Targeting renal purinergic signalling for the treatment of lithium-induced nephrogenic diabetes insipidus.

Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development of nephrogenic diabetes insipidus (NDI), a debilitating condition. Lithium-induced NDI is due to resistance of the kidney to arginine vasopressin (AVP), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides (ATP/UTP), acting via P2Y receptors, opposes the action of AVP on renal collecting duct (CD) by decreasing the cellular cAMP and thus AQP2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP/UTP-activated P2Y2 receptor in lithium-induced NDI in rats and showed that P2Y2 receptor knockout mice are significantly resistant to Li-induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP-activated P2Y12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulphate (Plavix(®)) ameliorates Li-induced NDI in rodents. Parallel in vitro studies showed that P2Y12 receptor blockade by the reversible antagonist PSB-0739 sensitizes CD to the action of AVP. Thus, our studies unravelled the potential beneficial effects of targeting P2Y2 or P2Y12 receptors to counter AVP resistance in lithium-induced NDI. If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti-AVP effects to those that enhance the sensitivity of the kidney to AVP action.

The nucleobase adenine as a signalling molecule in the kidney.

In 2002, the first receptor activated by the nucleobase adenine was discovered in rats. In the past years, two adenine receptors (AdeRs) in mice and one in Chinese hamsters, all of which belong to the family of G protein-coupled receptors (GPCRs), were cloned and pharmacologically characterized. Based on the nomenclature for other purinergic receptor families (P1 for adenosine receptors and P2 for nucleotide, e.g. ATP, receptors), AdeRs were designated P0 receptors. Pharmacological data indicate the existence of G protein-coupled AdeRs in pigs and humans as well; however, those have not been cloned so far. Current data suggest a role for adenine and AdeRs in renal proximal tubules. Furthermore, AdeRs are suggested to be functional counterplayers of vasopressin in the collecting duct system, thus exerting diuretic effects. We are only at the beginning of understanding the significance of this new class of purinergic receptors, which might become future drug targets.

Expression of salt and urea transporters in rat kidney during cisplatin-induced polyuria.

BACKGROUND: Cisplatin (CP) induced polyuria in rats is associated with a reduction in medullary hypertonicity, normally generated by the thick ascending limb (TAL) salt transporters, and the collecting duct urea transporters (UT). To investigate the molecular basis of this abnormality, we determined the protein abundance of major salt and UT isoforms in rat kidney during CP-induced polyuria. METHODS: Male Sprague-Dawley rats received either a single injection of CP (5 mg/kg, N = 6) or saline (N = 6) intraperitoneally five days before sacrifice. Urine, blood, and kidneys were collected and analyzed. RESULTS: CP-treated rats developed polyuric acute renal failure as assessed by increased blood urea nitrogen (BUN), urine volume and decreased urine osmolality. Western analysis of kidney homogenates revealed a marked reduction in band density of the bumetanide-sensitive Na-K-2Cl cotransporter in cortex (60% of control values, P < 0.05), but not in outer medulla (OM) (106% of control values). There were no differences in band densities for the renal outer medullary potassium channel (ROMK), the type III Na-H exchanger (NHE3), the alpha-subunit of Na,K-ATPase in the OM; or for UT-A1, UT-A2 or UT-A4 in outer or inner medulla. However, the band pattern of UT-A2 and UT-A4 proteins in the OM of CP-treated rats was different from the control rats, suggesting a qualitative modification of these proteins. CONCLUSIONS: Changes in the abundance of outer or inner medullary salt or urea transporters are unlikely to play a role in the CP-induced reduction in medullary hypertonicity. However, qualitative changes in UT proteins may affect their functionality and thus may have a role.

Extracellular nucleotide signaling along the renal epithelium.

