Aquaporins in Urinary Extracellular Vesicles (Exosomes).

Since the successful characterization of urinary extracellular vesicles (uEVs) by Knepper's group in 2004, these vesicles have been a focus of intense basic and translational research worldwide, with the aim of developing novel biomarkers and therapeutics for renal disease. Along with these studies, there is growing evidence that aquaporins (AQPs), water channel proteins, in uEVs have the potential to be diagnostically useful. In this review, we highlight current knowledge of AQPs in uEVs from their discovery to clinical application.

Diuretic effect of Lagopsis supina fraction in saline-loaded rats is mediated through inhibition of aquaporin and renin-angiotensin-aldosterone systems and up-regulation of atriopeptin.

Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. has been used as a diuretic agent in China for centuries with limited scientific evidence. This study investigated the diuretic efficacy and underlying mechanism of a macroporous adsorption resin with 30% ethanol elution fraction from L. supina (LSC) in saline-loaded rats and to identify its phytochemicals by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS). As a result, 18 phenylpropanoids, 14 flavonoids and 15 others were identified in LSC, among which stachysoside A and acteoside could be the main bio-active constituents responsible for the diuretic effect. In parallel, the daily administration of LSC (16, 32 and 64 mg/kg) markedly promoted urinary excretion after 2 h of treatment. Moreover, LSC had no effect on urinary Na+ and K+ concentrations, as well as on serum Na+-K+-ATPase activity. Meanwhile, LSC significantly decreased the serum levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), aldosterone (ALD), aquaporin (AQP) 1, AQP2 and AQP3, suppressed renal AQP1, AQP2, and AQP3 mRNA expressions, down-regulated AQP1, AQP2 and AQP3 protein levels, and up-regulated serum atriopeptin (ANP) level in a dose-dependent manner. These findings suggest that LSC has acute and prolonged diuretic effects by inhibiting the AQPs, RAAS, and upregulation of atriopeptin in saline-loaded rats, and this finding support LSC as a novel diuretic agent.

Exaggerated urinary excretion of aquaporin-2 in the pathological state of impaired water excretion dependent upon arginine vasopressin.

The present study was undertaken to determine whether urinary excretion of aquaporin-2 (UAQP-2) is of value to diagnose the pathological state of water retention and hyponatremia. UAQP-2 under ad libitum water drinking was 429 fmol/mg creatinine in the patients with water retention, a value significantly greater than that of 153 fmol/mg creatinine in the normal subjects. An acute oral water load test (20 mL/kg BW) was performed in 7 normal subjects (22-25 yr old) and 10 patients with water retention and hyponatremia (55-75 yr old). The percent excretion of the water load was only 30% in the patient group compared with 70% in the control group (P < 0.01). In the control group, minimal urinary osmolality was as low as 131 mosmol/kg H2O, which was responsible for the decrease in plasma arginine vasopressin (AVP) levels after the reduction in plasma osmolality. In the patient group, minimal urinary osmolality was 320 mosmol/kg H2O, and free water clearance remained below 0.6 mL/min after the water load. This impaired water excretion was consistent with the nonsuppressible levels of plasma AVP despite hypoosmolality. The nadir of UAQP-2 was obtained at 60-90 min. The minimal UAQP-2 was reduced to 284 fmol/mg creatinine, a value significantly greater than that of 76 fmol/mg creatinine in the control group. Similar results were obtained in the 6 patients with hypopituitarism, who had impaired water excretion and marked hyponatremia. Water excretion was totally normalized after the replacement of hydrocortisone (excretion of water load, 31% vs. 102%; P < 0.01). Hydrocortisone replacement also significantly reduced the minimal UAQP-2 from 225 to 49 fmol/mg creatinine after the acute oral water load, a value comparable to that in the control subjects. These results indicate that UAQP-2 is a potent marker to diagnose the pathological state of impaired water excretion and hyponatremia, dependent upon AVP, in patients with water retention and hypopituitarism.

Urinary excretion of aquaporin-2 water channel differentiates psychogenic polydipsia from central diabetes insipidus.

