Urinary Excretion Of Aquaporin-2 Research Articles

PS-BPB05-3: SERINE PROTEASE INHIBITION EXERTS AQUARETIC EFFECT AND DECREASES URINARY EXOSOMAL AQP2 LEVELS

Objective: Serine proteases (SPs) play pivotal roles in various physiological processes. In the kidney, it is reported that the epithelial sodium channel (ENaC) is activated via the proteolytic cleavage of α and γ subunits by SPs. We previously reported that a synthetic SP inhibitor camostat mesilate (CM) suppressed the cleavage of γENaC, thereby decreasing sodium reabsorption and blood pressure in aldosterone-infused rats. These results suggest the important roles of SPs within the kidney in the regulation of sodium homeostasis and blood pressure. Here, we aimed to identify novel physiological roles of SPs in the renal tubule and understand the mechanism of actions of SP inhibitors by administering CM into healthy rats. Design and method: Six-week-old male Sprague-Dawley rats were divided into control and CM groups. CM group rats were subcutaneously implanted with sustained-release pellets of CM (14 mg/day). After 24 h urine collection was performed, rats were sacrificed at day 7 to obtain blood and kidney samples. Urine volume was measured during the experimental period, and blood and urinary electrolytes, blood vasopressin, and renal tissue osmolality were measured. Protein expression of SPs and aquaporin-2 (AQP2) was analyzed by Western blotting. Results: CM significantly increased urine volume by approximately two-fold throughout the experimental period [urine volume (mL/day): control, 15.5 ± 2.4; CM, 30.6 ± 5.8], in a urinary sodium and osmolyte excretion-independent manner, which indicated that free water excretion had occurred. The vasopressin, potassium, and calcium levels in the blood and osmolality in the inner medulla, which affect free water reabsorption in the renal tubule, remained unchanged after CM administration. CM decreased urinary exosomal AQP2 excretion, suggesting that the AQP2 activity was suppressed in the collecting duct. These changes were reversed by the infusion of desmopressin. Such water diuretic action caused by CM was independent of its action on prostasin and TMPRSS4, which are serine proteases associated with sodium and water homeostasis, respectively. Conclusion: Our results revealed the association of SP inhibition with free water diuresis in the kidney and demonstrated that CM administration resulted in the downregulation of AQP2. The current findings suggest that SP inhibitors could become a new class of aquaretic drugs.

Camostat mesilate, a serine protease inhibitor, exerts aquaretic effects and decreases urinary exosomal AQP2 levels

Serine proteases (SPs) play physiological roles in the kidney. We previously reported that a synthetic SP inhibitor, camostat mesilate (CM), suppressed sodium reabsorption in the renal tubule and showed natriuretic effects in aldosterone-infused rats. Here, we aimed to explore novel physiological roles of SPs in the renal tubule and understand the mechanism of actions of SP inhibitors, by administering CM to healthy rats. Sprague–Dawley rats were classified into control and CM (subcutaneous sustained-release pellet) groups and sacrificed on day 7. CM significantly increased urine volumes by approximately two-fold in a urinary sodium- and osmolyte excretion-independent manner, indicating the occurrence of free water excretion. Serum vasopressin, potassium, and calcium levels and the osmolality in the renal medulla, which all affect free water reabsorption in the renal tubule, remained unchanged after CM administration. CM decreased urinary exosomal AQP2 excretion, suggesting suppression of AQP2 activity in the collecting duct. These changes were reversed by desmopressin infusion. Water diuresis caused by CM was independent of its action on prostasin or TMPRSS4. Our results revealed the association of SP inhibition with free water handling and demonstrated that CM administration exerted diuretic effects with AQP2 downregulation, suggesting SP inhibitors as a new class of aquaretic drugs.

Renal expression and urinary excretion of aquaporin-2 in postobstructive uropathy in rats.

