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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.

Excess lipid accumulation is associated with obesity-related chronic kidney disease, but the mechanisms of fatty acid (FA) uptake have been poorly understood. To this end, we investigated how FAs are taken up by tubular epithelial cells (TECs) in mice by using in vivo FA tracing and histological methods. Immunohistochemistry showed that CD36, which is a well-known FA transporter, was abundantly expressed on the basolateral side of proximal TECs (PTECs). The uptake of 125I-BMIPP (a radiolabeled FA tracer) was significantly reduced in CD36-knockout kidneys at 1 min after injection. In vivo imaging with multiphoton microscopy revealed that BODIPY-C12 (a fluorescence-labeled FA tracer) accumulated on both the basolateral and apical sides of PTECs. Numerous lipid droplets accumulated in PTECs after accelerated lipolysis. Furthermore, PTEC-specific injury via diphtheria toxin (DT) injection in transgenic mice expressing the DT receptor resulted in a compensatory increase in lipid accumulation in downstream TECs. Importantly, urinary FAs were undetectable, even in mice and humans with remarkable albuminuria. Our data demonstrate that renal TECs take up FAs from blood (CD36-dependent) and primary urine (CD36-independent) and can store excess FAs as neutral lipids. The results further show that renal tubules have hitherto largely unappreciated mechanisms by which the excretion of FAs into the urine is avoided.

In general, we need a lot of data for improving the accuracy of machine learning. However, the number of biological samples what we can obtain are not enough for machine learning. This problem exists in the classification of glomerular epithelial cells with the progress of disease, and its accuracy is contrary to our intuitive impression. Therefore, we would like to improve the accuracy by generating a lot of fake images using Generative Adversarial Nets (GANs). About podocyte cells, it was difficult to obtain an arbitrary disease by previous method. In this paper, we propose the model with restriction of learning by shapes information based on ACGANs, and we investigate how much fake images generated by our method are similar to real images. According to the results, the passage number of fake images by our method is 17% higher than conventional method.

Radiation therapy for head and neck cancer frequently causes salivary gland dysfunction. Pilocarpine is a clinically approved and effective drug that induces saliva secretion, thereby keeping the oral mucosa moist and reducing discomfort in patients, but the effect is transient. We expected that this drug also has beneficial long-term effects that maintain the integrity of salivary glands by reducing, for instance, apoptosis. Here, we examined the effects of long-term pilocarpine administration in irradiated mice. The results indicated that long-term pilocarpine administration significantly improved salivary flow in irradiated mice, suggesting the potential beneficial effects of long-term administration. To elucidate the underlying mechanism, we analyzed the histology, apoptosis, and proliferation of acinar cells, and the expression of functional membrane proteins such as transmembrane member 16A, aquaporin-5, and Na-K-Cl cotransporter. Long-term pilocarpine treatment seemed to decrease irradiation-induced apoptosis, although the change was not statistically significant. The present results indicated that long-term administration of pilocarpine has beneficial effects on salivary flow in irradiated mice, and suggested that long-term administration possibly decreases apoptosis in irradiated salivary glands.

