Expression patterns of aquaporins 1, 3, 5 in canine mammary gland carcinomas.

Aquaporins (AQPs) are water channel proteins, and the expression of AQPs in carcinoma cells has received much attention over the last 15 years. In the veterinary field, however, little is known about the expression of AQPs. In the present study using immunohistochemistry, we examined the expression of AQP1, AQP3, and AQP5 in canine mammary gland carcinomas. The 27 samples comprised 10 grade I, 12 grade II, and 5 grade III samples (See Materials and Methods section for grade classification method). AQP1 was expressed in only 2 of the grade III carcinomas, and the expression was limited to spindle-shaped cells in the solid structure and on the outside of the solid mass. AQP3-positive cells were observed in 20 of 22 grade I and II samples. On the other hand, among grade III carcinomas, AQP3 was expressed only in spindle-shaped cells in 1 sample. AQP5 was expressed in all grade I and II carcinomas but not in the grade III tumors. In addition, enhanced expression of basolateral AQP3 and apical AQP5 was observed in lobular hyperplastic cells. These results suggest that the expression patterns of AQP3 and AQP5 can be of help for judging the grading of canine mammary tumors and that AQP1 is likely to be involved in metastasis. Moreover, AQP3 and AQP5 might be relevant to lactation in female dogs.

Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK.

BACKGROUND/AIM: Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren's syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry 'leaf' water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry 'stem' water extract (BStEx) on lacrimal hyposecretion in NOD mice. MATERIALS AND METHODS: Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. RESULTS: After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. CONCLUSION: BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

Rhabdomyolysis, myoglobinuric nephrosis, and crystalline nephropathy in a captive bottlenose dolphin.

A 5-y-old female bottlenose dolphin (Tursiops truncatus) from an aquarium in Japan had clinical signs of anorexia, vomiting, and bradykinesia. Enrofloxacin and lactated Ringer solution were administered for treatment of bacterial infection and for rehydration. Elevations of creatine kinase and aspartate aminotransferase activities were detected on day 4 of treatment, indicating that rhabdomyolysis had developed on day 3. On day 5, serum creatinine and urea concentrations increased and remained high throughout the remaining treatment; the dolphin died on day 16. Postmortem examination revealed massive necrosis of the longissimus dorsi muscles. Histologic examination revealed extensive necrosis of skeletal myofibers, multifocal renal tubular necrosis with intratubular casts and crystals, and suppurative bronchopneumonia. The renal casts labeled positively with anti-myoglobin antibody; expression of aquaporin-1 was decreased in renal tubules compared to normal kidney tissue. To our knowledge, this description of clinicopathologic findings of rhabdomyolysis leading to acute kidney injury with concomitant crystalline nephropathy has not been reported previously in a bottlenose dolphin.

Upregulation of NADPH Oxidase 2 Contributes to Renal Fibrosis in pcy Mice: An Experimental Model of Nephronophthisis.

BACKGROUND: DBA/2FG-pcy (pcy) mice harbor a homozygous Nphp3 missense mutation and develop nephronophthisis with renal interstitial fibrosis. Previous studies have shown that aberrant oxygen homeostasis contributes to the renal pathology in pcy mice, but the underlying molecular mechanism remains largely unknown. METHODS: pcy mice and a control strain, DBA/2N (DBA) mice, were used. Renal levels of 62 mRNAs involved in oxygen homeostasis were investigated by real-time PCR, and the resulting data were used for extraction of pathological pathways. On the basis of the genes found to be upregulated and pathway analysis, further studies were performed using immunoblotting, immunohistochemistry, and pharmacological intervention. RESULTS: In comparison with DBA mice, the levels of 18 mRNAs were altered by >2-fold in pcy mice. Pathway analysis extracted molecular pathways related to oxidative stress, inflammation, and cell adhesion. As the levels of mRNAs relevant to the NADPH oxidase 2 (NOX2) pathway were prominently (4 genes >5-fold) increased in pcy mice, we further analyzed the molecules related to this pathway. A time course study suggested that the pathway was gradually activated in pcy mice from at least 5 weeks of age. Immunohistochemistry study revealed that NOX2 protein was colocalized with a macrophage marker protein in the renal interstitium. Moreover, treatment of pcy mice with apocynin, an inhibitor of the NOX2 pathway, ameliorated the renal fibrosis. CONCLUSION: Our findings suggest that the activation of the NOX2 pathway, possibly mediated by macrophage infiltration, plays a pivotal role in progressive renal fibrosis in pcy mice.