This work assessed the time course of water renal management together with aquaporin-2 (AQP2) kidney expression and urinary AQP2 levels (AQP2u) in obstructive nephropathy. Adult male Wistar rats were monitored after 1, 2, and 7days of bilateral ureteral release (bilateral ureteral obstruction (BUO); BUO-1, BUO-2 and BUO-7). Renal water handling was evaluated using conventional clearance techniques. AQP2 levels were assessed by immunoblotting and immunohistochemical techniques. AQP2 expression in apical membranes was downregulated in BUO-1 rats and upregulated both in BUO-2 and BUO-7 animals. AQP2 protein expression in whole cell lysate fraction from kidney cortex and medulla were significantly decreased in all the experimental groups. Concomitantly, mRNA levels of AQP2 decreased in renal medulla of all groups and in renal cortex from BUO-1; however, in renal cortex from BUO-2 and BUO-7 a recovery and an increase in the level of AQP2 mRNA were, respectively, observed. BUO-7 group showed a significant increase in AQP2u. The alterations observed in apical membranes AQP2 expression could explain, at least in part, the evolution time of water kidney management in the postobstructive phase of BUO. Additionally, the AQP2u increase after 7 days of ureteral release may be postulated as a biomarker of improvement in the kidney function.

Time course effects of methotrexate on renal handling of water and electrolytes in rats. Role of aquaporin-2 and Na-K-2Cl-cotransporter

Methotrexate (MTX) is a derivate of folic acid, commonly used as an anchor drug for the treatment and management of malignant diseases and autoimmune disorders. However, nephrotoxicity is an important drawback of MTX therapy. Unfortunately, there are not enough studies reporting the nature of the renal failure induced by MTX. Thus, the aim of this study was to evaluate the time course of renal handling of water and electrolytes in male Wistar rats, after the exposure to a unique dose of MTX (80 mg/kg b.w.). Experiments were carried out at day 2, day 4, day 8 and day 14 after MTX administration. Several parameters of kidney function related to water and electrolytes handling were evaluated. Renal expression and urinary excretion of aquaporin-2 (AQP2) and Na-K-2Cl-cotransporter (NKCC2) were determined by Western blotting. MTX produced alterations on water handling on the second day after treatment, showing a significant increase in solute free water reabsorption which might be mediated by the increased expression of AQP2 in apical membranes. On the other hand, MTX produced alterations on electrolytes handling on the fourth day after treatment, showing a significant decrease of sodium chloride excretion, mediated at least in part, by the increase renal expression of NKCC2. These results provide valuable information to clinical practice in order to be able to find therapeutic targets that diminish adverse effects and health deterioration. Moreover, MTX treatment altered AQP2 and NKCC2 urinary excretion allowing postulating these transporters as potential biomarkers of MTX induced nephrotoxicity.

The relationship between Aquaporin-2 protein levels in amniotic fluid and the fetal kidney in humans

ObjectivesAquaporin-2 (AQP2) is an important water channel protein that is expressed in the renal collecting duct and plays a key role in urine concentration and body water homeostasis. It has been demonstrated that the urinary excretion of AQP2 correlates strongly with its expression in the kidney in adult humans and rats. However, there have been no studies on the urinary excretion of AQP2 in human fetuses during development. Fetal urine is the main source of the amniotic fluid; we speculate that the level of AQP2 in the amniotic fluid could reflect the expression level of the AQP2 protein in the fetal kidney. The purpose of the present study was to explore the relationship between AQP2 in the amniotic fluid and that in the fetal kidney. MethodsIn the present study, the concentration of the AQP2 protein in human amniotic fluid was measured by Enzyme-linked immunosorbent assay (ELISA) and its expression level in human fetal kidneys were examined by wastern blot and immunohistochemistry. ResultsBoth the expression level of AQP2 in the fetal kidney (F = 195.9, P < 0.001) and the concentration of AQP2 in the amniotic fluid increased with gestational age (F = 1098, P < 0.001). Moreover, the concentration of AQP2 in the amniotic fluid was positively correlated with its expression level in the fetal kidney (r = 0.872, P < 0.0001). ConclusionsOur research indicates that AQP2 levels in the amniotic fluid may be used as a marker for AQP2 expression in the fetal kidney.

