Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na+ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCDC14) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCDC14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (< or =24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10-9 m) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.     

Identification of vasopressin-induced genes in AQP2-transfected MDCK cells by suppression subtractive hybridization

Arginine vasopressin (AVP) plays a major role in the modulation of water reabsorption in mammalian kidney. In addition to short-term regulation of aquaporin 2 (AQP2) trafficking, AVP also has long-term effects to regulate the expression of AQP2 in renal collecting duct. However, the detailed mechanism of the long-term effects of AVP in kidney remains to be elucidated. We have searched for genes induced by AVP using the polymerase chain reaction-based suppression subtractive hybridization technique in AVP-responsive AQP2-transfected MDCK cells. We found that the expression of the genes such as VIP17/MAL, annexin II, stimulatory GTP binding protein, tubulin, and mitochondrial ATP synthase was induced by AVP treatment for 4h. These results suggest that AVP might induce the expression of several genes related to the apical targeting of newly synthesized AQP2 as well as that of AQP2 for the long-term modification of water permeability in renal collecting duct.     

COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.     

mPGES-1 deletion potentiates urine concentrating capability after water deprivation

PGE(2) plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE(2) output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V(2) receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE(2) reduces urine concentrating ability through suppression of renal medullary expression of V(2) receptors and AQP2 but may enhance it by mediating the central AVP response.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.     

Prenatal exposure to ethanol causes partial diabetes insipidus in adult rats

Chronic consumption of ethanol in adult rats and humans leads to reduced AVP-producing neurons, and prenatal ethanol (PE) exposure has been reported to cause changes in the morphology of AVP-producing cells in the suprachiasmatic nucleus of young rats. The present studies further characterize the effects of PE exposure on AVP in the young adult rat, its hypothalamic synthesis, pituitary storage, and osmotically stimulated release. Pregnant rats were fed a liquid diet with 35% of the calories from ethanol or a control liquid diet for days 7-22 of pregnancy. Water consumption and urine excretion rate were measured in the offspring at 60-68 days of age. Subsequently, the offspring were infused with 5% NaCl at 0.05 ml.kg(-1).min(-1) with plasma samples taken before and at three 40-min intervals during infusion for measurement of AVP and osmolality. Urine output and water intake were approximately 20% greater in PE-exposed rats than in rats with no PE exposure, and female rats had a greater water intake than males. The relationship between plasma osmolality and AVP in PE-exposed rats was parallel to, but shifted to the right of, the control rats, indicating an increase in osmotic threshold for AVP release. Pituitary AVP was reduced by 13% and hypothalamic AVP mRNA content was reduced by 35% in PE-exposed rats. Our data suggest that PE exposure can cause a permanent condition of a mild partial central diabetes insipidus.     

Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice

Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.     

Angiotensin-(1-7) serves as an aquaretic by increasing water intake and diuresis in association with downregulation of aquaporin-1 during pregnancy in rats

We previously demonstrated that kidney and urine levels of angiotensin-(1-7) [ANG-(1-7)] were increased in pregnancy. To explore the role of ANG-(1-7) on fluid and electrolyte homeostasis during pregnancy, we evaluated the effect of the ANG-(1-7) antagonist D-alanine-[ANG-(1-7)] (A-779) on kidney function. Virgin and pregnant rats received infusion of vehicle or A-779 (48 microg.kg(-1).h(-1)) for 8 days by osmotic minipumps. Metabolic studies were done on treatment day 7-8. Virgin and pregnant rats at day 15 and 19 were killed, and blood and kidneys were collected. Kidneys were prepared for Western blot analysis for aquaporin-1 (AQP1) and aquaporin-2. In virgin female rats, A-779 increased urine volume and decreased urinary osmolality and AQP1 with no change in water intake. In 19-day pregnant rats, A-779 significantly decreased water intake and urine volume and increased urinary osmolality and kidney AQP1 expression. Only in late gestation did A-779 treatment decrease the difference between intake and output (balance). A-779 treatment increased plasma vasopressin in late gestation but did not change vasopressin in virgins. In virgin and pregnant animals, A-779 administration had no effect on blood pressure, plasma volume, blood volume, or urinary electrolytes. These results suggest that ANG-(1-7) produces antidiuresis associated with upregulation of AQP1 in virgin rats, whereas ANG-(1-7) produces diuresis in late gestation with downregulation of AQP1. ANG-(1-7) contributes to the enhanced water intake during pregnancy, allowing maintenance of the normal volume-expanded state despite diuresis produced in part by decreased AVP and AQP1.     