Reduced urinary release of AQP1- and AQP2-bearing extracellular vesicles in patients with advanced chronic kidney disease.

Although several studies have shown that release of water channel proteins, aquaporin 1 (AQP1) and AQP2 in urinary extracellular vesicles (uEV-AQP1 and -AQP2), were altered in experimental kidney injury models, their release in human chronic kidney disease (CKD) has been largely unexplored. The aim of the present study was to clarify whether the release of uEV-AQP1 and -AQP2 is altered in patients with CKD. Urine samples were collected from 15 healthy volunteers (normal group) and 62 CKD patients who were categorized into six glomerular filtration rate (GFR) categories (G1, G2, G3a, G3b, G4, and G5) in between 2005 and 2016 at Miyazaki Prefectural Miyazaki Hospital, Japan. uEV-proteins were evaluated by immunoblot analysis. The release of AQP1 and AQP2 were significantly decreased in patients with both CKD G4 and G5, in comparison with the normal group. The area under the receiver operating characteristic (ROC) curve (AUC) values for AQP1 and AQP2 in patients with CKD G4 and G5 were 0.926 and 0.881, respectively. On the other hand, the AUC values in patients with CKD G1-G3 were 0.512 for AQP1 and 0.680 for AQP2. Multiple logistic regression analysis showed that AQP1 and AQP2 in combination were useful for detecting CKD G4 and G5, with a higher AUC value of 0.945. These results suggest that the release of uEV-AQP1 and -AQP2 was decreased in patients with CKD G4 and G5, and these proteins might be helpful to detect advanced CKD.

Onion (Allium cepa L.)-Derived Nanoparticles Inhibited LPS-Induced Nitrate Production, However, Their Intracellular Incorporation by Endocytosis Was Not Involved in This Effect on RAW264 Cells.

The aim of this study was to evaluate the involvement of nanoparticles prepared from Allium cepa L. as anti-inflammatory agents. In the present study, we identified nanoparticles from Allium cepa L. using the ultracentrifugation exosome purification method. The nanoparticles were referred to as 17,000× g and 200,000× g precipitates, and they contained quercetins, proteins, lipids, and small-sized RNA. The nanoparticles inhibited nitric oxide production from lipopolysaccharide (LPS)-stimulated RAW264 cells without cytotoxic properties. Cellular incorporation was confirmed by laser microscopic observation after PKH26 staining. The inhibition of caveolae-dependent endocytosis and macropinocytosis significantly prevented the incorporation of the nanoparticles but had no effect on the inhibition of nitric oxide in RAW264 cells. Collectively, the identified nanoparticles were capable of inhibiting the LPS response via extracellular mechanisms. Taken together, the way of consuming Allium cepa L. without collapsing the nanoparticles is expected to provide an efficient anti-inflammatory effect.

Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model.

Urinary exosomes, small extracellular vesicles present in urine, are secreted from all types of renal epithelial cells. Aquaporin-2 (AQP2), a vasopressin-regulated water channel protein, is known to be selectively excreted into the urine through exosomes (UE-AQP2), and its renal expression is decreased in nephrotic syndrome. However, it is still unclear whether excretion of UE-AQP2 is altered in nephrotic syndrome. In this study, we examined the excretion of UE-AQP2 in an experimental rat model of nephrotic syndrome induced by the administration of puromycin aminonucleoside (PAN). Rats were assigned to two groups: a control group administered saline and a PAN group given a single intraperitoneal injection of PAN (125 mg/kg) at day 0. The experiment was continued for 8 days, and samples of urine, blood, and tissue were collected on days 2, 5, and 8. The blood and urine parameters revealed that PAN induced nephrotic syndrome on days 5 and 8, and decreases in the excretion of UE-AQP2 were detected on days 2 through 8 in the PAN group. Immunohistochemistry showed that the renal expression of AQP2 was decreased on days 5 and 8. The release of exosomal marker proteins into the urine through UEs was decreased on day 5 and increased on day 8. These data suggest that UE-AQP2 is decreased in PAN-induced nephrotic syndrome and that this reflects its renal expression in the marked proteinuria phase after PAN treatment.