Low aquaporin-2 excretion in the nephrotic syndrome: an escape from the vasopressin regulating effect

PurposeExperimental studies suggest that the nephrotic syndrome is associated with “vasopressin escape”, characterized by low aquaporin-2 (AQP2) expression in the collecting duct despite high vasopressin secretion. We investigated this phenomenon in patients with the nephrotic syndrome.Patients and methodsWe recruited 47 patients with proteinuric kidney disease who were distributed into the following four groups: 1) nephrotic syndrome with kidney dysfunction (n=10); 2) nephrotic syndrome with normal kidney function (n=16); 3) partial remission of nephrotic syndrome (n=10); and 4) minimal proteinuria (n=11). Nine healthy volunteers comprised a control group. Serum copeptin level (as a marker of vasopressin secretion) and urinary AQP2 were measured using ELISA.ResultsNephrotic syndrome was associated with a significant increase in serum copeptin levels compared with those in the other groups (all P<0.05). In patients with nephrotic syndrome and a partial remission of nephrotic syndrome combined, there was more than a ten-fold decrease in the median urinary AQP2 excretion (0.03 ng/mL) compared with healthy volunteers (0.41 ng/mL; P<0.001) and more than a five-fold decrease compared with patients with minimal proteinuria (0.21 ng/mL; P<0.05). Unlike copeptin levels, the median urinary AQP2 excretion in patients with minimal proteinuria also decreased but less significantly than in those with nephrotic syndrome. There was a negative correlation between the urinary AQP2 excretion and daily proteinuria (R=−0.41; P=0.005).ConclusionOur clinical study was the first to demonstrate low AQP2 excretion in nephrotic syndrome that may indicate an escape from the vasopressin regulating effect.

Renal phenotype in Bardet-Biedl syndrome: a combined defect of urinary concentration and dilution is associated with defective urinary AQP2 and UMOD excretion.

The renal phenotype in Bardet-Biedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. Five of 41 patients (12%) showed an estimated glomerular filtration rate < 60 ml·min-1·1.73 m-2 Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients, respectively. Four of 41 patients showed no renal anomalies on ultrasound. Twenty of 34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. dDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients, suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca, but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling.

Urinary excretion of the water channel aquaporin2 correlated with the pharmacological effect of tolvaptan in cirrhotic patients with ascites.

The water channel aquaporin2 (AQP2) at the apical membrane of renal collecting duct cells mediates water reabsorption. The expression of AQP2 at the apical membrane is tightly regulated by vasopressin and was quantitated by measurement of the urinary form by a recently developed ELISA. Tolvaptan, an antagonist of vasopressin type 2 receptor, inhibits water reabsorption in cirrhosis. The aim of this study was to determine the correlation between the pharmacological effect of tolvaptan and the dynamics of urinary AQP2 levels. Tolvaptan was administered to 41 cirrhotic patients with ascites unresponsive to standard diuretic therapy. Urinary excretion of AQP2 and urinary osmolarity were measured at the baseline and at 4, 8, and 24h after administration of tolvaptan. At the baseline, urinary AQP2/creatinine ratios were significantly higher in cirrhotic patients with ascites than in healthy controls (P<0.0001). After administration of tolvaptan, urinary AQP2/creatinine ratios decreased by 45.0% at 4h and 77.0% at 8h. Similarly, urinary osmolarity decreased by 42.0% at 4h and 41.5% at 8h. Urinary AQP2 levels and urinary osmolarity significantly correlated at the baseline and at all time points after tolvaptan administration. The degree of the decrease in urinary AQP2 levels and degree of the decrease in urinary osmolarity correlated significantly at 4h (r=0.452, P=0.009) and 8h (r=0.384, P=0.030) after tolvaptan administration. These results indicate that the vasopressin-AQP2 system plays a major role in fluid retention in cirrhosis and that the pharmacological effect of tolvaptan to inhibit water reabsorption can be monitored by measurement of the dynamics of urinary AQP2 levels.