Immunofluorescence microscopy is a powerful method for analysis of the subcellular localization of the protein of interest. The use of fluorescence is very effective for multiple labeling and for higher magnification observation with a laser confocal microscope. A basic protocol of the immunofluorescent staining in cultured cells with some useful suggestions are introduced in the present paper. The author describes the following contents. 1) Coverslips: coverslips are necessary to be coated with coating reagent such as collagen to improve the attachment and growing of the cells. Commercially available glass slides for cell culture are also useful. Permeable support filters are convenient for establishing the apical and basolateral compartments when epithelial cells are cultured. 2) Fixation: basic fixative is 4% paraformaldehyde in phosphate buffer. Ethanol including 1% acetic acid or pure ethanol or methanol are also effective for some antigens. 3) Permeabilization: treatment with Triton X-100 or saponin before and sometimes during the antibody incubation is required to improve the antibody accessibility. 4) Blocking: nonspecific binding of antibodies is blocked with 5% serum from the animal species as same as that of the secondary antibody you use. 5) Antibody incubations: antibodies are diluted in the blocking solution and incubated with samples. For multi-labeling with primary antibodies derived from different animal species, the specimen can be sequentially incubated with a mixture of primary antibodies and a mixture of secondary antibodies. 6) Choice of secondary antibodies: secondary antibodies which do react specifically to the target without cross-reaction and appropriate fluorescent dyes for multiple labeling are required. 7) Sample storage: the fluorescence can be kept for long period like several months to years in specimens which are mounted with anti-fading mounting medium and stored at -20°C. 8) Trouble shootings: some trouble shootings are also shown. The author hopes that this paper would help the readers to obtain better results in immunofluorescence.

Background Systemic sclerosis (SSc) is a connective tissue disorder characterized by the development of fibrosis in the skin and internal organs. Increasing evidence suggests that mesenchymal stem cells (MSCs) can be used to a treatment for fibrotic diseases. Recent studies have demonstrated that some of the biological effects of MSCs are due to the secretion of exosomes. However, the precise mechanisms underlying MSCs-derived exosomes in skin fibrosis are not well understood. Objective We aimed to elucidate the effect of MSCs-derived exosomes on skin fibrosis in SSc and the mechanism underlying their inhibitory action on fibrosis. Methods Exosome was collected from MSCs by ultracentrifugation method. We examined the suppressive effect of MSCs-derived exosome on skin fibrosis in bleomycin-induced SSc mouse model. Skin samples from the injected site were collected for further examination, and micro-RNA analysis of MSCs-derived exosome was performed. Results Injection of MSCs-derived exosomes significantly inhibited bleomycin-induced dermal fibrosis in mice. MSCs-derived exosomes significantly reduced the amount of collagen and the number of α-SMA+ myofibroblasts and CD68+ macrophages in lesional skin. They also reduced the expression of type I collagen and TGF-β receptor 1 in fibroblasts in vitro. Moreover, micro-RNA analysis revealed that several microRNAs in MSCs-derived exosomes have antifibrotic potential. We confirmed that overexpression of miR-196b-5p in fibroblasts significantly suppressed collagen type I alpha 2 expression. Conclusion This study demonstrated that inhibition of collagen type I expression by miR-196b-5p in exosomes might be one of the mechanisms by which MSCs suppress skin fibrosis in an SSc mouse model.

Pachychoroid spectrum diseases have been described as a new clinical entity within the spectrum of macular disorders. “Pachychoroid” is defined as choroidal thickening associated with dilated outer choroidal vessels often showing retinal pigment epithelium (RPE) degeneration. Although various clinical studies on the pachychoroid spectrum diseases have been conducted, the pathophysiology of pachychoroid has yet to be fully elucidated. In this study, we attempted to establish a mouse model of pachychoroid. We sutured vortex veins in eyes of wild type mice to imitate the vortex vein congestion in pachychoroid spectrum diseases. Fundus photography and ultra-widefield indocyanine green angiography showed dilated vortex veins from the posterior pole to the ampulla in eyes after induction of choroidal congestion. Optical coherence tomography and tissue sections presented choroidal thickening with dilatation of choroidal vessels. The RPE-choroid/retina thickness ratios on the tissue sections in the treated day 1 and day 7 groups were significantly greater than that in the control group (0.19±0.03 and 0.16±0.01 vs. 0.12±0.02, P