During the past two decades, several cell membrane receptors, which preferentially bind extracellular nucleotides, and their analogs have been identified. These receptors, collectively known as nucleotide receptors or "purinergic" receptors, have been characterized and classified on the basis of their biological actions, their pharmacology, their molecular biology, and their tissue and cell distribution. For these receptors to have biological and physiological relevance, nucleotides must be released from cells. The field of extracellular ATP release and signaling is exploding, as assays to detect this biological process increase in number and ingenuity. Studies of ATP release have revealed a myriad of roles in local regulatory (autocrine or paracrine) processes in almost every tissue in the body. The regulatory mechanisms that these receptors control or modulate have physiological and pathophysiological roles and potential therapeutic applications. Only recently, however, have ATP release and nucleotide receptors been identified along the renal epithelium of the nephron. This work has set the stage for the study of their physiological and pathophysiological roles in the kidney. This review provides a comprehensive presentation of these issues, with a focus on the renal epithelium.

Molecular physiology of urinary concentration defect in elderly population.

It is estimated that by the year 2050 one in five Americans will be 65 years or older. This mandates the growing need for clinical and basic research in the field of geriatric medicine to understand age-related maladies. The most prominent abnormality in renal function in the aging population is the inability to handle water, frequently resulting in hypo- or hyperosmolar states, and the associated electrolyte imbalances. During the past decade, thanks to the advent of powerful molecular techniques, rapid strides have been made in the approaches employed to understand and dissect the physiology of renal function in general and the urinary concentration mechanism in particular. Using an integrated approach of clinical observations, experimental model systems, molecular analysis, and functional genomics, a more comprehensive picture of the interplay of physiological systems in the genesis of urinary concentration defect in the elderly is beginning to emerge. Much remains to be deciphered regarding the complex interactions between the role of environment, genetics, diet, pharmacological agents and the general effects of aging on kidney function. The emerging importance of socio-economic and quality of life issues surrounding geriatric medicine encourage public and private support and funding for research in the area of age-related diseases, especially as they are related to the kidney.

Developmental expression of aquaporin 2 in the mouse inner ear.

OBJECTIVES: The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial expression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear. STUDY DESIGN: A systematic immunohistochemical study of Aqp2 protein expression was performed on embryonic mouse inner ears ranging from embryonic day 10 (otocyst stage) to embryonic day 18 (just before birth). METHODS: Serial cryosections of embryonic mouse inner ears were used for immunohistochemical experiments. A rabbit polyclonal antisera raised against a synthetic Aqp2 peptide was used with a standard nickel intensified 3,3-diaminobenzidine reaction protocol for immunolocalization of Aqp2 in tissue sections. RESULTS: Aquaporin 2 is expressed diffusely in the early otocyst, then becomes progressively restricted as the inner ear matures. During early cochlear duct formation (embryonic days 12 and 13), expression of Aqp2 is homogeneous; later, it becomes restricted to specific regions of the endolymphatic compartment (embryonic days 15 and 18). Similar restriction of expression patterns could be noted for the vestibular structures. Endolymphatic duct and sac and stria vascularis expression of Aqp2 was noted to occur fairly late during development but demonstrated a distinct pattern of immunolabeling. CONCLUSIONS: Aquaporin 2 shows an early and specific pattern of expression in the developing mouse inner ear, suggesting a significant role for this water channel protein in the development of endolymph homeostasis and meriting further functional studies of Aqp2 in the inner ear.

Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria.