Urinary aquaporin-2 excretion before and after transjugular intrahepatic portosystemic shunt insertion for refractory ascites

Objective. The role of renal aquaporin-2 (AQP2) water channel turnover in patients with liver cirrhosis, portal hypertension and water retention remains unclear. Transjugular intrahepatic portosystemic shunt (TIPS) insertion reduces portal hypertension, improves water excretion and lowers plasma vasopressin. The aim of this study was to establish whether TIPS insertion decreases urinary AQP2 excretion (uAQP2) in parallel with improved water excretion. Material and methods. Fourteen cirrhosis patients with refractory ascites were studied before TIPS insertion and 4 and 12 weeks after insertion. A 24-h urine collection was followed by an oral water load (20 ml/kg body weight) with a 4-h blood and urine sampling. Results. TIPS reduced the portal pressure gradient from a median 18(4) (25–75% InterQuartile-range) to 7(2) mmHg, p < 0.05 and the need for diuretics (p < 0.05). TIPS increased plasma sodium from 136(6) mmol/l to 139(4), (p < 0.05) and diuresis from 1650(1043) ml/24 h to 2230(560) (p < 0.05), although the 24-h urinary sodium excretion did not change. There was no change in the baseline uAQP2 before 274(249) ng/(mmol creatinine/24 h) and 12 weeks after TIPS 242(201). There were no systematic changes in uAQP2, plasma vasopressin or other vasoactive substances during the water loads, before or after TIPS. Conclusion. The effective amelioration of portal hypertension improved the patient’s water excretion and plasma sodium, but there was no change in renal AQP2 trafficking or vasopressin. These findings do not support a primary role for renal AQP2 water channels in portal hypertensive water retention.

Aquaporin 2: Identification and analysis of expression in calves’ urine during their first month of life

Michałek, K., Dratwa-Chałupnik, A., Ciechanowicz, A. K. and Malinowski, E. 2014. Aquaporin 2: Identification and analysis of expression in calves’ urine during their first month of life. Can. J. Anim. Sci. 94: 653–659. Aquaporin 2 (AQP2) is a vasopressin-regulated water channel of the renal collecting duct and is excreted into urine. Renal excretion of AQP2 has been studied in humans, dogs, rats and mice. The aim of this study was to identify and analyze expression of AQP2 in calves’ urine. Short and controlled diarrhea was induced in the calves in order to determine any change in renal excretion of AQP2 and to evaluate a possible role of this channel in water balance during the neonatal period. For this purpose, an additional portion of lactose was administered to the calves with a milk replacer. The present work shows that after transient diarrhea associated with excessive supply of lactose in the diet, a significant increase in renal AQP2 excretion was reported in the first 3 wk of the calves’ lives. Renal excretion of this protein in the urine increased significantly despite the absence of changes in the concentration of vasopressin or the osmotic pressure of the blood plasma. The increase observed in the expression of the protein might be associated with the reduction of the total Ca2+concentration in the blood plasma of calves. The results clearly indicate a different mechanism regulating urinary excretion of AQP2 in newborn calves, hitherto unknown. Although elevated renal excretion of AQP2 was observed, there was no increase in the osmolality of the urine in the calves tested.

Urinary excretion pattern of exosomal aquaporin-2 in rats that received gentamicin.

Urinary exosomes are nano-sized vesicles secreted into urine from all types of renal epithelial cells and are known to contain possible biomarker proteins for renal diseases. Gentamicin has been reported to decrease the level of renal aquaporin (AQP)2, which is known to be mainly expressed in renal collecting ducts and excreted into the urine via exosomes. In the present study, we investigated whether urinary exosomal AQP2 could serve as a potential biomarker for gentamicin-induced nephrotoxicity, especially collecting duct cell dysfunction. Gentamicin was given to rats intraperitoneally once every day starting on day 0. Gentamicin significantly increased the plasma creatinine concentration from day 5 and beyond. Also, gentamicin induced polyuria and a defective urine concentration mechanism on day 7, suggesting gentamicin-induced collecting duct cell dysfunction. Immunoblot analysis showed that gentamicin significantly increased urinary exosomal AQP2 excretion on day 1 but decreased it on day 7 compared with the control group. Similarly, increased excretion of exosomal tumor susceptibility gene 101 protein, frequently used as an exosome marker protein, was observed on day 1. However, gentamicin did not significantly affect the urinary excretion of exosomal tumor susceptibility gene 101 on day 7. Gentamicin slightly decreased renal AQP2 expression on day 2 and markedly decreased it on day 8. These data strongly suggest that the use of urinary exosomal AQP2 as a biomarker may allow detection of gentamicin-induced collecting duct cell dysfunction. Furthermore, urinary exosomal AQP2 might also be useful for the early detection of gentamicin-induced renal injury in addition to collecting duct injury.