Circadian rhythms are a fundamental biological phenomena that control various physiological functions. The suprachiasmatic nucleus (SCN) is a master clock that integrates various peripheral clocks. Recently, the choroid plexus (CP) was reported to be one such peripheral clock, a circadian oscillator that might conversely affect the SCN. Hence, the principle aim of our study was to unravel the circadian oscillator within the CP. Quantitative PCR against rPer1, rPer2, and rBmal1 showed that CP in the lateral ventricle (CP-LV) and fourth ventricle (CP–4V) has a robust circadian oscillator. The phases of the CP oscillator are between those of the pineal gland (PG) and SCN. Bioluminescence monitoring of explants showed that the intrinsic circadian period of CP-LV and CP-4V was approximately 21 h, which is shorter than SCN and PG. It is possible that interaction between oscillators of the CP-LV, CP-4V, PG, and SCN ensures the SCN adopts a stable 24 h rhythm, with each of the regions having an intrinsic oscillator with different phases and periods. In situ hybridization analysis revealed that dusk-to-dawn variation of rPer2 expression was found in epithelial cells of the CP only. Furthermore, the CP circadian oscillator might control cerebrospinal fluid secretion. However, no dusk-to-dawn variation in expression of the water channel, aquaporin 1, was observed. Further investigations are needed to clarify the involvement of circadian rhythm on CP.

The membrane water channel aquaporin (AQP) family is composed of 13 isoforms in mammals, eight of which are reportedly expressed in the kidney: AQP1, 2, 3, 4, 6, 7, 8, and 11. These isoforms are differentially expressed along the renal tubules and collecting ducts. AQP1 and 7 are distributed in the proximal tubules, whereas AQP2, 3, and 4 occur in the collecting duct system. They play important roles in the reabsorption of water and some solutes across the plasma membrane. In contrast to other aquaporins found in the kidney, AQP6, 8, and 11 are localized to the cytoplasm rather than to the apical or basolateral membranes. It is therefore doubtful that these isoforms are directly involved in water or solute reabsorption. AQP6 is localized in acid-secreting type A intercalated cells of the collecting duct. AQP8 has been found in the proximal tubule but its cellular location has not yet been defined by immunohistochemistry. AQP11 seems to be localized in the endoplasmic reticulum (ER) of proximal tubule cells. Interestingly, polycystic kidneys develop in AQP11-null mice. Many vacuole-like structures are seen in proximal tubule cells in kidneys of newborn AQP11-null mice. Subsequently, cysts are generated, and most of the mice die within a month due to severe renal failure. Although ER stress and impairment of polycystin-1, the product of the gene mutated in autosomal-dominant polycystic kidney disease, are possible causes of cystogenesis in AQP11-null mice, the exact mechanism of pathogenesis and the physiological function of AQP11 are yet to be resolved.

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.

Neurons co-expressing kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus, named KNDy neurons, are directly affected by sex hormones, and are well known for regulating the secretion of gonadotropin-releasing hormone. However, recent studies have shown that KNDy neurons also project and terminate to tuberoinfundibular dopaminergic (TIDA) neurons, suggesting a role in prolactin secretion. Moreover, there is a possibility that other neurosecretory dopaminergic neurons regulating prolactin secretion, such as periventricular hypophyseal dopaminergic (PHDA) neurons, may also be innervated by KNDy neurons. In the present study, by means of double immunohistochemistry and retrograde neural tracer, we examined whether KNDy neurons project directly to PHDA neurons that project to blood vessels, as well as to TIDA neurons. The results revealed that KNDy neurons are widely projecting to neurosecretory dopaminergic neurons of the PHDA and TIDA neurons in rats and mice. Secondary, presence of a major receptor for NKB, neurokinin-3 receptor (NK3R), in PHDA and TIDA neurons was examined and it appeared that most TIDA and PHDA neurons possess NK3R. These findings indicate that, in rodents, KNDy neurons widely project to neurosecretory dopaminergic neurons distributed in the hypothalamus, and may affect them via the NKB-NK3R signaling pathway.

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.