Release of urinary aquaporin-2-bearing extracellular vesicles is decreased in pregnant Japanese Black cattle.

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays an important role in renal water homeostasis. It has been reported that the level of AQP2 in human urine is altered during pregnancy. However, little is known about the level of urinary AQP2 in pregnant cattle. In this study, we examined the level of AQP2-bearing extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in both heifers and cows during the gestational and postpartum periods. The level of uEV-AQP2 was significantly decreased during gestation in comparison with the other cattle examined. Similarly, the levels of EV marker proteins in uEVs, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (ALIX), were significantly decreased during gestation. There were significant correlations between the levels of uEV-AQP2 and uEV-TSG101, or uEV-ALIX. Immunohistochemistry data from pregnant and non-pregnant cattle supported the notion that the level of uEV-AQP2 was decreased during gestation. These data indicate that the level of uEV-AQP2 is decreased in pregnant cattle, possibly through a decrease in both the number of EVs released into the urine and renal AQP2 expression.

Urinary extracellular vesicular release of aquaporins in patients with renal transplantation.

BACKGROUND: Diuresis has been observed within a week following renal transplantation, suggesting that the procedure causes acute disturbance of renal water homeostasis. Aquaporin (AQP) 1 and AQP2, important proteins for renal water reabsorption, have been identified in urinary extracellular vesicles (uEV-AQP1 and -AQP2), and experimental studies have shown that the presence of uEV-AQP1 and -AQP2 may be an indicator of their levels of expression in the kidney. However, the release patterns of uEV-AQP1 and -AQP2 during the acute phase following renal transplantation are largely unknown. METHODS: In this study, we examined the release of uEV-AQP1 and -AQP2 in recipients until 6 days (day 6) after renal transplantation. At Miyazaki prefectural Miyazaki Hospital, Japan, uEVs were obtained from 7 recipients, all of whom had received renal allografts from living donors. uEVs were isolated by differential centrifugation. RESULTS: Immunoblotting analysis showed that the release of uEV-AQP2 was significantly decreased on day 1 in comparison with a control sample (from 3 healthy volunteers), accompanied by high urine output and low urine osmolality. Thereafter, the level increased gradually to the control level by day 6. The release pattern of uEV-AQP1 was similar to that of uEV-AQP2, but the levels did not reach statistical significance in comparison with the control level at any of the time points examined. Evaluation of the relationship between urinary osmolality and uEV-AQPs revealed a significant correlation for uEV-AQP2, but not for uEV-AQP1. CONCLUSION: These results indicate that acute diuresis after renal transplantation might be due to a decrease in the renal expression of AQP2, whose level can be estimated from the amount released in uEVs.

A bell-shaped pattern of urinary aquaporin-2-bearing extracellular vesicle release in an experimental model of nephronophthisis.

The DBA/2-FG pcy (pcy) mouse is a model of human nephronophthisis, a recessive cystic kidney disease. Renal expression of aquaporin-2 (AQP2), a water channel protein, has been shown to be altered in pcy mice. However, the relationship between the renal expression and its release in urinary extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in pcy mice has remained largely unknown. In this study, we examined age-related alterations of this relationship in pcy mice. In comparison with control mice, pcy mice after the age of 14 weeks showed defective urinary concentration ability with an increase in urinary volume. Interestingly, the release of uEV-AQP2 increased progressively up to the age of 16 weeks, but at 21 weeks the release did not significantly differ from that in control mice (i.e., a bell-shaped pattern was evident). Similar results were obtained for uEV marker proteins, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (Alix). Immunoblot analysis revealed that renal AQP2 expression increased progressively from 11 weeks, and immunohistochemistry showed that this increase was possibly due to an increase in the number of AQP2-positive cells. Analysis of mRNAs for seven types of AQP expressed in the kidney supported this notion. These data suggest that the level of uEV-AQP2 does not simply mirror the renal expression of AQP2 and that the altered release of uEV-AQP2 in pcy mice depends on the numbers of both renal AQP2-positive cells and EVs released into the urine.

miRNA profiling of urinary exosomes to assess the progression of acute kidney injury.