Effects of atorvastatin on systemic and renal NO dependency in patients with non‐diabetic stage II–III chronic kidney disease

Clinical trials suggest that statins have beneficial effects on the cardiovascular system independent from their cholesterol lowering properties. In patients with chronic kidney disease stage II-III, we tested the hypothesis that atorvastatin increased systemic and renal nitric oxide (NO) availability using L-N(G) -monomethyl arginine (L-NMMA) as an inhibitor of NO production. In a randomized, placebo-controlled, crossover study patients were treated with atorvastatin for 5 days with standardized diet and fluid intake. Glomerular filtration reate (GFR), fractional excretions of sodium (FENa ), urinary excretion of aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ ), vasoactive hormones (renin, angiotensin II, aldosterone, arginine vasopressin, endothelin-1 and brain natriuretic peptide) and central blood pressure (BP) estimated by applanation tonometry were measured before and after systemic administration of the NO inhibitor L-NMMA. Atorvastatin caused a significant reduction in U-ENaCγ , but sodium excretion, C H 2 O , FENa and u-AQP2 were not changed by atorvastatin. L-NMMA reduced renal effect variables, including GFR, FENa and u-ENaCγ and increased brachial BP and central BP to a similar extent during both treatments. Vasoactive hormones were changed in the same way by L-NMMA during atorvastatin and placebo treatment. During, atorvastatin and placebo treatment, inhibition of nitric oxide synthesis induced the same response in brachial and central blood pressure, GFR, renal tubular function and vasoactive hormones. Thus, the data do not support that atorvastatin changes nitric oxide availability in patients with mild nephropathy. The reduced u-ENaC may reflect changes in sodium absorption in the nephron induced by atorvastatin.

Sclederma of Poria cocos exerts its diuretic effect via suppression of renal aquaporin-2 expression in rats with chronic heart failure

Ethnopharmacological relevanceSclederma of Poria cocos (Hoelen) has been used as a diuretic in traditional Asian medicine. However, the underlying mechanism by which Sclederma of Poria cocos (hoelen) exerts its diuretic effect has not been well identified. The aim of the present study was to evaluate the effects of Sclederma of Poria cocos (hoelen) in rats with chronic heart failure (CHF) induced by acute myocardial infarction and to investigate the underlying mechanisms. Materials and methodsAn aqueous extract of Sclederma of Poria cocos (hoelen) (2.4g/kg/d, 1.2g/kg/d or 0.6g/kg/d) or furosemide (20mg/kg/d) was administered orally to male Sprague-Dawley rats starting on the day of coronary ligation. The urine output of all rats was quantified and collected every day for 1 or 4 weeks. The expression of aquaporin-2 (AQP2) was examined after treatment for 1 or 4 weeks. ResultsUrinary output increased significantly and urinary osmolality decreased after oral administration of Sclederma of Poria cocos (hoelen) for both 1 and 4 weeks. Sclederma of Poria cocos (hoelen) caused less electrolyte disorder than furosemide. Furthermore, Sclederma of Poria cocos (hoelen) reduced the levels of plasma BNP in CHF rats, whereas furosemide had no effect. Importantly, both mRNA and protein expression of AQP2 were down-regulated and urinary excretion of AQP2 was decreased after administration of Sclederma of Poria cocos (hoelen) to CHF rats. Similarly, Sclederma of Poria cocos (hoelen) reduced plasma arginine vasopressin (AVP) level and down-regulated vasopressin type 2 receptor (V2R) mRNA expression. ConclusionsSclederma of Poria cocos (hoelen) exerts its diuretic effect and improves cardiac function in CHF rats via the AVP-V2R-AQP2 axis.