Aquaporins are water channel proteins which enable rapid water movement across the plasma membrane. Aquaporin-5 (AQP5) is the major aquaporin and is expressed on the apical membrane of salivary gland acinar cells. We examined the effects of repeated administration of pilocarpine, a clinically useful stimulant for salivary fluid secretion, and isoproterenol (IPR), a stimulant for salivary protein secretion, on the abundance of AQP5 protein in rat salivary glands by immunofluorescence microscopy and semi-quantitative immunoblotting. Unexpectedly AQP5 was decreased in pilocarpine-administered salivary glands, in which fluid secretion must be highly stimulated, implying that AQP5 might not be required for fluid secretion at least in pilocarpine-administered state. The abundance of AQP5, on the other hand, was found to be significantly increased in IPR-administered submandibular and parotid glands. To address the possible mechanism of the elevation of AQP5 abundance in IPR-administered animals, changes of AQP5 level in fasting animals, in which the exocytotic events are reduced, were examined. AQP5 was found to be decreased in fasting animals as expected. These results suggested that the elevation of cAMP and/or frequent exocytotic events could increase AQP5 protein. AQP5 expression seems to be easily changed by salivary stimulants, although these changes do not always reflect the ability in salivary fluid secretion.

Niu D, Kondo T, Nakazawa T, Yamane T, Mochizuki K, Kawasaki T, Matsuzaki T, Takata K & Katoh R (2012) Histopathology 61, 543–551 Expression of aquaporin3 in human neoplastic tissues Aims: Aquaporin3 (AQP3) is distributed widely in mammalian tissues and plays an important role in fluid homeostasis. The aim of this study was to investigate the pattern of expression of AQP3 in a variety of human neoplastic tissues and to explore its diagnostic implications. Methods and results: We studied 798 neoplastic tissues using immunohistochemistry with anti-AQP3 antibody. We demonstrated a high positive frequency of AQP3 immunoreactivity in pituitary adenomas, salivary gland tumours, thymic tumours, adenocarcinoma of the lung and prostate, squamous cell carcinomas of the skin, oesophagus and uterine cervix, apocrine carcinoma of the breast, germinal cell tumours of the ovary and testis and urothelial carcinoma of the bladder. None of the sarcomas or central nervous system tumours showed AQP3 immunoreactivity. Most tumours with a high frequency of AQP3 positivity had corresponding or surrounding normal cells that also expressed AQP3. AQP3 was not a specific marker for benign or malignant epithelial neoplasms. Conclusion: AQP3 protein is expressed in a variety of epithelial tumours limiting its use as a diagnostic marker. Furthermore, AQP3 expression in tumour cells reflected the expression status of AQP3 in the corresponding normal cells. Our data suggest that water metabolism through AQP3 is maintained during neoplastic transformation in most human tissues.

The pituitary gland is composed of the adenohypophysis and neurohypophysis. The adenohypophysis contains endocrine cells, folliculo-stellate (FS) cells, and marginal layer cells, whereas the neurohypophysis mainly comprises axons and pituicytes. To understand the molecular nature of water transfer in the pituitary gland, we examined the immunohistochemical localization of the membrane water channels aquaporin-4 (AQP4) and AQP5 in rat tissue. Double immunofluorescence analysis of AQP4 and S100 protein, a known marker for FS cells, marginal layer cells, and pituicytes, clearly revealed that FS cells and marginal layer cells in the adenohypophysis and the pituicytes in pars nervosa are positive for AQP4. AQP5 was found to be localized at the apical membrane in some marginal layer cells surrounding the Rathke’s residual pouch, in which AQP4 was observed to be localized on the basolateral membranes. These results suggest the following possibilities: 1) FS cells especially require water for their functions and 2) transepithelial water transfer could occur between the lumen of Rathke’s residual pouch and the interstitial fluid in the adenohypophysis through the AQP4 and AQP5 channels in the marginal layer cells.

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.

