OBJECTIVETo study the interaction among aquaporin1 (AQP), aquaporin2 (AQP2) and antisecretory factor( AF) , and their expression in the rat inner ear for furthur understanding of Meniere' s disease.

METHODSInner ear tissue section of six healthy male Sprague-Dawley rats was performed and Envision immunochemical staining was applied to detect the expression of AF, AQP1 and AQP2 in the rat inner ear. Vestibular and cochlear tissues of twenty healthy male Sprague-Dawley rats were dissected. Coimmunoprecipitation and Western Blot were used to specifically immunoprecipitate AF protein in the vestibular and cochlear tissues with monoclonal antibodies against AQP1 and polyclonal antibodies antibodies against AQP2 to detect the above precipitate with specific antibodies against AF.

RESULTS(1) AF was widely distributed in the inner ear, such as marginal cells of stria vascularis , five classes of spiral ligament fibrocyte , Reissner's membrane, basilar membrane, ampullar crest and so on with mild or moderate staining. In addition, round membrane was moderately or markedly stained. Positive immunostaining was found in the cochlear spiral ganglion, vestibular nerve and cochlear nerve. AQP1 was distributed in the intermediate cells in stria vascularis, type III fibrocyte of spiral ligament, basilar membrane and round membrane with moderate to marked degree of immunostaining intensity. AQP2 was mainly localized to the type II, IV, and V fibrocyte of spiral ligament, with moderate to marked degree of immunostaining intensity, round membrane was weakly stained. (2) No band was observed in the control and a single immunoreactive band of 60 000 was observed, which was equal to the molecular mass of AF.

CONCLUSIONS(1) AF, AQP1 and AQP2 have its individual specific localization in the rat inner ear, which was close to the parts of endolymph, so regulating water of the endolymph may be possible. (2) The range of localization of AF overlapped the distribution of AQP1 and AQP2. The results showed the existence of AF protein in the immunoprecipitate using co-immunoprecipitation combined with Western Blot. It suggested that the interaction between AQP1, AQP2 and AF might be possible.

Animals ; Aquaporin 1 ; metabolism ; Aquaporin 2 ; metabolism ; Cochlea ; metabolism ; Ear, Inner ; metabolism ; Male ; Neuropeptides ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUND: The present study was aimed at investigating whether there is a mechanism exerted by endogenous nitric oxide(NO) in the regulation of aquaporin(AQP) water channels in the kidney. METHODS: Male Sprague-Dawley rats were treated with N(G)-nitro-L-arginine methyl ester(L-NAME, 40 mg/L drinking water) to inhibit the endogenous generation of nitric oxide. Four weeks later, total abundance and shuttling of AQP2 proteins were determined in different regions of the kidney. RESULTS: Chronic inhibition of NO synthesis increased the expression of AQP2 channels in cortex, outer medulla, and inner medulla of the kidney. The AQP2 shuttling was not significantly altered, as evidenced by an unaltered ratio of AQP2 expression in the membrane fraction versus that in the cytoplasmic fraction. CONCLUSION: It is suggested that endogenous NO activity plays a tonic inhibitory role in the expression of AQP2 channels in the kidney.
Animals ; Aquaporin 2* ; Aquaporins* ; Cytoplasm ; Drinking ; Humans ; Kidney* ; Male ; Membranes ; Nitric Oxide* ; Rats* ; Rats, Sprague-Dawley

Vasopressin, a neurohypophyseal peptide hormone, is the endogenous agonist at V1a, V1b, and V2 receptors. The most important physiological function of vasopressin is the maintenance of water homeostasis through interaction with V2 receptors in the kidney. Vasopressin binds to V2 receptor and increases the number of aquaporin-2 at the apical plasma membrane of collecting duct principal cells. That induces high water permeability across the membrane. Several non-peptide vasopressin receptor antagonists have been developed and are being studied primarily for treating conditions characterized by hyponatremia and fluid overload. Further studies are needed to determine how they are best used in these situations.
Aquaporin 2 ; Cell Membrane ; Homeostasis ; Hyponatremia ; Kidney ; Membranes ; Permeability ; Receptors, Vasopressin ; Vasopressins

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP (400 muM) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.
Animals ; Aquaporin 2 ; Cell Line ; Embryonic Structures ; Fibroblasts ; Luciferases ; Mice* ; Osmolar Concentration ; Promoter Regions, Genetic

Prostaglandin E2 (PGE2), a bioactive lipid mediator, is one of the most important locally acting factors involved in a variety of physiological and pathophysiological processes. PGE2 binds with four EP receptors (EP1-4) to activate G protein-coupled receptor signaling responses. Recent functional and molecular studies have revealed that PGE2 plays an essential role in regulation of renal fluid transport via a variety of mechanisms. The water balance mainly depends on the regulation of aquaporin-2 (AQP2) by arginine vasopressin (AVP) in renal collecting duct principal cells. In recent years, increasing evidence suggests that PGE2 plays an important role in renal water reabsorption in the collecting ducts. In this paper, we reviewed the role of PGE2 and its receptors in the regulation of water reabsorption in the kidney, which may provide a new therapeutic strategy for many diseases especially nephrogenic diabetes insipidus.
Aquaporin 2/metabolism* ; Biological Transport ; Diabetes Insipidus, Nephrogenic ; Dinoprostone ; Humans ; Water/metabolism*