The present study was undertaken to determine whether urinary excretion of aquaporin-2 (AQP-2) water channel under ad libitum water intake is of value to differentiate polyuria caused by psychogenic polydipsia from central diabetes insipidus. A 30-min urine collection was made at 0900 h in 3 groups of: 11 patients with central diabetes insipidus (22-68 yr old), 10 patients with psychogenic polydipsia (28-60 yr old), and 15 normal subjects (21-38 yr old). In the patients with central diabetes insipidus, the plasma arginine vasopressin level was low despite hyperosmolality, resulting in hypotonic urine. Urinary excretion of AQP-2 was 37 +/- 15 fmol/mg creatinine, a value one-fifth less than that in the normal subjects. In the patients with psychogenic polydipsia, plasma arginine vasopressin and urinary osmolality were as low as those in the patients with central diabetes insipidus. However, urinary excretion of AQP-2 of 187 +/- 45 fmol/mg creatinine was not decreased, and its excretion was equal to that in the normal subjects. These results indicate that urinary excretion of AQP-2, under ad libitum water drinking, participates in the differentiation of psychogenic polydipsia from central diabetes insipidus.

Close association of urinary excretion of aquaporin-2 with appropriate and inappropriate arginine vasopressin-dependent antidiuresis in hyponatremia in elderly subjects.

The present study was undertaken to determine whether urinary excretion of aquaporin-2 (AQP-2) participates in the involvement of arginine vasopressin (AVP) in hyponatremia less than 130 mmol/L in 33 elderly subjects (> or =65 yr old) during the last 5-yr period. Subjects were separated into euvolemic hyponatremia groups: 13 with hypopituitarism, 8 with syndrome of inappropriate secretion of antidiuretic hormone (SIADH), 8 with mineralocorticoid-responsive hyponatremia of the elderly, and 4 with miscellaneous diseases. Approximately 40% of those with hyponatremia was derived from hypopituitarism, but severe hyponatremia was found in the patients with SIADH and mineralocorticoid-responsive hyponatremia of the elderly. Plasma AVP levels remained relatively high despite hypoosmolality and were tightly linked with exaggerated urinary excretion of AQP-2 and antidiuresis in the 3 groups of patients, except for one miscellaneous one. An acute water load test verified the impairment in water excretion, because the percent excretion of the water load was less than 42% and the minimal urinary osmolality was not sufficiently diluted. Also, plasma AVP and urinary excretion of AQP-2 were not reduced after the water load. The inappropriate secretion of AVP was evident in the patients with SIADH and hypopituitarism, and hydrocortisone replacement normalized urinary excretion of AQP-2 and renal water excretion in those with hypopituitarism. In contrast, the appropriate antidiuresis seemed to compensate loss of body fluid in the patients with mineralocorticoid-responsive hyponatremia of the elderly, who lost circulatory blood volume by 7.3% (mean). Fludrocortisone acetate increased renal sodium handling and body fluid, resulting in the reduction in AVP release and urinary excretion of AQP-2 in mineralocorticoid-responsive hyponatremia of the elderly. These findings indicate that urinary excretion of AQP-2 may be a more sensitive measure of AVP effect on renal collecting duct cells than are plasma AVP levels, and that increased urinary excretion of AQP-2 shows exaggerated AVP-induced antidiuresis in hyponatremic subjects in the elderly. In addition, mineralocorticoid-responsive hyponatremia of the elderly has to be carefully differentiated from SIADH in elderly subjects.

Urinary aquaporin-2 excretion in nocturnal enuresis.

OBJECTIVE: To evaluate the role of the arginine vasopressin (AVP)-aquaporin-2 (AQP-2) axis in the pathogenesis of nocturnal enuresis. STUDY PARTICIPANTS: Twelve children (seven male and five female), aged 11.6+/-4.3 (6.7-15.6) years, suffering from primary monosymptomatic nocturnal enuresis and 12 healthy children, matched for sex and age. Enuretic children were further subdivided into responders and non-responders to treatment with 1-desamino-8-d-AVP (DDAVP). METHODS: Serum concentrations of AVP, and plasma and urine osmolality were measured at night (0100, 0400 and 0700 h), together with nocturnal urinary excretion of AQP-2 (2000-0800 h). Magnetic resonance imaging (MRI) of the pituitary gland was carried out to evaluate the amount of AVP stored in the posthypophysis. RESULTS: Mean AVP serum concentrations were similar in patients and controls. Urinary AQP-2 was also similar in patients and controls, but responders had a significantly lower level of AQP-2 than non-responders (P<0.005). Plasma osmolality was greater in patients than in controls (P<0.001), whereas urinary osmolality was similar in both groups. No difference in the ratio of the signal intensity of the posterior lobe of the hypophysis to that of the pons (AVP content) was found between patients and controls or between responders and non-responders. CONCLUSION: A decreased urinary excretion of AQP-2 is associated with, and seems to have a role in, nocturnal enuresis, at least in some children, and this could also explain why only some of them respond to DDAVP treatment.

