[Clinicopathologic features with collecting duct carcinoma of kidney: report of 10 cases].

Objective: To study the pathological features, immunophenotypes, differential diagnoses and prognostic parameters of collecting duct carcinoma of the kidney (CDC). Methods: Clinical imaging, histopathology, immunohistochemistry, and survival data of 10 patients at First Affiliated Hospital of Nanjing Medical University from January 2009 to August 2017 were retrospectively analyzed along with a review of literatures. Results: The clinical symptoms of CDC were not specific, and image examinations showed space-occupying mass lesions. Tumors were mainly located in renal medulla with grey and firm cut face and the presence of focal hemorrhage and necrosis. Microscopically, there were predominant tubular or tubular-papillary structures with associated focal sarcomatoid areas, desmoplastic stromal reaction and lymphoplasmacytic cells infiltration. Tumor cells had marked cytological atypia with high grade nuclei, conspicuous nucleolus and numerous mitoses. Immunohistochemically, tumor cells were strongly positive for CK19, E-cadherin, vimentin, HCK, CK7 and PAX8. The main treatment was radical nephrectomy in the patients. Seven cases died of CDC with median survival of 10 months. Conclusions: CDC is a rare, highly aggressive malignancy of kidney with poor prognosis. Definitive diagnosis should be made by histology and immunohistochemistry. Differential diagnoses include papillary renal cell carcinoma(type Ⅱ), renal medullary carcinoma, infiltrating high grade urothelial carcinoma, renal pelvis adenocarcinoma and metastatic adenocarcinomas.

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 µM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 µM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 µM), but not fluoxetine (100 µM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 µM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD.

P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

Recombinant human erythropoietin pretreatment attenuates acute renal tubular injury against ischemia-reperfusion by restoring transient receptor potential channel-6 expression and function in collecting ducts.

OBJECTIVE: Acute renal tubular injury is a serious complication in the postoperative period, which is associated with high mortality and increased ICU stay. We aimed to demonstrate the protective effect of rhEPO against acute tubular injury induced by ischemia-reperfusion and to explore the mechanism of canonical transient receptor potential channel-6. DESIGN: Randomized laboratory animal study. SETTINGS: Animal research laboratory. INTERVENTIONS: Male Sprague-Dawley rats were randomly divided into three groups: the sham group, the control group, and the rhEPO group. Experimental acute tubular injury was established in rats by bilateral renal arterial occlusion for 30 minutes followed by reperfusion. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained for cystatin-C and neutrophil gelatinase-associated lipocalin measurements by enzyme-linked immunosorbance assays. Seventy-two hours after reperfusion, urine samples were collected for osmolality and fractional excretion of sodium (%) assays on a chemistry analyzer. Kidneys were harvested at 24, 48, and 72 hours after reperfusion. Transient receptor potential channel-6, aquaporin-2, and Na,K-ATPase expression in collecting ducts were studied by immunofluorescence and Western blot. Coimmunoprecipitations were also performed to identify the possible signalplex relation between transient receptor potential channel-6 and aquaporin-2 or Na,K-ATPase channels. RhEPO pretreatment significantly inhibited serum cystatin-C (2 hr: 453 ± 64 µg/L vs 337 ± 28 µg/L, p < 0.01), serum neutrophil gelatinase-associated lipocalin (72 hr: 1,175 ± 107 ng/L vs 1,737 ± 402 ng/L, p < 0.05), and urinary fractional excretion of sodium (%) increase (0.9 ± 0.1 vs 2.2 ± 0.8, p < 0.05) and alleviated the decrease of urinary osmolality (1,293 ± 101 mosmol/kg H2O vs 767 ± 91 mosmol/kg H2O, p < 0.05) induced by ischemia-reperfusion injury. Meanwhile, recombinant human erythropoietin greatly improved the ischemia-reperfusion-induced attenuation of transient receptor potential channel-6 expression (48 hr: 42% ± 2% vs 67% ± 2% and 72 hr: 55% ± 2% vs 66% ± 2%), as well as aquaporin-2 and Na,K-ATPase expression in collecting ducts. Transient receptor potential channel-6 functionally interacted with Na,K-ATPase but not aquaporin-2. CONCLUSIONS: Recombinant human erythropoietin pretreatment at the dose of 5,000 IU/kg potently prevented ischemia-reperfusion-induced acute tubular injury, which might be partly attributed to the restoring the effect of transient receptor potential channel-6 expression and collecting duct function.

Urinary tract obstruction in the mouse: the kinetics of distal nephron injury.