Aquaporins are membrane water channel proteins that function mainly in water transfer across cellular membranes. In our present study, we investigated the immunohistochemical distribution of aquaporin 1 (AQP1), AQP3, AQP4, and AQP5 in the mouse respiratory system by immunofluorescence, immunoperoxidase, and immunoelectron microscopy. AQP3, AQP4, and AQP5 are expressed in epithelial cells, whereas AQP1 is expressed in subepithelial connective tissues and capillaries. In the airway surface epithelia from the nasal cavity to the intrapulmonary bronchioles, AQP5 was found to be mainly localized to the luminal side and both AQP3 and AQP4 to the abluminal side. In the alveolar epithelium, AQP5 is localized to the apical membranes of both type I and type II alveolar cells. Compared with the previous studies on the rat respiratory system, in which AQP5 is restricted to the alveolar type I cells and absent from the airway surface epithelia, we found that AQP5 in the mouse is much more widely distributed throughout the surface epithelia. These results suggest that AQP5 has a critical role in water-handling, such as the maintenance of airway surface liquid and clearance of alveolar fluid in the mouse respiratory system.

Reversion-induced LIM protein (RIL) is a member of the ALP (actinin-associated LIM protein) subfamily of the PDZ/LIM protein family. RIL serves as an adaptor protein and seems to regulate cytoskeletons. Immunoblotting suggested that RIL is concentrated in the astrocytes in the central nervous system. We then examined the expression and localization of RIL in the rat central nervous system and compared it with that of water channel aquaporin 4 (AQP4). RIL was concentrated in the cells of ependyma lining the ventricles in the brain and the central canal in the spinal cord. In most parts of the central nervous system, RIL was expressed in the astrocytes that expressed AQP4. Double-labeling studies showed that RIL was concentrated in the cytoplasm of astrocytes where glial fibrillary acidic protein was enriched as well as in the AQP4-enriched regions such as the endfeet or glia limitans. RIL was also present in some neurons such as Purkinje cells in the cerebellum and some neurons in the brain stem. Differential expression of RIL suggests that it may be involved in the regulation of the central nervous system.

Phosphorylation of serine 256 (S256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-PK1cells that express point mutations of AQP2 S261 and S256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 (S261A) and AQP2 (S261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, S261A, S261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of S256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of S261. Finally, blocking AQP2 endocytosis by methyl-β-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of S256 and S261, although as previously reported, the S256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of S261. Together, our data indicate that the phosphorylation state of AQP2 at S261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of S261 phosphorylation/dephosphorylation in vasopressin action remains to be determined.

Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood-brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2.

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

Vasopressin is the main hormone that regulates water conservation in mammals and one of its major targets is the principal cells in the renal collecting duct. Vasopressin increases the apical water permeability of principal cells, mediated by apical accumulation of aquaporin-2 (AQP2), a water channel protein, thus facilitating water reabsorption by the kidney. The mechanisms underlying the accumulation of AQP2 in response to vasopressin include vesicular trafficking from intracellular storage vesicles expressing AQP2 within several tens of minutes (short-term regulation) and protein expression of AQP2 over a period of hours to days (long-term regulation). This chapter reviews vasopressin signaling in the kidney, focusing on the molecular mechanisms of short- and long-term regulations of AQP2 expression.

A GFP-labeled sodium-dependent glucose transporter SGLT1 (SGLT-GFP) was transfected into MDCK cells. SGLT-GFP was localized at the apical membrane in confluent cells. When cellular cholesterol was depleted by methyl-beta-cyclodextrin (MbetaCD) treatment, the localization of SGLT-GFP gradually switched from apical to whole plasma membrane. Time-lapse microscopy revealed that the effect of MbetaCD appeared within 30 min, and that the transition of SGLT-GFP to the whole plasma membrane was completed within 2 hr after the administration. Immunofluorescence microscopy revealed that the tight junction framework remained steady during this process. The effect of MbetaCD on SGLT-GFP localization was counter-balanced by the addition of cholesterol into the culture medium. Disruption of microtubules by colcemid also perturbed SGLT-GFP localization. SGLT-GFP localized to the whole plasma membrane by colcemid treatment, and apical localization was restored within 1 hr after -removal of colcemid. Inhibition of protein synthesis by cycloheximide had no effect on the transition of SGLT-GFP induced by the MbetaCD or colcemid. These results indicated that the apical localization of SGLT-GFP is maintained by cellular cholesterol and microtubules, possibly with an apical recycling machinery.

Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.

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

We examined the expression and immunolocalization of water-channel aquaporins in the mammary gland by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry. RT-PCR and immunoblotting revealed the expression of aquaporin-1 (AQP1) and AQP3 in the lactating rat mammary gland. AQP3 was detected in the alveolar epithelium and duct system whereas AQP1 was found in the capillaries and venules. AQP3 was present in the basolateral membrane of secretory epithelial cells and intralobular and interlobular duct epithelial cells. The main duct near the orifice in the nipple, which is comprised of a stratified epithelium, bore AQP3 in its basal and intermediate layers. AQP1 was located in both the apical and basolateral membranes of capillary and venule endothelia. AQP3 was not detected in virgin females. AQP3 was found in some differentiating mammary epithelial cells in the pregnant rat. AQP1 was present in capillaries and venules in the differentiating mammary gland of the pregnant rat and in the mammary fat pad of virgin females. We found a similar distribution of AQP1 and AQP3 in the mouse. AQP1 and AQP3 seem to play roles in the synthesis and/or secretion of milk.

Aquaporin 1 (AQP1) is an isoform of membrane water channels. We examined the distribution of AQP1 in peripheral nerves. Immunofluorescence microscopy with specific antibodies to AQP1 was performed in the tissue sections of the rat tongue, esophagus, trachea, and sciatic nerve. AQP1 was present in the perineurium of nerve fiber bundles in the sciatic nerve and nerve fiber bundles in the tongue, esophagus, and trachea. Laser confocal microscopy of the double-labeled specimens for AQP1 and sugar transporter GLUT1 revealed that AQP1 was localized in the outer layer(s) of the perineurial cell layers, whereas GLUT1 was present throughout the entire perineurial cell layers. AQP1 and GLUT1 were also present in blood vessels inside the nerve fiber bundle. They were rarely found in the same blood vessels, namely, AQP1 was hardly detectable in GLUT1-positive blood vessels and GLUT1 was hardly detectable in AQP1-positive vessels. AQP1 in these sites of the blood-nerve barrier may participate in the water transfer across the barrier and contribute to the maintenance of the milieu of the nerve. In addition, we showed the presence of AQP1 in the perichondrium of the tracheal cartilage suggesting a possible role in water transfer between the cartilage and surrounding connective tissues.

Aquaporins (AQPs) are water channel proteins of cellular membranes serving in the permeation of water across the membrane. AQP families are found virtually in all types of life ranging from bacteria to plant and animal cells. In mammals, at least 13 isoforms of AQPs have been identified. They are classified into three subtypes: classical aquaporins, aquaglyceroporins, and superaquaporins. These AQPs are differentially expressed in a wide variety of cells and tissues in the body, and play important roles in water metabolism. In the kidney, at least 6 isoforms of AQPs, namely AQP1, AQP2, AQP3, AQP4, AQP6, and AQP7, are reported to be expressed. Water transfer occurs mainly in the proximal tubules and collecting ducts in the kidney. In the proximal tubules, AQP1 and AQP7 are expressed, among which AQP1 plays a major role in water reabsorption. In the collecting ducts, AQP2, AQP3, AQP4, and AQP6 are expressed. AQP3 and AQP4 are localized at the basolateral membrane. AQP2 is stored in the cytoplasmic vesicles and is translocated to the apical plasma membrane in response to antidiuretic hormone. Mutations of AQP2 lead to either loss of channel function or mistrafficking and result in nephrogenic diabetes insipidus, the inability to concentrate urine.

Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.