A decrease in aquaporin 2 excretion is associated with bed rest induced high calciuria

BackgroundExposure to microgravity or immobilization results in alterations of renal function, fluid redistribution and bone loss, which couples to a rise of urinary calcium excretion. We recently demonstrated that high calcium delivery to the collecting duct reduces local Aquaporin-2 (AQP2) mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration and reducing calcium saturation. To investigate renal water balance adaptation during bed rest, a model to mimic the effects of microgravity on earth, the effect of changes in urinary calcium on urinary AQP2 excretion were assessed.MethodsTen healthy men (aged 21-28 years) participated in the experiment. Study design included 7 days of adaptation and 35 days of continuous bed rest (days -6 to 0 and 1 to 35, respectively) under controlled diet. Food records and 24-hour urine samples were collected daily from day -3 to 35. Changes in blood hematocrit were used as an indirect index of plasma volume changes. AQP2 excretion was measured by ELISA.ResultsBed rest induced bone demineralization and a transient increase in urinary calcium followed by transient decrease in AQP2 excretion, which can reduce the urine concentrating ability causing plasma volume reduction. The return of calciuria to baseline was followed by a recovery of AQP2 excretion, which allows for a partial restoration of plasma volume.ConclusionsThese results further support the view that urinary calcium can modulate the vasopressin-dependent urine concentration through a down-regulation of AQP2 expression/trafficking. This mechanism could have a key role in the prevention of urine super-saturation due to hypercalciuria.

Effect of atorvastatin on renal NO availability and tubular function in patients with stage II-III chronic kidney disease and type 2 diabetes

Background: Statins have beneficial effects on cardiovascular morbidity and mortality independently of reduction of plasma cholesterol. Purpose and methods: In patients with type 2 diabetes and nephropathy, chronic kidney disease stage II-III, we tested the hypothesis that atorvastatin increased systemic and renal nitric oxide (NO) availability using L-NMMA as an inhibitor of NO production. We performed a randomized, placebo-controlled, crossover study, using atorvastatin/placebo treatment for five days with a standardized diet and fluid intake. We measured brachial BP (bBP), central BP (cBP), GFR, urinary output (OU), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary excretion of albumin (UAER and UACR), AQP2 (u-AQP2) and ENaC (u-ENaCγ) and plasma concentrations of vasoactive hormones: renin, angiotensin II, aldosterone, arginine vasopressin, endothelin-1 and brain natriuretic peptide. Results: During atorvastatin and placebo treatment, L-NMMA infusion, changed the effect variables significantly, but to the same extent, i.e. an increase in bBP and cBP, and a decrease in GFR, OU, CH2O, FENa, u-AQP2 and u-ENaCγ. In addition, renin and angiotensin II was reduced, aldosterone increased, and vasopressin, endothelin-1 and brain natriuretic hormone unchanged. Conclusion: During, atorvastatin and placebo treatment, inhibition of nitric oxide synthesis induced the same response in brachial and central blood pressure, GFR, renal tubular function and vasoactive hormones. Thus, atorvastatin did not change nitric oxide availability in type 2 diabetics with nephropathy.

Puberty alters renal water handling

We investigated the influence of sex and puberty stage on circadian urine production and levels of antidiuretic hormone [arginine vasopressin (AVP)] in healthy children. Thirty-nine volunteers (9 prepuberty boys, 10 prepuberty girls, 10 midpuberty boys, and 10 midpuberty girls) were included. All participants underwent a 24-h circadian inpatient study under standardized conditions regarding Na(+) and fluid intake. Blood samples were drawn every 4 h for measurements of plasma AVP, serum 17-β-estradiol, and testosterone, and urine was fractionally collected for measurements of electrolytes, aquaporin (AQP)2, and PGE2. We found a marked nighttime decrease in diuresis (from 1.69 ± 0.08 to 0.86 ± 0.06 ml·kg(-1)·h(-1), P < 0.001) caused by a significant nighttime increase in solute-free water reabsorption (TcH2O; day-to-night ratio: 0.64 ± 0.07, P < 0.001) concurrent with a significant decrease in osmotic excretion (day-to-night ratio: 1.23 ± 0.06, P < 0.001). Plasma AVP expressed a circadian rhythm (P < 0.01) with a nighttime increase and peak levels at midnight (0.49 ± 0.05 pg/ml). The circadian plasma AVP rhythm was not influenced by sex (P = 0.56) or puberty stage (P = 0.73). There was significantly higher nighttime TcH2O in prepuberty children. This concurred with increased nighttime urinary AQP2 excretion in prepuberty children. Urinary PGE2 exhibited a circadian rhythm independent of sex or puberty stage. Levels of serum 17β-estradiol and testosterone were as expected for sex and puberty stage, and no effect on the AVP-AQP2-TcH2O axis was observed. This study found a circadian rhythm of plasma AVP independent of sex and puberty stage, although nighttime TcH2O was higher and AQP2 excretion was more pronounced in prepuberty children, suggesting higher prepuberty renal AVP sensitivity.

