duct, vasopressin induces the cycling of vesicles that are thought to carry water channels to and from the apical plasma membrane of principal cells, thus modulating the water permeability of this membrane. In the intercalated cells of the collecting duct, hydrogen ion secretion is controlled by the recycling of vesicles carrying proton pumps to and from the plasma membrane. In both cell types, "coated" carrier vesicles are involved, but whereas clathrin-coated vesicles participate in water channel recycling, the vesicles in intercalated cells are coated with the cytoplasmic domains of proton pumps. Following a brief outline of membrane recycling in general, this review summarizes previous data on membrane recycling in the collecting duct and related transporting epithelia and discusses some selected points relating to the role of membrane recycling and cell-specific function in the collecting duct.

scholarly journals Compensatory increase in AQP2, p-AQP2, and AQP3 expression in rats with diabetes mellitus

2001 ◽  
Vol 280 (4) ◽  
pp. F715-F726 ◽  
Author(s):  
Lene N. Nejsum ◽  
Tae-Hwan Kwon ◽  
David Marples ◽  
Allan Flyvbjerg ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Plasma Membrane ◽  
Sodium Excretion ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Urinary Sodium Excretion ◽  
Apical Plasma Membrane ◽  
Compensatory Increase ◽  
Intracellular Vesicles

Diabetes mellitus (DM) is associated with osmotic diuresis and natriuresis. At day 15, rats with DM induced by streptozotocin ( n = 13) had severe hyperglycemia (27.1 ± 0.4 vs. 4.7 ± 0.1 mM in controls) and had a fivefold increase in water intake (123 ± 5 vs. 25 ± 2 ml/day) and urine output. Semiquantitative immunoblotting revealed a significant increase in inner medullary AQP2 (201 ± 12% of control rats, P < 0.05) and phosphorylated (Ser256) AQP2 (p-AQP2) abundance (299 ± 32%) in DM rats. Also, the abundance of inner medullary AQP3 was markedly increased to 171 ± 19% of control levels (100 ± 4%, n = 7, P < 0.05). In contrast, the abundance of whole kidney AQP1 (90 ± 3%) and inner medullary AQP4 (121 ± 16%) was unchanged in rats with DM. Immunoelectron microscopy further revealed an increased labeling of AQP2 in the apical plasma membrane of collecting duct principal cells (with less labeling in the intracellular vesicles) of DM rats, indicating enhanced trafficking of AQP2 to the apical plasma membrane. There was a marked increase in urinary sodium excretion in DM. Only Na+/H+ exchanger NHE3 was downregulated (67 ± 10 vs. 100 ± 11%) whereas there were no significant changes in abundance of type 2 Na-phosphate cotransporter (128 ± 6 vs. 100 ± 10%); the Na-K-2Cl cotransporter (125 ± 19 vs. 100 ± 10%); the thiazide-sensitive Na-Cl cotransporter (121 ± 9 vs. 100 ± 10%); the α1-subunit of the Na-K-ATPase (106 ± 7 vs. 100 ± 5%); and the proximal tubule Na-HCO3 cotransporter (98 ± 16 vs. 100 ± 7%). In conclusion, DM rats had an increased AQP2, p-AQP2, and AQP3 abundance as well as high AQP2 labeling of the apical plasma membrane, which is likely to represent a vasopressin-mediated compensatory increase in response to the severe polyuria. In contrast, there were no major changes in the abundance of AQP1, AQP4, and several major proximal and distal tubule Na+ transporters except NHE3 downregulation, which may participate in the increased sodium excretion.

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