Congenital urinary tract obstruction is the single most important cause of childhood chronic kidney disease. We have previously demonstrated that human and primate fetal obstruction impairs the development, differentiation, and maturation of the kidney. Research using postnatal rodent models has primarily focused upon the role of proximal tubular injury, with few reports of collecting duct system pathology or the suitability of the postnatal models for examining injury to the distal nephron. We have employed the mouse unilateral ureteric obstruction (UUO) model and examined time points ranging from 1 to 14 days of obstruction. Many of the key features of fetal collecting duct injury are replicated in the postnatal mouse model of obstruction. Obstruction causes a sixfold increase in myofibroblast accumulation, two- to threefold dilatation of tubules of the distal nephron, 65% reduction of principal cell aquaporin 2 expression, 75% reduction of collecting duct intercalated cell abundance, and disruption of E-cadherin- and ßcatenin-mediated collecting duct epithelial adhesion. Notably, these features are shared by the distal and connecting tubules. This work confirms that distal nephron pathology is a significant component of postnatal mouse UUO. We have highlighted the utility of this model for investigating collecting duct and distal tubule injury and for identifying the underlying mechanisms of the distal nephron's contribution to the repair and fibrosis.

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination.

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

Phosphorylation of aquaporin-2 regulates its water permeability.

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.

Proteomic analysis of lithium-induced nephrogenic diabetes insipidus: mechanisms for aquaporin 2 down-regulation and cellular proliferation.

Lithium is a commonly prescribed mood-stabilizing drug. However, chronic treatment with lithium induces numerous kidney-related side effects, such as dramatically reduced aquaporin 2 (AQP2) abundance, altered renal function, and structural changes. As a model system, inner medullary collecting ducts (IMCD) isolated from rats treated with lithium for either 1 or 2 weeks were subjected to differential 2D gel electrophoresis combined with mass spectrometry and bioinformatics analysis to identify (i) signaling pathways affected by lithium and (ii) unique candidate proteins for AQP2 regulation. After 1 or 2 weeks of lithium treatment, we identified 6 and 74 proteins with altered abundance compared with controls, respectively. We randomly selected 17 proteins with altered abundance caused by lithium treatment for validation by immunoblotting. Bioinformatics analysis of the data indicated that proteins involved in cell death, apoptosis, cell proliferation, and morphology are highly affected by lithium. We demonstrate that members of several signaling pathways are activated by lithium treatment, including the PKB/Akt-kinase and the mitogen-activated protein kinases (MAPK), such as extracellular regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38. Lithium treatment increased the intracellular accumulation of beta-catenin in association with increased levels of phosphorylated glycogen synthase kinase type 3beta (GSK3beta). This study provides a comprehensive analysis of the proteins affected by lithium treatment in the IMCD and, as such, provides clues to potential lithium targets in the brain.

Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS.

Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as "transcription factor" proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5'-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5'-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS(3) technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

H-Ras, R-Ras, and TC21 differentially regulate ureteric bud cell branching morphogenesis.

The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras-expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras-expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.

Site-specific expression of IQGAP1 in human nephrons.

IQGAP1 is a multifunctional, 190-kDa scaffolding protein that plays an important role in the regulation of cell adhesion, migration, proliferation, differentiation, polarization and cytoskeletal remodeling. IQGAP1 is ubiquitously expressed in human organs and is highly expressed in the kidney. Currently, the site-specific expression of IQGAP1 in the human nephrons is unclear. We performed Western blotting analysis, immunohistochemistry and double-immunolabeling confocal microscopic analysis of IQGAP1 with specific biomarkers of each nephron segment to study the expression and distribution of IQGAP1 in human nephrons. We found that IQGAP1 was strongly expressed in human podocytes and glomerular endothelial cells, but weakly expressed in glomerular mesangial cells. In human renal tubules, IQGAP1 was strongly expressed in the collecting duct, moderately expressed in the proximal tubule, medullary loop, distal convoluted tubule and connecting tubule. IQGAP1 staining was much stronger in the apical membrane in the proximal tubule, thick descending limb and thick ascending limb of medullary loop and collecting duct. However, the expression of IQGAP1 was mainly in the basolateral membrane of the connecting tubule, and diffusely in the thin limb of medullary loop and distal convoluted tubule. The interaction between IQGAP1 and F-actin suggested that cytoskeleton regulation may be the underlying mechanism mediating the effect of IQGAP1 in human nephrons. To the best of our knowledge, this is the first report of specific expression and differential subcellular location of IQGAP1 in human nephrons. The site-specific expression pattern of IQGAP1 suggests that IQGAP1 may play diverse roles in various human nephron segments.

AKAP220 colocalizes with AQP2 in the inner medullary collecting ducts.