The present study analyzed the effect of Citri Reticulatae Pericarpium on endogenous metabolites in spleen deficiency and phlegm dampness syndrome by metabolomics, and explored the underlying mechanism of Citri Reticulatae Pericarpium in the treatment of spleen deficiency and phlegm dampness syndrome.The model of spleen deficiency and phlegm dampness syndrome was induced in rats by the multi-factor modeling method.The intervention effects of Citri Reticulatae Pericarpium on rats with spleen deficiency and phlegm dampness syndrome were preliminarily evaluated by observing the pathological changes of rat liver tissues and measuring the plasma content of pathological and biochemical indexes such as triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C).Immunohistochemistry was used to detect the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland, and the effect of Citri Reticulatae Pericarpium on aquaporin expression in rats with spleen deficiency and phlegm dampness syndrome was evaluated.Furthermore, UHPLC-ESI-MS/MS was used to analyze the metabolic profiles of rat plasma samples.Multiple methods, such as principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used for pattern recognition.Differential metabolites were screened out by t-test and variable importance in projection(VIP), followed by pathway analysis based on MetaboAnalyst 5.0.As revealed by experimental results, Citri Reticulatae Pericarpium could improve the pathological changes of liver tissues, increase the levels of HDL-C in the plasma, reduce the levels of TC, TG, and LDL-C, and enhance the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland of rats with spleen deficiency and phlegm dampness syndrome.In addition, 87 differential metabolites of spleen deficiency and phlegm dampness syndrome were screened out by UHPLC-ESI-MS/MS(the levels of 39 metabolites increased significantly and the levels of 48 metabolites decreased significantly), with the representatives of glycine, L-isoleucine, N-acetyl-L-tyrosine, xanthine, hypoxanthine, and trigonelline.The differential metabolites were mainly enriched in the pathways of steroid hormone biosynthesis, linoleic acid metabolism, and purine metabolism.This study distinguished and revealed the characteristic metabolic pattern of spleen deficiency and phlegm dampness syndrome by metabolomics.The preliminary construction of the OPLS-DA model provides an objective basis for the differentiation of spleen deficiency and phlegm dampness syndrome in traditional Chinese medi-cine(TCM), as well as ideas and methods for exploring the biological basis of TCM syndrome from the molecular level and the overall level.
Animals ; Aquaporin 2 ; Cholesterol, LDL ; Citrus/chemistry* ; Drugs, Chinese Herbal ; Metabolomics ; Rats ; Spleen ; Tandem Mass Spectrometry

Ubiquitination is known to be important for endocytosis and lysosomal degradation of aquaporin-2 (AQP2). Ubiquitin (Ub) is covalently attached to the lysine residue of the substrate proteins and activation and attachment of Ub to a target protein is mediated by the action of three enzymes (i.e., E1, E2, and E3). In particular, E3 Ub-protein ligases are known to have substrate specificity. This minireview will discuss the ubiquitination of AQP2 and identification of potential E3 Ub-protein ligases for 1-deamino-8-D-arginine vasopressin (dDAVP)-dependent AQP2 regulation.
Aquaporin 2 ; Deamino Arginine Vasopressin ; Endocytosis ; Kidney ; Kidney Tubules, Collecting ; Ligases ; Lysine ; Proteins ; Substrate Specificity ; Ubiquitin ; Ubiquitination ; Vasopressins

When the concentration of sodium (Na+) in arterial plasma (P(Na)) declines sufficiently to inhibit the release of vasopressin, water will be excreted promptly when the vast majority of aquaporin 2 water channels (AQP2) have been removed from luminal membranes of late distal nephron segments. In this setting, the volume of filtrate delivered distally sets the upper limit on the magnitude of the water diuresis. Since there is an unknown volume of water reabsorbed in the late distal nephron, our objective was to provide a quantitative assessment of this parameter. Accordingly, rats were given a large oral water load, while minimizing non-osmotic stimuli for the release of vasopressin. The composition of plasma and urine were measured. The renal papilla was excised during the water diuresis to assess the osmotic driving force for water reabsorption in the inner medullary collecting duct. During water diuresis, the concentration of creatinine in the urine was 13-fold higher than in plasma, which implies that ~8% of filtered water was excreted. The papillary interstitial osmolality was 600 mOsm/L > the urine osmolality. Since 17% of filtered water is delivered to the earliest distal convoluted tubule micropuncture site, we conclude that half of the water delivered to the late distal nephron is reabsorbed downstream during water diuresis. The enormous osmotic driving force for the reabsorption of water in the inner medullary collecting duct may play a role in this reabsorption of water. Possible clinical implications are illustrated in the discussion of a case example.
Animals ; Aquaporin 2 ; Aquaporins ; Creatinine ; Diuresis ; Membranes ; Nephrons ; Osmolar Concentration ; Permeability ; Phenobarbital ; Plasma ; Polyuria ; Punctures ; Rats ; Sodium ; Vasopressins ; Water