Renal water channel expression in newborns: measurement of urinary excretion of aquaporin-2.

Aquaporin-2 (AQP-2) encodes the vasopressin-regulated "water channels" of the renal collecting duct and is excreted in human urine. We measured urinary excretion of AQP-2 by radioimmunoassay in 15 term and 10 preterm infants on day 1 and day 4 of life to determine the molecular basis of water balance during the newborn period. AQP-2 was detectable in the urine of term and preterm newborns, but AQP-2 excretion was severalfold less than the reported level in normal adults. Urinary excretion of AQP-2 significantly decreased postnatally, in parallel with a reduction in urine osmolality and arginine vasopressin (AVP) excretion. Urinary AQP-2 correlated positively and significantly with urine osmolality on days 1 and 4 and with AVP on day 1 in both groups. No significant differences were detected in AQP-2 levels between term and preterm newborns. Our findings suggest that vasopressin-regulated water channels are expressed in the renal collecting duct of both term and preterm newborns, although to a lesser extent as compared with adults, and these channels encoded by AQP-2 contribute to the urine concentrating power of the newborn kidney.

Urinary concentrating defect in rats given Shiga toxin: elevation in urinary AQP2 level associated with polyuria.

Shiga toxin (Stx) plays a central role in the etiology of hemolytic uremic syndrome (HUS) associated with Stx-producing Escherichia coli infection. The deposition of Stx2 in the renal collecting duct epithelial cells of rats administered Stx2 intravenously has been demonstrated by immunohistochemistry, and these rats were shown to develop substantial morphological changes in the kidney tubules, associated with polyuria. Severe polyuria was observed as an early event with no other obvious sequelae after Stx administration, in parallel with elevated urinary level of aquaporin 2 (AQP2) water channel protein that was determined by a sandwich EIA assay. Immunoblotting revealed that Stx treatment markedly induced an elevation in urinary AQP2 level and reduction in AQP2 protein in the renal plasma membranes. Elevated urinary AQP2 level was a more sensitive marker to assess Stx-induced renal tubular damage than urinary beta2-microglobulin or N-acetyl-beta-D-glucosaminidase in rats. Stx2 caused more severe renal tubular impairment than Stx1. Change in urinary AQP2 level by Stx1 and Stx2 at non-lethal doses of 40 ng/kg and 10 ng/kg, respectively, was reversed at 7 days in association with recovery of urinary concentrating ability, suggesting that there is a causative link.

Effect of exercise on natriuretic peptides in plasma and urine in chronic heart failure.

BACKGROUND: Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are elevated in chronic heart failure (CHF). ANP is known to be increased during exercise in healthy subjects and CHF, while the response in BNP during exercise is less clear and does not exist in C-type natriuretic peptide (CNP) and aquaporin-2 (AQP2) in either healthy subjects or CHF. METHODS: Eleven patients with CHF and eleven healthy subjects performed a maximal aerobic exercise test. ANP and BNP in plasma were determined every 3 min and at maximum exercise by radioimmunoassay (RIA) and CNP and AQP2 in urine were determined before and after the exercise test by RIA. RESULTS: The absolute increase in BNP during exercise was higher in patients with CHF (CHF: 4.1 pmol/l; healthy subjects: 1.3 pmol/l, P<0.05) and was positively correlated to BNP at rest (P<0.05), while the absolute increase in ANP during exercise was the same in the two groups (CHF: 4.2 pmol/l; healthy subjects: 6.8 pmol/l, not significant, NS). In CHF, exercise did not change either u-CNP excretion (rest: 9.8 ng/mmol creatinine; after exercise: 8.8 ng/mmol, NS) or u-AQP2 (rest: 466 ng/mmol creatinine; after exercise: 517 ng/mmol creatinine, NS) as well as in healthy subjects where u-CNP (rest: 9.7 ng/mmol creatinine; after exercise: 9.2 ng/mmol creatinine) and u-AQP2 (rest: 283 ng/mmol creatinine; after exercise: 307 ng/mmol creatinine) were the same at rest and after exercise. CONCLUSION: The absolute increase in BNP during exercise is higher in patients with CHF compared to healthy subjects. It is suggested that this is a compensatory phenomenon to improve the exercise capacity in CHF, and that BNP is a more important factor in cardiovascular homeostasis during exercise in CHF than ANP.