BACKGROUND: Cisplatin (CP)-induced polyuria in rats is attributed to decreased medullary hypertonicity and/or an end-organ resistance to vasopressin. However, the roles of renal aquaporins (AQPs) have not yet been explored. METHODS: Male Sprague-Dawley rats (230 to 245 g) received either a single injection of CP (5 mg/kg, N = 4) or saline (N = 4) intraperitoneally five days before sacrifice. Urine, blood, and kidney samples were analyzed. RESULTS: Platinum accumulated in the cortex and outer medulla of CP-treated rats (39.05 +/- 7.50 and 36.48 +/- 12.44 microg/g vs. 2.52 +/- 0.43 and 1.87 +/- 0.84 microg/g dry tissue in controls, respectively). Histologically, tubular damage and decreased AQP1 immunolabeling were detected in the S3 segment of proximal tubules. CP treatment caused 4.4- and 4.8-fold increases, respectively, in blood urea nitrogen and urine volume, and a 4. 4-fold decrease in urine osmolality. Immunoblots showed that AQP2 and AQP3 were significantly reduced to 33 +/- 10% (P < 0.001) and 69 +/- 11% (P < 0.05), respectively, in the inner medulla of CP-treated rats. Immunocytochemical analysis showed a decrease in AQP2 labeling in the inner medulla of CP-treated rats. Northern hybridization revealed a 33 +/- 11% (P < 0.002) decrease in AQP2 mRNA expression in the inner medulla of CP-treated rats. AQP1 protein expression levels were modestly (67 +/- 7%, P = 0.057) and significantly (53 +/- 13%, P < 0.007) decreased in outer and inner medullae, respectively, of CP-treated rats. CONCLUSIONS: CP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla.

Cellular localization of P2Y(2) purinoceptor in rat renal inner medulla and lung.

Physiological and pharmacological studies have demonstrated that extracellular ATP, acting through P2Y(2) purinoceptor, modulates water permeability of renal medullary collecting duct cells and the secretion of ions, mucin, and surfactant phospholipids by respiratory epithelia. Here we provide direct molecular evidence for the expression of P2Y(2) purinoceptor in these cells. RT-PCR confirmed P2Y(2) purinoceptor mRNA expression in rat lung and kidney and demonstrated expression in renal collecting ducts. Northern analysis showed that both lung and kidney express one 3.6-kb P2Y(2) purinoceptor mRNA transcript. Immunoblots using peptide-derived polyclonal antibody to P2Y(2) purinoceptor showed that inner medullary collecting ducts (IMCD) express two distinct and specific products (47 and 105 kDa) and account for the majority of the receptor expression in inner medulla, whereas the 105-kDa form is predominant in lung. Immunoperoxidase labeling on cryosections showed localization of receptor protein in the apical and basolateral domains of IMCD principal cells and in the secretory cells (Clara cells and goblet cells) of the terminal respiratory bronchioles.

SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles.

Vesicle targeting proteins ("SNAREs") have been proposed to direct vasopressin-induced trafficking of aquaporin-2 water channels in kidney collecting ducts. A newly identified SNARE protein, SNAP-23, is proposed to mediate vesicle targeting to the plasma membrane in diverse tissues. The current studies were done to determine whether SNAP-23 is expressed in collecting ducts with an intracellular distribution compatible with a role in aquaporin-2 trafficking. RT-PCR demonstrated SNAP-23 mRNA in microdissected collecting ducts and other tubular segments including the proximal tubule and thick ascending limb. Immunoblotting using a polyclonal antibody raised against a COOH-terminal peptide revealed a solitary band at an apparent molecular mass of 30 kDa in renal medullary membrane fractions and inner medullary collecting duct suspensions. Differential centrifugation revealed that SNAP-23 is present in membrane fractions including the low-density fraction enriched in intracellular vesicles. Immunocytochemistry revealed SNAP-23 labeling at both the apex and the cytoplasm of collecting duct principal cells. Immunoblotting of intracellular vesicles immunoisolated using an aquaporin-2 antibody revealed the presence of both SNAP-23 and synaptobrevin-2 (VAMP-2) in aquaporin-2-bearing vesicles. We conclude that SNAP-23 is strongly expressed in collecting duct principal cells, consistent with a role in vasopressin-regulated trafficking of aquaporin-2. However, localization of SNAP-23 in both intracytoplasmic vesicles and plasma membranes suggests a function different from that originally proposed for SNAP-25 in synaptic vesicle targeting.

Expression of synaptotagmin VIII in rat kidney.

