Aquaporin-4 Protein Research Articles

Is aquaporin-3 involved in water-permeability changes in the killifish during hypoxia and normoxic recovery, in freshwater or seawater?

Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance, to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavorable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs-the gills and intestine. Using immunofluorescence, we localized aquaporin-3 (AQP3) protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial AQP3 messenger-RNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter AQP3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish AQP3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater and facilitates intestinal permeability in seawater and freshwater.

Aquaporin 4 in the chicken oviduct during a pause in laying induced by food deprivation

In the present study we hypothesize that aquaporin 4 (AQP4) expression in the chicken oviduct would change during a pause in egg laying that was induced by fasting. Accordingly, the aim of this investigation was to examine the AQP4 mRNA and protein expression, and immunolocalization in the chicken oviduct during the course of regression. The experiment was carried out on laying hens subjected to a pause in laying that was induced by food deprivation for 5 days. Control hens were fed ad libitum. The birds were sacrificed on day 6 of the experiment and all segments of the oviduct were isolated, including the infundibulum, magnum, isthmus, shell gland, and vagina. Subsequently, the gene and protein expressions of AQP4 in the tissues were tested by real-time PCR and Western blot, respectively. The relative mRNA expression of AQP4 was the highest in the infundibulum and vagina and the lowest, and least detectable, in the magnum. The level of AQP4 protein was the highest in the infundibulum and the lowest in the magnum. Fasting resulted in a decrease of the AQP4 mRNA expression () in the infundibulum, a decrease in protein abundance () in the shell gland, and an increase in protein level () in the vagina. Immunohistochemistry demonstrated tissue- and cell-dependent localization of AQP4 protein in the oviductal wall. The intensity of staining was as follows: the infundibulum shell gland vagina isthmus ≫ magnum. In the control hens, the immunoreactivity for AQP4 in the vagina was similar, whereas in other oviductal segments, the immunoreactivity was stronger when compared with the chickens subjected to a pause in laying. In summary, these findings suggest that the AQP4 is an essential protein involved in the regulation of water transport required to create a proper microenvironment for fertilization and egg formation in the hen oviduct.

Renomedullary Interstitial Cell Endothelin A Receptors Regulate BP and Renal Function.

The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro. To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age. BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE2 excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE2 excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1-induced renomedullary interstitial cell contraction was observed. Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

Hypoxia-Induced Aquaporin-3 Changes Hepatocellular Carcinoma Cell Sensitivity to Sorafenib by Activating the PI3K/Akt Signaling Pathway.

PurposeHypoxia-induced changes are primarily activated in patients with hepatocellular carcinoma (HCC) and long-term sorafenib exposure, thereby reducing the sensitivity to the drug. Aquaporin-3 (AQP3), a member of the aquaporin family, is a hypoxia-induced substance that affects the chemosensitivity of non-hepatocellular tumors. However, its expression and role in the sensitivity of hypoxic HCC cells to sorafenib-induced apoptosis remain unclear. The purpose of this study was to detect changes in AQP3 expression in hypoxic HCC cells and to determine whether these changes alter the sensitivity of these cells to sorafenib.Materials and MethodsHuh7 and HepG2 hypoxic cell models were established and AQP3 expression was detected using quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Furthermore, the role of AQP3 in cell sensitivity to sorafenib was evaluated via flow cytometry, Western blotting, and a CCK-8 assay.ResultsThe results of qPCR and Western blotting showed that AQP3 was overexpressed in the Huh7 and HepG2 hypoxic cell models. Furthermore, AQP3 protein levels were positively correlated with hypoxia-inducible factor-1α (HIF-1α) levels. Compared with cells transfected with lentivirus-GFP (Lv-GFP), hypoxic cells transfected with lentivirus-AQP3 (Lv-AQP3) were less sensitive to sorafenib-induced apoptosis. However, the sensitivity to the drug increased in cells transfected with lentivirus-AQP3RNAi (Lv-AQP3RNAi). Akt and Erk phosphorylation was enhanced in Lv-AQP3-transfected cells. Compared with UO126 (a Mek1/2 inhibitor), LY294002 (a PI3K inhibitor) attenuated the AQP3-induced insensitivity to sorafenib observed in hypoxic cells transfected with Lv-AQP3. Combined with LY294002-treated cells, hypoxic cells transfected with Lv-AQP3RNAi were more sensitive to sorafenib.ConclusionThe study results show that AQP3 is a potential therapeutic target for improving the sensitivity of hypoxic HCC cells to sorafenib.

Aquaporin-Containing Proteopolymersomes in Polyelectrolyte Multilayer Membranes.