Because exosomes have gained attention as a source of biomarkers, we investigated if miRNAs in exosomes (exo-miRs) can report the disease progression of organ injury. Using rat renal ischemia-reperfusion injury (IRI) as a model of acute kidney injury (AKI), we determined temporally-released exo-miRs in urine during IRI and found that these exo-miRs could reliably mirror the progression of AKI. From the longitudinal measurements of miRNA expression in kidney and urine, we found that release of exo- miRs was a regulated sorting process. In the injury state, miR-16, miR-24, and miR-200c were increased in the urine. Interestingly, expression of target mRNAs of these exo-miRs was significantly altered in renal medulla. Next, in the early recovery state, exo-miRs (miR-9a, miR-141, miR-200a, miR-200c, miR-429), which share Zeb1/2 as a common target mRNA, were upregulated together, indicating that they reflect TGF-ß-associated renal fibrosis. Finally, release of exo-miRs (miR-125a, miR-351) was regulated by TGF-ß1 and was able to differentiate the sham and IRI even after the injured kidneys were recovered. Altogether, these data indicate that exo-miRs released in renal IRI are associated with TGF-ß signaling. Temporal release of exo-miRs which share targets might be a regulatory mechanism to control the progression of AKI.

An Early Decrease in Release of Aquaporin-2 in Urinary Extracellular Vesicles After Cisplatin Treatment in Rats.

Aquaporin-1 (AQP1) and AQP2 are important proteins involved in the regulation of renal water handling. Both AQPs have been found in urinary extracellular vesicles (uEVs) (uEV-AQP1 and -AQP2). Cisplatin, an antineoplastic agent, is known to down-regulate renal AQP1 and AQP2. However, the effect of cisplatin on the release of uEV-AQP1 and -AQP2 is largely unknown. In this study, we examined whether treatment of rats with cisplatin affected the release of uEV-AQP1 and -AQP2. Blood tests indicated that renal function was little altered at 24 h after cisplatin treatment but thereafter decreased dramatically at all of the other time points examined. Release of uEV-AQP1 was slightly increased at 24 h and decreased at 168 h. On the other hand, release of uEV-AQP2 was decreased dramatically at 24 h, and the decrease was maintained during the experimental period. These data suggest that uEV-AQP2 can be used to detect early renal impairment due to cisplatin. Furthermore, a combination of uEV-AQP2 and -AQP1 may be useful for estimation of cisplatin-induced renal injury in a stage-dependent manner.

Involvement of the NADPH oxidase 2 pathway in renal oxidative stress in Aqp11 -/- mice.

Aquaporin-11 (AQP11) is an intracellular AQP. Several studies with Aqp11 -/- mice have shown that AQP11 has a role in normal development of the kidney after birth. Our previous studies have suggested that alteration of oxygen homeostasis may be involved in the kidney injury caused by AQP11 deficiency, although the underlying mechanism is largely unknown. To clarify this issue, we examined genes that are related to oxygen homeostasis in Aqp11 -/- mice. Among 62 genes that are involved in oxygen homeostasis, 35 were upregulated by more than 2-fold in Aqp11 -/- mice in comparison with wild-type mice. Pathway analysis using these genes extracted the pathway responsible for production of reactive oxygen species in macrophages. As expression of the genes involved in the NADPH oxidase 2 (NOX2) complex was dramatically increased by more than 14-fold, we further analyzed NOX2 at the protein level. Immunoblotting analysis demonstrated a dramatic increase of NOX2 protein in the kidney of Aqp11 -/- mice, and immunohistochemistry showed that NOX2 protein and a marker protein for macrophages were increased in the renal interstitium. These results indicate that NOX2-induced oxidative stress accompanied by macrophage infiltration plays an important role in alteration of oxygen homeostasis in Aqp11 -/- mice.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats.