The synaptotagmins are a family of integral membrane proteins proposed to function as regulators of both exocytosis and endocytosis. Here, we have used immunochemical techniques and RT-PCR to assess sites of renal expression of synaptotagmin VIII. A polyclonal antibody was raised to a synthetic peptide corresponding to the carboxy-terminal 21 amino acids of mouse synaptotagmin VIII. On immunoblots of membrane fractions from renal cortex and medulla (and in several other tissues), the antibody labeled a 52-kDa band (absent with preimmune IgG). Immunofluorescence localization was carried out in tissue sections from rat kidney. The synaptotagmin VIII antibody labeled early proximal tubules, thin ascending limbs, thick ascending limbs, connecting tubules, and collecting ducts. In collecting ducts, both type A and B intercalated cells exhibited basolateral labeling, whereas principal cells were labeled chiefly in the apical and subapical portion of the cells. Thick ascending limbs were labeled in both the basolateral and apical regions. RT-PCR experiments using total RNA extracted from cortex and medulla or microdissected inner medullary collecting ducts gave a single band of appropriate size. Sequencing of the PCR product confirmed that the amplified target is synaptotagmin VIII. We conclude that synaptotagmin VIII is broadly expressed among renal tubule epithelia, raising the possibility that it is involved in regulation of transport and/or cell remodeling at several sites in the nephron and collecting duct.

Expression of syntaxins in rat kidney.

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henle's loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.

Evidence that aquaporin-1 mediates NaCl-induced water flux across descending vasa recta.

Outer medullary descending vasa recta (OMDVR) were perfused in vitro, and volume efflux was measured by driving water movement with transmural gradients of NaCl or albumin. Consistent with mediation by water channels, p-chloromercuribenzenesulfonic acid (pCMBS) markedly inhibited volume flux induced by NaCl. Dithiothreitol reversed the inhibition, pCMBS did not significantly alter water flux induced by albumin. Osmotic water permeability (Pf) of the pCMBS-sensitive pathway of glutaraldehyde-fixed and nonfixed OMDVR was 1,102 +/- 449 and 1,257 +/- 718 microns/s (means +/- SD), respectively. pCMBS reduced Pf to near zero, whereas diffusional water permeability in the same vessels was only slightly inhibited. Immunoreactive aquaporin-1 (AQP1) measured by enzyme-linked immunosorbent assay in collagenase-treated and untreated OMDVR was 5.2 +/- 1.0 and 4.2 +/- 0.4 fmol/mm, respectively, values that account well for the experimental Pf. We conclude that OMDVR water flux driven by NaCl gradients is most likely mediated by the AQP1 water channel and that NaCl and urea gradients drive water efflux in vivo by this route.

Ultramicrodetermination of vasopressin-regulated urea transporter protein in microdissected renal tubules.

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called "UT-1") responsible for facilitated urea transport across the apical plasma membrane of inner medullary collecting duct (IMCD) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.

Quantitation of aquaporin-2 abundance in microdissected collecting ducts: axial distribution and control by AVP.

Aquaporin-2 (AQP-2) is the arginine vasopressin-regulated water channel of the renal collecting ducts. Using an improved version of a fluorescence-based enzyme-linked immunosorbent assay (Y. Maeda, B. L. Smith, P. Agre, and M. A. Knepper. J. Clin. Invest. 95: 422-428, 1995), we quantified AQP-2 protein abundance in microdissected renal collecting ducts from normal Sprague-Dawley (SD) rats and vasopressin-deficient Brattleboro rats. Standard curves were linear in the range of 0-200 fmol/well and were highly reproducible from day to day (lower limit of detection 2.3 fmol; coefficient of variation 6-9%). In SD rats thirsted for 24 h, the measured quantities of AQP-2 were as follows (x 10(9) molecules/mm): cortical collecting ducts (CCD), 4.3 +/- 0.5; outer medullary collecting ducts (OMCD), 10.1 +/- 1.7; initial one-third of inner medullary collecting duct (IMCD-1), 9.2 +/- 1.1; middle one-third of the IMCD (IMCD-2), 7.5 +/- 0.8; terminal one-third of the IMCD (IMCD-3), 3.3 +/- 0.6; n = 7-12. In IMCD-2 this corresponds to 11.8 +/- 1.3 x 10(6) AQP-2 molecules per cell. Thus AQP-2 is extremely abundant in collecting duct cells. AQP-2 levels were decreased in untreated Brattleboro rats relative to the parent strain Long-Evans (LE) by 68% in IMCD-2 and 44% in CCD. Following vasopressin infusion by osmotic minipumps, AQP-2 levels in IMCD-2 of Brattleboro rats rose gradually, reaching levels equivalent to those seen in LE rats after 5 days. A similar rise was seen in the CCD, indicating that the vasopressin-induced increase was not dependent on a large increase in the interstitial tonicity. Thus a rise in circulating vasopressin concentration increases the level of AQP-2 protein expression in collecting ducts, presumably via a direct action of vasopressin.