Aquaporin-2 (AQP2) is one of the membrane water channel proteins expressed in principal cells of the kidney collecting ducts. In the basal state, AQP2 resides in the storage vesicles localized in the subapical cytoplasm. Upon stimulation with vasopressin, AQP2 is translocated to the apical plasma membrane by the exocytic fusion of the storage vesicles with the apical membrane. This translocation enables the transepithelial reabsorption of water from the lumen to the interstitium via AQP2 at the apical membrane and AQP3/AQP4 at the basolateral membrane. AQP2-storage vesicles are distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and lysosomes. The early endosomal marker EEA1 is colocalized with some of AQP2 vesicles. Further analyses in Madin-Darby canine kidney (MDCK) cells transfected with AQP2 revealed that subapical Rab11-positive/EEA1-negative smaller vesicles constitute part of the AQP2 storage vesicles for the translocation to the apical membrane. Termination of stimulation results in the retrieval of AQP2 to the larger EEA1-positive early endosomal compartment. AQP2 is then transferred to the subapical storage compartment in a PI3-kinase-dependent manner. GLUT4 is an isoform of glucose transporters whose localization is also regulated by vesicular trafficking induced by insulin stimulation. Comparison of the intracellular localization of AQP2 with GLUT4 suggests distinct regulation of AQP2 trafficking. Keywords:: water channel, aquaporin-2, kidney, collecting duct, endosome

Aim: To clone new phosphate transporters from the kidney. Methods: EST bank was screened for new members of Na-dependent inorganic phosphate (Pi) co-transporter. To obtain a full-length clone, a rat kidney cDNA library was screened with the EST clone as a probe. Results: We isolated a cDNA that encodes a type I Na/Pi co-transporter. Based on the sequence homology and tissue distribution, it will be the rat ortholog of human NPT4 (Solute Carrier family 17, member A3, SLC17A3) and is designated rNPT4. When expressed in Xenopus oocytes, Na/Pi co-transport activity was demonstrated. In Northern blotting analysis, rNPT4 was selectively expressed in the liver and kidney. In immunohistochemical analysis, rNPT4 was localized at the apical membrane of the proximal tubules (S2, S3) in the rat kidney. The electron microscopic studies confirmed this finding. Conclusion: The present study revealed the expression of a new type I Na/Pi co-transporter at the brush border membrane of the proximal tubules in the kidney.

Immunolabeling of cultured cell lines of epithelial origin is often performed to study cell polarity. To explore the localization of cellular molecules in detail, we devised an efficient and convenient method to prepare semithin and ultrathin cryosections of cells cultured on permeable support. Multiple specimens were piled up to obtain many vertical images of cells in a single section. Freezing them in cooled n-hexane resulted in clear and homogeneous frozen specimens, from which semithin and ultrathin cryosections of epitherial cells on permeable support were easily obtained. With this method, it is possible to prepare many cryosections of good quality for histochemical examination of vertical images of cells.

cDNAs encoding facilitated-diffusion glucose transporters GULT1, GULT3, GULT4, and GULT5 were introduced into the rat pancreas by in-vivo electroporation method, and their expression and localization in pancreatic acinar cells were examined immunohistochemically. GLUT1 was localized at the basolateral membrane, whereas GLUT3 and GLUTS were at the apical membrane. Restriction of GLUT3 and GLUTS to the apical membrane makes a marked contrast to their localization to the entire plasma membrane when expressed in hepatocytes in situ. Such differential localization may be due to differential apical targeting mechanism: direct targeting in acinar cells and indirect transcytotic delivery in hepatocytes in situ. GLUT4 was present in the membrane of zymogen secretory granules in the cytoplasm in acinar cells. This observation suggests that GLUT4 is segregated to the regulated secretory pathway. Expression of glucose transporters in situ is a useful method in analyzing the targeting mechanism of membrane proteins in cells of tissues in situ.