Urinary aquaporin-2 excretion in dogs: a marker for collecting duct responsiveness to vasopressin.

In humans, the urinary aquaporin-2 (U-AQP2) excretion closely parallels changes in vasopressin (VP) action and has been proposed as a marker for collecting duct responsiveness to VP. This report describes the development of a radioimmunoassay for the measurement of U-AQP2 excretion in dogs. In addition, the localization of AQP2 in the canine kidney was investigated by immunohistochemistry. Basal U-AQP2 excretion was highly variable among healthy dogs. Two hours after oral water loading, the mean U-AQP2/creatinine ratio decreased significantly from (231 +/- 30) x 10(-9) to (60 +/- 15) x 10(-9) (P = 0.01), while the median plasma VP concentration decreased from 4.2 pmol/l (range 2.2-4.8 pmol/l) to 1.2 pmol/l (range 1.0-1.9 pmol/l). Subsequent intravenous administration of desmopressin led to a significantly increased mean U-AQP2/creatinine ratio of (258 +/- 56) x 10(-9) (P = 0.01). Two hours of intravenous hypertonic saline infusion (20% NaCl, 0.03 ml/kg body weight/min) significantly increased the mean U-AQP2/creatinine ratio from (86 +/- 6) x 10(-9) to (145 +/- 23) x 10(-9) (P = 0.045), while the median plasma VP concentration increased significantly from 2.2 pmol/l (range 1.1-6.3 pmol/l) to 17.1 pmol/l (range 8.4-67 pmol/l) (P < 0.001). Immunohistochemistry revealed extensive labeling for AQP2 in the kidney collecting duct cells, predominantly localized in the apical and subapical region. As in humans, U-AQP2 excretion in dogs closely reflects changes in VP exposure. Urinary AQP2 excretion may become a diagnostic tool in dogs for the differentiation of polyuric conditions such as (partial) central or nephrogenic diabetes insipidus, primary polydipsia, and inappropriate VP release.

Urinary excretion of aquaporin-2 water channel protein in chronic heart failure rats.

OBJECTIVE: To study the urinary excretion of aquaporin-2 (AQP2) water channel protein, and the relationship between urine AQP2 concentration and renal AQP2 gene expression in chronic heart failure (CHF) rats. METHODS: Male Sprague-Dawley rats (200 g-250 g) underwent either a left coronary artery ligation, a model of CHF, or a sham-operation. Nine weeks after surgery, urinary AQP2 concentrations and renal AQP2 protein levels were measured by Western blot. RESULTS: The urinary concentration of AQP2 water channel protein increased significantly in CHF rats as compared with sham-operated rats (365.6% +/- 102.9% vs 98.5% +/- 47.6%, P < 0.01). There was positive correlation between urinary AQP2 concentration and renal AQP2 protein expression (r = 0.89, P < 0.01). CONCLUSION: The urinary concentration of AQP2 water channel protein increases significantly in chronic heart failure rats.

[Urinary excretion of aquaporin-2 in water metabolism disorders].

Water metabolism plays an essential role in the homeostasis of body fluids in animals and humans. It is regulated by arginine vasopressin (AVP), renal function and water drinking. Disorders of water metabolism result in an increase or decrease in a body water or fluid, which manifest as hyponatremia, hypernatremia, polyuria, dehydration or edema. In the pathogenesis of such pathological conditions AVP is either directly or indirectly involved. Aquaporin-2 (AQP-2) is an AVP-dependent water channel in renal collecting duct cells. Approximately 3% of AQP-2 is excreted into the urine, which is measurable by RIA or Western blot using a specific antibody against AQP-2. There was positive relationship between urinary excretion of AQP-2 (UAQP-2) and plasma AVP levels in normal subjects. UAQP-2 varied in a wide range under ad libitum water drinking. The level of UAQP-2 was one eighth less in patients with central diabetes insipidus than in normal subjects, and it was 2.8-fold greater in patients with water retention. A hypertonic saline infusion test manifested the difference in the UAQP-2 response to an increase in plasma osmolality between the patients with central diabetes insipidus and the normal subjects. Acute oral water load clarified the impaired water excretion and the persistent elevation of UAQP-2 in patients with water retention. Such increased UAQP-2 was linked to nonsuppressible levels of plasma AVP despite hypoosmolality. These results indicate that UAQP-2 is a useful marker to diagnose disorders of water metabolism.

Enzyme-linked immunosorbent assay for urinary aquaporin-2 water channel protein measurement.