Poly-D-glutamic acid induces an acute lysosomal thesaurismosis of proximal tubules and a marked proliferation of interstitium in rat kidney.

Renal damage caused by polycationic peptides is well documented, but renal damage caused by polyanionic peptides is not. During our attempts to inhibit the nephrotoxicity of aminoglycoside antibiotics by polyanionic peptides, we discovered that poly-D-glutamic acid (molecular weight, 20 kd; 250 mg/kg/day subcutaneously for 1 to 4 days) produces an acute thesaurismosis in the proximal tubular cells associated with a marked proliferation of peritubular interstitial cells in rat kidney. Thesaurismotic bodies were easily visualized by light microscopy at the basal pole of proximal tubular cells with the cationic stain Giemsa. By electron microscopy, these bodies appeared membrane-limited, frequently distorted, filled with heterogeneous granular material, accessible to injected peroxidase (a tracer of the endocytic pathway), and generally stainable for the lysosomal enzyme arylsulfatase. Specimens obtained 3 hours after injection of poly-D-glutamic acid and horseradish peroxidase suggested an impairment of endosome and/or lysosome fission, but not fusion. By histoautoradiographic examination after 3H-thymidine incorporation, global labeling indices of cortical cells were increased 11- to 18-fold in poly-D-glutamic acid-treated rats as compared with controls, with > 80% of labeled cells localized in the interstitium. Distal tubular and glomerular cells also showed a moderate proliferation, but proximal tubular cells showed no significant necrosis or proliferation. Although tubular thesaurismosis persisted, interstitial cell proliferation resolved within 7 days after cessation of treatment. We suggest that poly-D-glutamic acid is a convenient tool to induce a rapid and sustained lysosomal storage disorder. It could also help clarify the relationship between insults to tubular cells and proliferation of peritubular cells, two features frequently associated in tubulointerstitial disorders. The mechanism of the thesaurismosis and of the interference with the dynamics of fusion-fission of the endocytic apparatus are addressed in the companion paper.

Mechanism of the thesaurismosis and altered lysosomal dynamics induced by poly-D-glutamic acid in kidney proximal tubular cells.

In the companion paper, we report that a single injection of poly-D-glutamic acid causes an acute lysosomal storage condition and apparently impairs the lysosomal fission dynamics. The present paper addresses the mechanisms of these two alterations using a combination of in vivo and in vitro biochemical approaches. After a single intravenous injection, 14C-poly-D-glutamic acid was rapidly cleared from the plasma and appeared in the urine. Yet, a small but sizable fraction of the injected polymer was taken up by the kidney cortex through a saturable process (Kuptake, 150 mg/kg body wt; uptakemax 96 micrograms/g cortex). Analytical subcellular fractionation of cortex homogenates demonstrated that at initial stages, the 14C label was predominantly associated with subcellular particles of intermediate size and low equilibrium density, and was therefore slowly transferred to larger particles equilibrating at high density, then codistributing with the lysosomal hydrolases. At a concentration of 10 mg/ml (equivalent to its estimated concentration in lysosomes), poly-D-glutamic acid formed micronic aggregates ( > or = 10 microns) when brought to solution at pH < or = 6 in relation to its decreased ionization (pKa of lateral chains approximately equal to 4.25). Finally, 1 day after the injection of poly-D-glutamic acid, the activities of several lysosomal enzymes (hexosaminidase, cathepsin B, acid sphingomyelinase, and sulfatase B), but not of all of them (eg, acid phosphatase), were increased in the kidney cortex. We propose that poly-D-glutamic acid reaches lysosomes by adsorptive endocytosis and becomes concentrated within these organelles because its withstands hydrolysis until it forms aggregates or precipitates, causing a decrease in the fluidity or the deformability ("gelling") of the lysosomal matrix. This should alter the dynamics of intercommunication of these organelles by impairing their fission without a proportionate effect on their fusion properties. In addition, the data suggest that the presence of poly-D-glutamic acid directly or indirectly slows down the degradation of several lysosomal enzymes.