Aquaporin-5 (AQP5), an isoform of membrane water channel aquaporins, is expressed in the salivary and lacrimal glands. We surveyed the expression and immunohistochemical localization of AQP5 in the rat digestive system. RT-PCR analysis revealed that AQP5 is expressed in the submandibular gland, tongue, gastric corpus, pyloric region, duodenum, and liver. Immunofluorescence microscopy using AQP5-specific antibodies showed that AQP5 protein is present in the minor salivary glands of the tongue, the pyloric glands, and duodenal glands. To distinguish apical and basolateral domains of the plasma membrane of epithelial cells, double-immunofluorescence staining for AQP5 and tight junction protein occludin was performed. In the minor salivary gland, AQP5 was present in both the serous and mixed secretory end portions. AQP5 was found in the apical membrane of the secretory cells including intercellular secretory canaliculi demarcated with occludin. At higher magnifications, omega-shaped indentations of AQP5 labeling were seen along the apical membrane, suggesting a dynamic process for the apical membrane in exocytosis. Only weak labeling for AQP5 was detected in the basolateral domain. In the stomach, AQP5 was detected in the apical membrane of the pyloric gland secretory cells. In the duodenum, AQP5 was restricted to duodenal glands, where it was localized to the apical membrane. AQP5 was not detected in the intestinal glands or cells in the villi. These observations show that AQP5 is localized mainly in the apical membrane, including intercellular secretory canaliculi of secretory cells in the minor salivary glands, pyloric glands, and duodenal glands. AQP5 appears to play an important role in water transfer in these glands.

Expression of genes followed by the examination of the localization of their products by histochemical methods is one of important and powerful approaches to elucidate the role of genes of interest. We show here a simple and versatile electroporation method for the introduction of plasmids into cells cultured on coverslips for histochemical examination. Electroporation apparatus was assembled using a 35-mm plastic dish and aluminum wire. The apparatus was placed over the coverslip and plasmid DNA was applied onto it. Green fluorescent protein (GFP) was used as a reporter of transfection. Under the appropriate settings of voltage, duration, and pulse numbers, successful transfection was easily achieved in 3T3-L1 fibroblasts, HeLa cells, Cos7 cells, and MDCK cells. The electroporation method described here is simple, easy, rapid, and economical, and may be best suited for the screening of many genes for their histochemical examination.

Water channel proteins, aquaporins, are integral membrane proteins serving in the permeation of water and some other small molecules. Eleven isoforms of aquaporins have been identified from various tissues to date. They are expressed in tissue- and cell-specific manners, and are closely related to the specific functions of tissues and cells. Aquaporins are usually localized to the plasma membrane. Some isoforms are present in cytoplasmic compartments, and their translocation to the plasma membrane is crucial in the regulation of water transfer. This review focuses on the localization of aquaporins in mammalian tissues and discusses the physiological importance of water channels.

The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.

Background The aim of the present study was to clarify the anatomy between the left triangular ligament (LTL) and the appendix fibrosa hepatis (AFH) in order not to sever the AFH when dissecting the LTL. Methods Totals of 43 and 27 cadaveric livers were examined macroscopically and histologically, respectively. Results The LTL attached itself to the diaphragmatic surface of the AFH through almost all lengths of the AFH. This might be the reason why AFH is so often dissected together with the LTL. There were two types of relation between the LTL and the AFH; in one type, the starting point of the LTL existed on the left liver and in the other type, it was on the AFH. Twenty-five of 27 AFH included remnants of the bile duct and 12 of 25 AFH had comparatively large bile ducts, which was unexceptionally accompanied by the well-developed peribiliary vascular plexus. AFH showed a variety of shapes, such as rectangular (6/43), long triangular (4/43), short triangular (7/43), triangular plus cordlike (11/43), cordlike (12/43) and bifurcated (3/43) types. Conclusions As AFH sometimes includes relatively large bile ducts, it is recommended for surgeons to sever the AFH not just simply by electrocautery but by ligating its stump securely.