During dehydration, protein kinase A phosphorylates aquaporin 2 (AQP2) at serine 256 and this is essential for apical membrane sorting of AQP2 in the collecting ducts. A-kinase anchoring proteins (AKAPs) bind protein kinase A and protein phosphatases conferring substrate specificity to these enzymes and localize them to the appropriate intracellular compartment. We found that AKAP220 bound to AQP2 in a yeast two-hybrid screen. Further, it was highly localized to the papilla compared to other regions of the kidney. Using double immunofluorescence and immunoelectron microscopy we found that AKAP220 co-localized with AQP2 in the cytosol of the inner medullary collecting ducts. Forskolin-mediated phosphorylation of AQP2, transiently expressed in COS cells, was increased by AKAP220 co-expression. Our results suggest that AKAP220 may be involved in the phosphorylation of AQP2 by recruiting protein kinase A.

Jouberin localizes to collecting ducts and interacts with nephrocystin-1.

Joubert syndrome and related disorders are autosomal recessive multisystem diseases characterized by cerebellar vermis aplasia/hypoplasia, retinal degeneration and cystic kidney disease. There are five known genes; mutations of which give rise to a spectrum of renal cystic diseases the most common of which is nephronophthisis, a disorder characterized by early loss of urinary concentrating ability, renal fibrosis, corticomedullary cyst formation and renal failure. Many of the proteins encoded by these genes interact with one another and are located at adherens junctions or the primary cilia and or basal bodies. Here we characterize Jouberin, a multi-domain protein encoded by the AHI1 gene. Immunohistochemistry with a novel antibody showed that endogenous Jouberin is expressed in brain, kidney and HEK293 cells. In the kidney, Jouberin co-localized with aquaporin-2 in the collecting ducts. We show that Jouberin interacts with nephrocystin-1 as determined by yeast-2-hybrid system and this was confirmed by exogenous and endogenous co-immunoprecipitation in HEK293 cells. Jouberin is expressed at cell-cell junctions, primary cilia and basal body of mIMCD3 cells while a Jouberin-GFP construct localized to centrosomes in subconfluent and dividing MDCK cells. Our results suggest that Jouberin is a protein whose expression pattern supports both the adherens junction and the ciliary hypotheses for abnormalities leading to nephronophthisis.

High frequency and wide range of human kidney papillary crystalline plugs.

Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.

Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct.

We recently cloned a cDNA of the collecting duct apical membrane water channel of rat kidney, which is important for the formation of concentrated urine (Fushima, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. 1993. Nature [Lond.]. 361:549-552). Since urine concentrating ability varies among mammalian species, we examined whether an homologous protein is present in human kidney. By screening a human kidney cDNA library, we isolated a cDNA clone, designated human aquaporin of collecting duct (hAQP-CD), that encodes a 271-amino acid protein with 91% identity to rat AQP-CD. mRNA expression of hAQP-CD was predominant in the kidney medulla compared with the cortex, immunohistochemical staining of hAQP-CD was observed only in the collecting duct cells, and the staining was dominant in the apical domain. Functional expression study in Xenopus oocytes confirmed that hAQP-CD worked as a water channel. Western blot analysis of human kidney medulla indicated that the molecular mass of hAQP-CD is 29 kD, which is the same mass expected from the amino acid sequence. Chromosomal mapping of the hAQP-CD gene assigned its location to chromosome 12q13. These results could be important for future studies of the pathophysiology of human urinary concentration mechanisms in normal and abnormal states.

Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts.

We investigated immunohistochemical localization of V2 vasopressin receptor along the nephron using a specific polyclonal antibody. Staining was observed in some of thick ascending limbs and all of principal and inner medullary collecting duct (IMCD) cells. Not only basolateral but also luminal membrane was stained in collecting ducts, especially in terminal IMCD (tIMCD). To learn the functional role of luminal V2 receptor in tIMCD, we studied the luminal effects of arginine vasopressin (AVP) on osmotic water permeability (Pf), urea permeability (Pu), and cAMP accumulation using isolated perfused rat tIMCD. In the absence of bath AVP, luminal AVP caused a small increase in cAMP accumulation, Pf and Pu, confirming the presence of V2 receptor in the lumen of tIMCD. In contrast, luminal AVP inhibited Pf and Pu by 30-65% in the presence of bath AVP by decreasing cAMP accumulation via V1a or oxytocin receptors and by an unknown mechanism via V2 receptors in the luminal membrane of tIMCD. These data show that V2 receptors are localized not only in the basolateral membrane but also in the luminal membrane of the distal nephron. Luminal AVP acts as a negative feedback system upon the basolateral action of AVP in tIMCD.

Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2 water channels.

Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney.

To examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD. Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884 +/- 245 mOsm/kg) was significantly higher than that in the control (938 +/- 91). In the V2R antagonist group, Uosm was significantly decreased to 249 +/- 29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor.

Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (mean +/- SE, fmol/mm): S-1 proximal, 10.8 +/- 2.1; S-2, 10.0 +/- 2.3; S-3, 21.3 +/- 3.1; type 1 thin descending limb (DTL), 12.9 +/- 4.6; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.