大耳猬血液、尿无机离子等指标测定及肾脏水通道蛋白1、2的表达

测定了大耳猬血清及尿中多种无机离子和尿素氮等指标,并应用免疫组织化学方法观察了AQP1、AQP2在肾脏的表达.大耳猬血清钠、氯含量较高;而尿液中以钾、钠、氯及尿素氮含量较高.尿液中主要离子浓度高于血清,较为浓缩,尿素氮、钾排泄能力较强.AQP1免疫反应阳性表达于近曲小管上皮和髓袢细段,AQP2主要表达于集合管上皮细胞.因此,AQP1、AQP2可能在大耳猬肾脏水重吸收及尿液浓缩过程中具有重要作用.     

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.     

Upregulation of collecting duct aquaporin-2 by metabolic acidosis: role of vasopressin

Metabolic acidosis is associated with alteration in fluid and electrolyte reabsorption in a number of nephron segments. However, the effects of metabolic acidosis on urine osmolality and aquaporin-2 (AQP-2) remain poorly understood. In these studies, we examined the effects of chronic metabolic acidosis on water handling by the kidney. Rats were placed in metabolic cages and subjected to water (control) or 280 mM NH₄Cl loading for 120 h to induce metabolic acidosis. The results indicated a significant increase in urine osmolality with no change in urine volume or urinary Na⁺ excretion in acid-loaded animals. This effect was independent of alteration in fluid intake or salt/Cl^- loading. Immunoblotting and Northern hybridization studies indicated that AQP-2 protein abundance and mRNA expression levels increased significantly along the collecting duct system of NH₄Cl-but not NaCl-loaded animals. RIA results indicated that metabolic acidosis was associated with a fourfold increase in circulating levels of vasopressin (AVP) and a significant increase in brain AVP mRNA expression levels. In conclusion, metabolic acidosis upregulates the expression levels of AQP-2 and increases urine osmolality, suggesting an adaptive increase in water reabsorption in the collecting duct. A concomitant increase in AVP synthesis and secretion likely plays an essential role in the adaptation of AQP-2 in metabolic acidosis. kidney; acid-base; urine osmolality; sodium excretion rate     

Synergistic effects of nitric oxide and prostaglandins on renal escape from vasopressin-induced antidiuresis

Recent results from our laboratories indicate that renal escape from AVP-induced antidiuresis is accompanied by marked downregulation of kidney aquaporin-2 (AQP2) and AVP V2 receptors. The present studies evaluated the effect of nitric oxide (NO) and PG synthesis blockade on escape from antidiuresis. dDAVP-infused rats were water loaded (WL) for 5 days. l-NAME, an NO synthesis inhibitor, or diclofenac, a cyclooxygenase inhibitor, was infused subcutaneously beginning 1 day before WL. As early as 2 days after WL, urine volume increased and urine osmolality decreased, indicating the onset of escape. Endogenous NO synthesis, measured as urinary NO2 + NO3 excretion, was significantly increased in the WL group compared with the non-WL controls during all 5 days of WL. l-NAME (20 mg. kg(-1). day(-1)) markedly decreased urine volume on days 4 and 5 of WL, indicating inhibition of the escape phenomenon. Kidney AQP2 protein was significantly increased by this dose of l-NAME as well. A lower dose of l-NAME (10 mg. kg(-1). day(-1)) or diclofenac (2.5 mg. kg(-1). day(-1)) did not significantly affect the escape phenomenon by itself, but the combination of l-NAME and diclofenac showed a marked inhibitory effect on the escape phenomenon, which was also accompanied by a significant increase in kidney AQP2 expression. These results therefore suggest that renal NO and PG both play important roles in escape from AVP-induced antidiuresis by acting synergistically to downregulate kidney AQP2 expression.     

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.     

Impaired water diuresis in a patient with pseudo-Bartter syndrome.

A 32-year-old man was diagnosed as having pseudo-Bartter syndrome due to surreptitious habitual vomiting and to maldigestion related to decayed teeth. His chief complaints were muscle pain and weakness. In this case, metabolic alkalosis, hypokalemia, hypochloremia, increased plasma renin activity and aldosterone levels were noticed with marked decreases in urinary chloride excretion. Creatinine clearance (GFR) and renal plasma flow (RPF) were also decreased. Blood pressure was normal, but the pressor response to angiotensin II was attenuated. Before treatment with 0.9% saline infusion, plasma vasopressin (AVP) was not suppressed sufficiently by lowering the plasma osmolality (Posm) with an oral water load (WL), but it normally responded to a rise in Posm due to hypertonic saline infusion. Moreover, plasma AVP was normally suppressed by WL after the replenishment of saline. Plasma atrial natriuretic peptide (ANP) was low before WL, but increased normally in response to WL. However, inconsistent with the normal response in this case, decreases in plasma AVP failed to dilute urinary osmolality and to increase urine flow, irrespective of the levels of plasma ANP. These results indicate that chronic inanition due to surreptitious vomiting causes impaired renal diluting ability through decreases in GFR and RPF, irrespective of the levels of plasma AVP and ANP.     