Effect of volume expansion with hypertonic- and isotonic saline and isotonic glucose on sodium and water transport in the principal cells in the kidney

BackgroundThe renal distal nephron plays an important role in the maintenance of sodium balance, extra cellular volume and blood pressure. The degree of water transport, via aquaporin2 water channels (AQP2), and sodium transport, via epithelial sodium channels (ENaC) in renal collecting duct principal cells are reflected by the level of urinary excretion of AQP2 (u-AQP2) and the γ-fraction of ENaC (u-ENaCγ). The effects of an acute intravenous volume load with isotonic saline, hypertonic saline and glucose on u-AQP2, u-ENaCγ and underlying mechanisms have never been studied in a randomized, placebo-controlled trial in healthy humans.MethodsWe studied the effects of 0.9% saline (23 ml/kg), 3% saline (7 ml/kg) and 5% glucose (23 ml/kg) on u-AQP2 and u-ENaCγ, fractional sodium excretion (FENa), free water clearance (CH2O), and plasma concentrations of vasopressin (AVP), renin (PRC), angiotensin II (ANG II) and aldosterone (Aldo) in a randomized, crossover study of 23 healthy subjects, who consumed a standardized diet, regarding calories, sodium and fluid for 4 days before each examination day.ResultsAfter isotonic saline infusion, u-AQP2 increased (27%). CH2O and u-ENaCγ were unchanged, whereas FENa increased (123%). After hypertonic saline infusion, there was an increase in u-AQP2 (25%), u-ENaCγ (19%) and FENa (96%), whereas CH2O decreased (-153%). After isotonic glucose infusion, there was a decrease in u-AQP2 (-16%), ENaCγ (-10%) and FENa (-44%) whereas CH2O increased (164%). AVP remained unchanged after isotonic saline and glucose, but increased after hypertonic saline (139%). PRC, AngII and p-Aldo decreased after isotonic and hypertonic saline infusion, but not after glucose infusion.ConclusionsVolume expansion with 3% and 0.9% saline increased u-AQP2, while isotonic glucose decreased u-AQP2. Infusion of hypertonic saline increased u-ENaCγ, whereas u-ENaCγ was not significantly changed after isotonic saline and tended to decrease after glucose. Thus, the transport of water and sodium is changed both via the aquaporin 2 water channels and the epithelial sodium channels during all three types of volume expansion to regulate and maintain water- and sodium homeostasis in the body.Trial registrationClinical Trial no: NCT01414088

Excretion of urinary exosomal AQP2 in rats is regulated by vasopressin and urinary pH

Urinary exosomes are small vesicles secreted into urine from all renal epithelial cell types and known to contain proteins that are involved in renal secretion and reabsorption. Among these proteins, urinary exosomal aquaporin-2 (AQP2) has been suggested to be useful for diagnosis of renal disease. However, the mechanisms underlying the excretion of urinary exosomal AQP2 are largely unknown. In this study, we examined the mechanisms of urinary exosomal AQP2 excretion in vivo, using diuretics including furosemide (FS), an inhibitor of the sodium-potassium-chloride symporter; acetazolamide (ACTZ), an inhibitor of carbonic anhydrase; OPC-31260 (OPC), a vasopressin type 2 receptor antagonist; and NaHCO3, a urinary alkalizing agent. Samples of urine from rats were collected for 2 h just after treatment with each diuretic, and urinary exosomes were isolated by ultracentrifugation. Urinary exosomal AQP2 excretion was dramatically increased by treatment with FS accompanied by urine acidification or with ACTZ accompanied by urine alkalization. Immunohistochemistry showed that apical localization of AQP2 was clearly evident and the plasma vasopressin level was increased after each treatment. Although treatment with OPC alone had no significant effect, coadministration of OPC completely inhibited the FS-induced and partially reduced the ACTZ-induced responses, respectively. Treatment with NaHCO3 increased the excretion of urinary exosomal AQP2 accompanied by urine alkalization. This increased response was partially inhibited by coadministration of OPC. These data suggest that an increased plasma level of vasopressin promoted the excretion of urinary exosomal AQP2 and that urine alkalinization also increased it independently of vasopressin.