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Renal aquaporins.

Aquaporins (AQPs) are a newly recognized family of transmembrane proteins that function as molecular water channels. At least four aquaporins are expressed in the kidney where they mediate rapid water transport across water-permeable epithelia and play critical roles in urinary concentrating and diluting processes. AQP1 is constitutively expressed at extremely high levels in the proximal tubule and descending limb of Henle's loop. AQP2, -3 and -4 are expressed predominantly in the collecting duct system. AQP2 is the predominant water channel in the apical plasma membrane and AQP3 and -4 are found in the basolateral plasma membrane. Short-term regulation of collecting duct water permeability by vasopressin is largely a consequence of regulated trafficking of AQP2-containing vesicles to and from the apical plasma membrane.

Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct.

The P2u class of nucleotide receptors is linked to mobilization of intracellular Ca2+ in many cell types, including the renal collecting duct cells. In the present studies, we examined the effects of nucleotides (ATP, UTP, and ADP; 10 microM each) on the arginine vasopressin (AVP, 0.1 nM)-stimulated osmotic water permeability (Pf) in in vitro perfused terminal inner medullary collecting ducts (IMCD) of rat. ATP or UTP, when added to the bath, decreased the AVP-stimulated Pf by approximately 40%. These effects were reversible upon withdrawal of the nucleotides. However, addition of ADP to the bath or sham exchange of the bath had no significant effect on the Pf. Furthermore, ATP did not have any significant effect on the Pf stimulated either by a membrane-permeant, nonhydrolyzable adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chlorophenylthio)-cAMP, 0.1 mM] o by forskolin (1 microM). In line with these findings, ATP decreased the AVP-stimulated cAMP levels in IMCD suspensions to approximately 68%. In addition, ATP did not exert an inhibitory effect on the AVP-stimulated Pf in the presence of calphostin C (150 nM), an inhibitor of protein kinase C. These results lead us to conclude the following: 1) agonist occupancy of the putative nucleotide receptor in the terminal IMCD causes an inhibition of AVP-stimulated Pf; and 2) this effect is due to a decrease in cellular cAMP levels, most likely resulting from activation of the phosphoinositide signaling pathway.

Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.

Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned whose expression appears to be limited to the collecting duct. Previously, we immunolocalized this water channel to the apical plasma membrane (APM) and to intracellular vesicles (IVs) of collecting duct cells. Here, we test the hypothesis that vasopressin increases cellular water permeability by inducing exocytosis of AQP-CD-laden vesicles, transferring water channels from IVs to APM. Rat collecting ducts were perfused in vitro to determine water permeability and subcellular distribution of AQP-CD in the same tubules. The collecting ducts were fixed for immunoelectron microscopy before, during, and after exposure to vasopressin. Vasopressin exposure induced increases in water permeability and the absolute labeling density of AQP-CD in the APM. In parallel, the APM:IV labeling ratio increased. Furthermore, in response to vasopressin withdrawal, AQP-CD labeling density in the APM and the APM:IV labeling ratio decreased in parallel to a measured decrease in osmotic water permeability. We conclude that vasopressin increases the water permeability of collecting duct cells by inducing a reversible translocation of AQP-CD water channels from IVs to the APM.