Appearance of specific proteins in the apical plasma membrane of cultured renal collecting duct principal cell epithelium after chronic administration of aldosterone and arginine vasopressin

Principal cell epithelium of renal collecting duct from neonatal rabbit kidney was cultured in the presence of aldosterone (1 x 10(-6) M) and arginine vasopressin (AVP; 1 x 10(-6) M) for 10 days to investigate, by immunohistochemical methods using specific monoclonal antibodies, whether the hormones influence the expression and insertion of plasma membrane proteins. The experiments demonstrated that aldosterone alone or aldosterone plus AVP significantly increased the number of epithelial cells reacting at the luminal and lateral plasma membrane with the antibodies CD 2 and 3, specific for renal collecting duct, as we have shown in the kidney. In cultures treated with aldosterone and aldosterone plus AVP, nearly all epithelial cells were labelled by the antibodies, while controls or AVP treatment showed 41% and 24% unreactive cells, respectively. These findings were complemented with electrophysiological experiments, in which epithelia pretreated by aldosterone or aldosterone plus AVP showed significantly hyperpolarized transepithelial voltage (Vte) and higher resistance (Rte) than controls or AVP-treated specimens. The experiments demonstrated that chronic administration of aldosterone or of aldosterone plus AVP to increase Na+-transport was paralleled by the appearance of collecting-duct-specific proteins in the epithelium. Consequently, this result indicates that aldosterone influences the functional maturity of the cultured epithelium.     

AQP1 gene expression is upregulated by arginine vasopressin and cyclic AMP agonists in trophoblast cells

Aquaporins (AQPs) are water channels that regulate water flow in many tissues. As AQP1 is a candidate to regulate placental fluid exchange, we sought to investigate the effect of arginine vasopressin (AVP) and cAMP agonists on AQP1 gene expression in first trimester-derived extravillous cytotrophoblasts (HTR-8/Svneo) and two highly proliferative carcinoma trophoblast-like cell lines but with a number of functional features of the syncytiotrophoblast namely; JAR and JEG-3 cells. Our data demonstrated that AVP (0.1 nM) significantly increased the expression of AQP1 mRNA at 10 h in HTR-8/SVneo and JEG-3 cells (P<0.05). Both SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP, and forskolin, an adenylate cyclase stimulator significantly increased AQP1 mRNA expression in all cell lines after 2 h in a dose-dependent manner (P<0.05) with a parallel increase in protein expression. In the time course study, 5 microM of either SP-cAMP or forskolin significantly stimulated AQP1 mRNA expression after 2 h in HTR-8/SVneo cells and after 10 h in JAR and JEG-3 cells. AQP1 protein expression was highest after 20 h in both HTR-8/SVneo and JEG-3 cells (P<0.05). AVP-stimulated cAMP elevation was blocked in the presence of 9-(tetrahydro-2'-furyl) adenine (SQ22536) (100 microM), a cell-permeable adenylate cyclase inhibitor (P<0.05). These results indicate that in trophoblasts-like cells AQP1 gene expression is upregulated by both AVP and cAMP agonists. Furthermore, our data demonstrate that a cAMP-dependent pathway is responsible for the AVP effect on AQP1. Thus, modulation of AQP1 expression by maternal hormones may regulate invasion and fetal-placental-amnion water homeostasis during gestation.     

Proteomic analysis of AQP11-null kidney: Proximal tubular type polycystic kidney disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by the mutation of polycystins (PC-1 or PC-2), in which cysts start from the collecting duct to extend to all nephron segments with eventual end stage renal failure. The cyst development is attenuated by a vasopressin V2 receptor antagonist tolvaptan which, however, will not affect proximal tubule cysts devoid of V2 receptor. Aquaporin-11 (AQP11) is expressed selectively in the proximal tubule of the kidney and AQP11-null kidneys have a disruptive PC-1 trafficking to the plasma membrane to develop polycystic kidneys. Here, we analyzed AQP11-null kidneys at the beginning of cyst formation by quantitative proteomic analysis using Tandem Mass Tag (TMT). Among ~ 1200 identified proteins, 124 proteins were differently expressed by > 1.5 or < 0.8 fold change. A pancreatic stone inhibitor or a growth factor, lithostathine-1 (Reg1) was most enhanced by 5 folds which was confirmed by western blot, while mitochondria-related proteins were downregulated. The identified proteins will be new target molecules for the treatment of proximal tubular cysts and helpful to explore the functional roles of AQP11 in the kidney.     