Acute kidney injury (AKI) is an important risk factor for the development of chronic kidney disease (CKD), and an alteration in renal water handling has been observed during the transition of AKI to CKD. Urinary exosomal release of aquaporin-1 (AQP1) and AQP2, important proteins for renal water handling, has recently been reported to predict their levels of renal expression. Therefore, we examined the patterns of urinary exosomal release of AQP1 and AQP2, and the exosomal marker proteins tumor susceptibility 101 protein (TSG101) and ALG-2 interacting protein X (Alix), in the acute and chronic phases following induction of AKI by renal bilateral ischemia/reperfusion (I/R) in rats. Blood tests and histological examinations indicated that AKI occurred before at 7 days after renal I/R ( day 7) and that renal fibrosis developed progressively thereafter. Immunoblotting demonstrated significant decreases in the urinary exosomal release of AQP1 and AQP2 during severe AKI. Urinary exosomal release of Alix and TSG101 was significantly increased on day 7. These data were also confirmed in rats with unilateral renal I/R causing more serious AKI. Urinary exosomal release of either the Ser-256- or Ser-269-phosphorylated form of AQP2, both of which are involved in apical trafficking of AQP2, was positively correlated with that of total AQP2. These results suggest that urinary exosomal release of AQP1 and AQP2 is reduced in I/R-induced AKI, whereas that of Alix and TSG101 is increased in the initial phase of renal fibrosis. Furthermore, apical trafficking of AQP2 appears to be related to urinary exosomal release of AQP2.

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.

Acetazolamide enhances the release of urinary exosomal aquaporin-1.

BACKGROUND: Renal aquaporin-1 (AQP1), a water channel protein, is known to be secreted into urine, conveyed by nano-sized extracellular vesicles called exosomes. A previous study has demonstrated that acetazolamide (AZ), a diuretic that inhibits carbonic anhydrases, alters the expression level of AQP1 in cultured cells. Here we investigated whether AZ alters the release of urinary exosomal AQP1 in vivo. METHODS: The effect of AZ on urinary exosomal AQP1 secretion was examined in rats and compared with furosemide (another diuretic), NaHCO3 (an alkalizing agent) and NH4Cl (an acidifying agent). Urine, blood and kidney samples were obtained 2 h after each treatment. Urinary exosomes were isolated by a differential centrifugation technique and urinary exosomal proteins were analyzed by immunoblotting. RESULTS: The release of exosomal AQP1 into urine was markedly increased after treatment with AZ, accompanied by alkaluria and metabolic acidosis. Immunohistochemistry clearly demonstrated that AZ increased the apical membrane expression of AQP1 in the proximal tubules. AZ did not affect the release of exosomal marker proteins (tumor susceptibility gene 101 protein and apoptosis-linked gene 2 interacting protein X). Treatment with furosemide did not change, whereas NaHCO3 and NH4Cl decreased the exosomal release of AQP1. CONCLUSION: The present findings indicate that AZ increases the release of exosomal AQP1 into urine in association with enhanced apical membrane expression of AQP1.

Arterial blood gas anomaly in canine hepatobiliary disease.

Arterial blood gas analysis is an important diagnostic and monitoring tool for respiratory abnormalities. In human medicine, lung complications often occur as a result of liver disease. Although pulmonary complications of liver disease have not been reported in dogs, we have frequently encountered hypoxemia in dogs with liver disorders, especially extrahepatic biliary obstruction. In addition, respiratory disorders account for 20% of perioperative fatalities in dogs. Therefore, in this study, we evaluated the respiratory status in dogs with hepatobiliary disease by arterial blood gas analysis. PaO2 and PaCO2 were measured. Alveolar-arterial oxygen difference (AaDO2), the indicator of gas exchange efficiency, was calculated. Compared to healthy dogs (control group), hepatobiliary disease dogs had significantly lower PaO2 and higher AaDO2. Hypoxemia (PaO2 of ≤80 mmHg) was observed in 28/71 dogs with hepatobiliary disease. AaDO2 was higher (≥30 mmHg) than the control group range (11.6 to 26.4 mmHg) in 32/71 hepatobiliary disease dogs. By classifying type of hepatobiliary disease, dogs with extrahepatic biliary obstruction and chronic hepatitis showed significantly lower PaO2 and higher AaDO2 than in a control group. Dogs with chronic hepatitis also had significantly lower PaCO2. The present study shows that dogs with hepatobiliary disease have respiratory abnormalities more than healthy dogs. Preanesthetic or routine arterial blood gas analysis is likely beneficial to detect the respiratory abnormalities in dogs with hepatobiliary disease, especially extrahepatic biliary obstruction and chronic hepatitis.

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.

The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

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.