Arginine vasopressin‐dependent and AVP‐independent mechanisms of renal fluid absorption during thirsting despite glucocorticoid‐mediated vasopressin suppression

Glucocorticoids seem to modify the release and effects of plasma arginine vasopressin (pAVP). However, underlying processes are not well understood. This study aimed to evaluate the mechanism of the modulating effects of glucocorticoids on pAVP and renal water reabsorption. Fluid deprivation tests were performed without (d0) and after one (d1) and five days (d5) of oral prednisolone (Pred) pretreatment in a dosage relevant to drug therapy (30 mg/day). Twelve healthy male volunteers participated in this trial. Plasma and urinary osmolality, pAVP, renin, aldosterone, plasma atrial natriuretic peptide (ANP) as well as urinary secretion of aquaporin-2 (AQP2) and prostaglandin E(2) (PGE2) were analysed. An appropriate rise in pAVP was observable during thirsting (P < 0.001), which was absent after Pred pretreatment. However, the plasma and urinary osmolality after Pred treatment did not differ when compared with the basal thirsting test. Unchanged urinary AQP2 excretion suggests AVP-independent mechanisms of renal fluid reabsorption. Plasma renin concentration as well as ANP was substantially increased after Pred intake at d1 and d5 (both P < 0.05), which may mediate such AVP-independent mechanisms. Urinary PGE2 secretion was not influenced by Pred pretreatment, making a PGE2-mediated effect on renal AQP2 translocation and water permeability unlikely. Increased efficacy of exogenous desmopressin at d1 and d5 indicates also a relative increase in AVP sensitivity of the tubular cells after Pred intake. The here presented data are compatible with an increased AVP sensitivity and a partially AVP-independent regulation of AQP2 translocation and renal fluid reabsorption during glucocorticoid treatment.

Effect of Amiloride and Spironolactone on Renal Tubular Function and Central Blood Pressure in Patients with Arterial Hypertension during Baseline Conditions and after Furosemide: A Double-Blinded, Randomized, Placebo-Controlled Crossover Trial

This study demonstrates that the increased potassium content in the body seems to change both the blood pressure and renal tubular function. We wanted to test the hypotheses that amiloride and spironolactone induced potassium retention reduces ambulatory blood pressure (ABP) and central blood pressure (CBP) during baseline conditions and after furosemide and that the tubular transport via the epithelial sodium channels (ENaCs) and aquaporin-2 (AQP2) water channels was increased by furosemide in arterial hypertension. Each of three 28-day treatment periods (placebo, amiloride, and spironolactone) was completed by a 4-day period with standardized diet regarding calories and sodium and water intake. At the end of each period, we measured pulse wave velocity (PWV), central systolic blood pressure (CSBP), central diastolic blood pressure (CDBP), glomerular filtration rate (GFR), free water clearance (CH2O), fractional excretion of sodium (FENa) and potassium (FEK), urinary excretion of AQP2 (u-AQP2), urinary excretion of γ-fraction of the ENaC (u-ENaCγ), and plasma concentrations of renin (PRC), angiotensin II (p-Ang II), and aldosterone (p-Aldo) at baseline conditions and after furosemide bolus. Ambulatory blood pressure and CBP were significantly lowered by amiloride and spironolactone. During 24-hour urine collection and at baseline, GFR, CH2O, FENa, FEK, u-AQP2 and u-ENaCγ were the same. After furosemide, CH2O, FENa, FEK, u-AQP2, u-ENaCγ, PRC, p-Ang II, p-Aldo, PWV and CDBP increased after all treatments. However, during amiloride treatment, FEK increased to a larger extent than after spironolactone and during placebo after furosemide, and CSBP was not significantly reduced. The increases in water and sodium absorption via AQP2 and ENaC after furosemide most likely are compensatory phenomena to antagonize water and sodium depletion. Amiloride is less effective than spironolactone to reduce renal potassium excretion.Trial registration: ClinicalTrials.gov identifier: NCT0138088.