AT1a receptor signaling is required for basal and water deprivation-induced urine concentration in AT1a receptor-deficient mice

It is well recognized that ANG II interacts with arginine vasopressin (AVP) to regulate water reabsorption and urine concentration in the kidney. The present study used ANG II type 1a (AT(1a)) receptor-deficient (Agtr1a(-/-)) mice to test the hypothesis that AT(1a) receptor signaling is required for basal and water deprivation-induced urine concentration in the renal medulla. Eight groups of wild-type (WT) and Agtr1a(-/-) mice were treated with or without 24-h water deprivation and 1-desamino-8-d-AVP (DDAVP; 100 ng/h ip) for 2 wk or with losartan (10 mg/kg ip) during water deprivation. Under basal conditions, Agtr1a(-/-) mice had lower systolic blood pressure (P < 0.01), greater than threefold higher 24-h urine excretion (WT mice: 1.3 ± 0.1 ml vs. Agtr1a(-/-) mice: 5.9 ± 0.7 ml, P < 0.01), and markedly decreased urine osmolality (WT mice: 1,834 ± 86 mosM/kg vs. Agtr1a(-/-) mice: 843 ± 170 mosM/kg, P < 0.01), without significant changes in 24-h urinary Na(+) excretion. These responses in Agtr1a(-/-) mice were associated with lower basal plasma AVP (WT mice: 105 ± 8 pg/ml vs. Agtr1a(-/-) mice: 67 ± 6 pg/ml, P < 0.01) and decreases in total lysate and membrane aquaporin-2 (AQP2; 48.6 ± 7% of WT mice, P < 0.001) and adenylyl cyclase isoform III (55.6 ± 8% of WT mice, P < 0.01) proteins. Although 24-h water deprivation increased plasma AVP to the same levels in both strains, 24-h urine excretion was still higher, whereas urine osmolality remained lower, in Agtr1a(-/-) mice (P < 0.01). Water deprivation increased total lysate AQP2 proteins in the inner medulla but had no effect on adenylyl cyclase III, phosphorylated MAPK ERK1/2, and membrane AQP2 proteins in Agtr1a(-/-) mice. Furthermore, infusion of DDAVP for 2 wk was unable to correct the urine-concentrating defects in Agtr1a(-/-) mice. These results demonstrate that AT(1a) receptor-mediated ANG II signaling is required to maintain tonic AVP release and regulate V(2) receptor-mediated responses to water deprivation in the inner medulla.     

Treatment with the vascular disrupting agent combretastatin is associated with impaired AQP2 trafficking and increased urine output

Combretastatin A-4 disodium phosphate (CA4P) is a vascular disrupting agent known to mediate its effects primarily on tumor blood vessels. CA4P has previously been shown to induce a significant increase in mean arterial blood pressure and in hemoglobin concentration in mice. In the present study, we examined whether this is associated with a general leakage of water into certain tissues or with changes in renal water handling. Munich-Wistar rats received either CA4P (30 mg/kg body wt) or saline intraperitoneally as a bolus injection. One hour later, hemoglobin concentration and mean blood pressure increased significantly. MRI showed no significant changes in tissue water content following CA4P administration. However, urine output and salt excretion increased 1 h after CA4P treatment, without changes in urinary and medullary osmolality. Aquaporin 2 (AQP2) mRNA levels in kidney inner medulla did not change 1 h after CA4P treatment, but semiquantitative confocal laser-scanning microscopy analysis demonstrated a decrease in phosphorylated AQP2 (pS256-AQP2) apical distribution within the collecting ducts of CA4P-treated rats compared with the characteristic apical localization in control rats. Furthermore, we demonstrated that CA4P cause disruption of microtubules and a weaker apical labeling of pS256-AQP2 in collecting duct principal cells within 1 h. In conclusion, our data indicate that water escapes from the vascular system after CA4P treatment, and it may take place primarily through a renal mechanism. The CA4P-mediated increase in urine output seems to be a local effect in the collecting ducts due to reduced AQP2 trafficking to the apical plasma membrane.