OBJECTIVE: To develop an efficient and stable enzyme-linked immunosorbent assay (ELISA) for detecting urinary aquaporin-2 (AQP2) water channel protein. METHODS: Rat AQP2 C-terminal peptides (CELHSPQSLPRGSKA) were synthesized and linked to KLH to prepare rabbit anti-AQP2 polyclonal antibodies, and IgG of the antibodies were labeled with horseradish peroxidase (HRP). Rat models of congestive heart failure (CHF) was established by ligation of the left coronary artery, in which both direct and sandwich ELISA for urinary AQP2 detection were tested. RESULTS: Double antibody sandwich ELISA was able to detect urinary AQP2 as low as 15.625 pmol/ml with intra-and inter-assay coefficients of variance (CVs) of 4.65% and 14.05% respectively. Urinary AQP2 concentration determined by this assay showed significant positive relation to that by Western blot analysis in CHF rats. CONCLUSION: Double antibody sandwich ELISA was successfully established to detect urine AQP2 in CHF rats, which is more efficient and simpler than Western blot analysis.

Urine concentration and avian aquaporin water channels.

Although birds and mammals have evolved from primitive tetrapods and advanced divergently, both can conserve water by producing hyperosmotic urine. Unique aspects in the avian system include the presence of loopless and looped nephrons, lack of the thin ascending limb of Henle's loop, a corticomedullary osmotic gradient primarily consisting of NaCl without contribution of urea, and significant postrenal modification of final urine. The countercurrent multiplier mechanism operates between the descending and ascending limbs of Henle via recycling of a single solute (NaCl) with no water accompaniment, forming an osmotic gradient along the medullary cone. Bird kidneys and developing rat kidneys share morphological and functional characteristics. Avian kidneys express aquaporin (AQP) 1, 2, and 4 homologues that share considerable homology with mammalian counterparts, but their distribution and function may not be the same. AQP2 expression in Japanese quail (q) evolves in the collecting duct of early metanephric kidneys and continues to increase in intensity and distribution during nephrogenesis and maturation. qAQP2 mRNA and protein are increased by arginine vasotocin (avian ADH), but vasotocin-induced enhancement of cAMP production and water permeability are less marked than in mammalian kidneys. Nephrogenesis is delayed by insufficient nutrition in avian embryos and newborns and results in fewer nephrons and an impaired water balance in adults. Diabetes insipidus quail with homozygous autosomal recessive mutation and an unaffected vasotocin system have low AQP2 expression, underdeveloped medullary cones. Comparative studies will provide important insight into integrative, cellular, and molecular mechanisms of epithelial water transport and its control by humoral, neural, and hemodynamic mechanisms.

Down-regulation of urinary AQP2 and unaffected response to hypertonic saline after 24 hours of fasting in humans.

BACKGROUND: In rats, 24 hours of fasting impairs urinary concentrating ability by down-regulation of aquaporin-2 (AQP2). We tested the hypothesis that 24 hours of fasting in humans reduces the capability to form AQP2 and impairs the antidiuretic response to hypertonic saline infusion. METHODS: In a crossover study of 14 healthy subjects, the effect of 24 hours of fasting was compared to a nonfasting control experiment on urinary excretion of AQP2 (u-AQP2), free water clearance (C(H(2)O)), plasma arginine vasopressin (AVP), urinary cyclic AMP (u-cAMP), and natriuretic peptides. The following response to 3% sodium infusion was measured using the same effect variables. U-AQP2, AVP, and u-cAMP were determined by radioimmunoassays. RESULTS: Fasting during 24 hours reduced u-AQP2 (14%), increased AVP (30%) despite a reduction in serum osmolality (P < 0.05), and depleted volume. C(H(2)O) and urine volume were not reduced, thus relatively increased after fasting. u-cAMP was not significantly different between the two procedures. Three percent saline resulted in the same relative increases in AVP, serum osmolality, u-AQP2, and u-cAMP and decreases in C(H(2)O) and urine volume independently of fasting. The reduced u-AQP2 and increased AVP after fasting were maintained during and after saline infusion. CONCLUSION: Twenty-four hours of fasting decreased u-AQP2 and reduced urine osmolality likely as a result of decreased sensitivity of collecting duct cells to AVP. Fasting-related insensitivity of collecting duct cells to AVP was restored by 3% saline infusion. Finally, after saline infusion, other factors such as the increased plasma atrial natriuretic peptide (p-ANP) levels could contribute to the u-AQP2 regulation.