BACKGROUND: Bilateral ureteral obstuction (BUO) has been known to decrease the expression of renal aquaporin-2 (AQP2) and Na-K-2Cl cotransporter (NKCC2). The polyuria and urinary concentration defect in postobstructive diuresis (POD) may be explained by these molecular changes. By contrast, chronic infusion of antidiuretic hormone (ADH) has been known to increase the expression of renal AQP2 and NKCC2, but there have been no studies regarding the chronic effect of ADH in molecular level when introducing to POD. We tried to identify the changes of renal expression of AQP2 and NKCC2 in 24 hour BUO rat at POD-7 day and the chronic effect of ADH to the expression of AQP2 and NKCC2 in sham operation rat and in 24 hour BUO rat, at sham operation 7 day and at POD-7 day, respectively. METHODS: Twenty four Spraugue-Dawley rats were divided into four groups. Group I (Control group): sham operation rats(n=6). Group II (BUO group): 24 hour BUO and release of it (n=6). Group III (dDAVP group): dDAVP (1-deamino-8d-arginine vasopressin: V2-receptor-selective agonist) infusion at rate of 20 ng/hour by osmotic minipump subcutaneously for 7 days in sham operation rats (n=6). Group IV (BUO+dDAVP group): dDAVP infusion at rate of 20 ng/hour by osmotic minipump by same method as Group III in 24 hour BUO rats (n=6). All rats were sacrificed at POD-7 day (Group II, Group IV) or sham operation-7 day (Group I, Group III) and renal expression of AQP2 and NKCC2 were analyzed by immunohistochemistry and by Western blot method. Blood and urinary chemistry examinations were done concurrently. RESULTS: BUO group showed increased urine output and decreased urine osmolality (p<0.05) and decreased expressions of AQP2 and NKCC2 compared with Control group {29.1 +/- 4.2% vs. 100 +/- 10.0% (p< 0.05); 40.2 +/- 5.4% vs. 100 +/- 7.9% (p<0.05) respectively}. dDAVP group had decreased urine output from POD-1 day to POD-5 day and increased urine osmolality (p<0.05) POD-1 day to POD-7 day during and increased expressions of AQP2 and NKCC2 compared with Control group {206.5 +/- 19.0% vs. 100 +/- 10.0% (p<0.05); 180.6 +/- 13.3% vs. 100 +/- 7.9% (p<0.05) respectively}. But BUO group showed no difference in urine output and urine osmolality and expressions of AQP2 and NKCC2 compared with BUO+dDAVP group {29.1 +/- 4.2% vs. 42.2 +/- 2.3% (p<0.84); 40.2 +/- 5.4 % vs. 47.9 +/- 4.3% (p<0.91) respectively}. CONCLUSION: BUO and POD show decreased expressions of AQP2 and NKCC2 and the unresponsiveness to chronic ADH infusion may be the pathophysiologic basis of POD such as increased urine output, decreased urine osmolality.
Animals ; Aquaporin 2* ; Blotting, Western ; Chemistry ; Deamino Arginine Vasopressin ; Diuresis ; Immunohistochemistry ; Osmolar Concentration ; Polyuria ; Rats* ; Ureter* ; Ureteral Obstruction* ; Vasopressins

Whether there exists a Sympathetic neural mechanism regulating the expression of aquaporin (AQP) water channels in the kidney was investigated. Male Sprague-Dawley rats were treated with reserpine (1 mg/kg, IP), and the expression of AQP1-4 proteins was determined in the kidney one day thereafter. Following the treatment with reserpine, the systolic blood pressure measured in a conscious state was significantly decreased in the experimental group compared with that in the control (83+/-8 vs 124+/-6 mmHg n=6 each, P<0.05). The expression of AQP2 proteins was decreased in the cortex, outer medulla, and inner medulla. The decrease of AQP2 proteins was in parallel in the membrane and the cytoplasmic fractions, suggesting a preserved AQP2 targeting. No significant changes were observed in the expression of AQP1, AQP3, or AQP4. Neither basal nor AVP-stimulated formation of cAMP was significantly altered. These results suggest that the sympathetic nervous system has a tonic stimulatory effect specifically on the expression of AQP2 water channels in the kidney.
Animals ; Aquaporin 2* ; Aquaporins* ; Blood Pressure ; Cytoplasm ; Humans ; Kidney* ; Male ; Membranes ; Rats* ; Rats, Sprague-Dawley ; Reserpine* ; Sympathetic Nervous System