The field of membranes saw huge developments in the last decades with the introduction of both polyelectrolyte multilayer (PEM)-based membranes and biomimetic membranes. In this work, we combine these two promising systems and demonstrate that proteopolymersomes (PP+) with the incorporated aquaporin protein can be distributed in a controlled fashion using PEMs, even on the inner surface of a hollow fiber membrane. In this way, various proteopolymersome multilayers (PPMs) are fabricated using PP+ as the positively charged species in combination with the polyanions poly(styrene 4-sulfonate) (PSS) and poly(acrylic acid) (PAA). It is shown by reflectometry through alternately adsorbing the polyanions and PP+ that, for both PAA and PSS, a good layer growth is possible. However, when the multilayers are imaged by SEM, the PAA-based PPMs show dewetting, whereas vesicular structures can only be clearly observed in and on the PSS-based PPMs. In addition, membrane permeability decreases upon coating the PPMs to 2.6 L∙m−2∙h−1∙bar−1 for PAA/PP+ and 7.7 L∙m−2∙h−1∙bar−1 for PSS/PP+. Salt retentions show that PAA/PP+ layers are defective (salt retentions <10% and high molecular weight cut-off (MWCO)), in line with the observed dewetting behavior, while PPMs based on PSS show 80% MgSO4 retention in combination with a low MWCO. The PSS/PP+ membranes show a Donnan-exclusion behavior with moderate MgCl2 retention (50%–55%) and high Na2SO4 retention (85%–90%) indicating a high amount of negative charge present within the PPMs. The corresponding PEMs, on the other hand, are predominately positively charged with MgCl2 retention of 97%–98% and Na2SO4 retention of 57%–80%. This means that the charge inside the multilayer and, thus, its separation behavior can be changed when PP+ is used instead of a polycation. When comparing the PPM membranes to the literature, similar performances are observed with other biomimetic membranes that are not based on interfacial polymerization, but these are the only ones prepared using a desired hollow fiber geometry. Combining PEMs and biomimetic approaches can, thus, lead to relevant membranes, especially adding to the versatility of both systems.

Epidemiology of Pediatric NMOSD in Germany and Austria.

Background: Neuromyelitis optica spectrum disorders (NMOSD) are severe inflammatory demyelinating disorders of the central nervous system mainly characterized by recurrent episodes of uni- or bilateral optic neuritis (ON), transverse myelitis (TM) and brainstem syndromes (BS). The majority of adult patients has serum antibodies directed against the water channel protein aquaporin 4 (AQP4-abs). In pediatric patients, AQP4-abs are less, while antibodies against myelin oligodendrocyte glycoprotein (MOG-abs) are more frequently detectable than in adults. Some children with NMOSD have neither AQP4- nor MOG-ab (double-seronegative).Objective: Evaluation of epidemiological data regarding incidence and prevalence of pediatric NMOSD in Germany and Austria.Methods: We recruited pediatric NMOSD patients between 1 March 2017 and 28 February 2019 with five different tools: (1) ESPED (Surveillance Unit for Rare Pediatric Disorders in Germany), (2) ESNEK (Surveillance for Rare Neurological Disorders during Childhood), (3) pediatric neurology working group within the Austrian Society of Pediatrics and Adolescent Medicine, (4) BIOMARKER Study and (5) NEMOS (Neuromyelitis optica Study Group). We requested data regarding clinical symptoms, antibody status, therapy regimen and response via a standardized questionnaire.Results: During the 2-year recruitment period, 46 (both incidental and prevalent) patients with a suspected diagnosis of NMOSD were brought to our attention. Twenty-two of these patients did not fulfill the inclusion criteria. Of the remaining 24 children, 22 had a median age at onset of 11 (range 3–17) years and 16/22 were female (72.7%) (no data in two patients). Sixteen of 24 patients were AQP4-ab positive (67%), 4/24 MOG-ab positive (16.7%), three children were double-seronegative and in one patient no antibody testing was done. We calculated an incidence rate of 0.022 per 100,000 person-years for Germany, while there was no incidental case in Austria during the recruitment period. The prevalence rate was 0.147 and 0.267 per 100,000 persons in Germany and Austria, respectively.Conclusion: Pediatric NMOSD, with and without associated antibodies, are very rare even considering the different limitations of our study. An unexpected finding was that a considerable proportion of patients was tested neither for AQP4- nor MOG-abs during diagnostic work-up, which should prompt to establish and disseminate appropriate guidelines.

Impacts of silicon and silicon nanoparticles on leaf ultrastructure and TaPIP1 and TaNIP2 gene expressions in heat stressed wheat seedlings

Heat stress is one of the most crucial factors affecting crop growth and productivity worldwide. So, searching for a potent eco-friendly heat stress alleviator is the main issue nowadays. The current study was conducted to assess the ameliorative effects of 1.5 mM potassium silicate (K2SiO3, further only Si) or 1.66 mM silicon dioxide nanoparticles (SiNPs) on wheat (Triticum aestivum L.) seedlings exposed to heat stress (45 °C, 4 h). The observations show that Si or SiNPs treatments significantly restored the heat stress-provoked ultrastructural distortions of cellular organelles, particularly chloroplasts and the nucleus. Further, both Si and SiNPs enhanced the photosynthetic capacity as revealed by increments in the photochemical efficiency of photosystem II and the performance index as well as the content of photosynthetic pigments. A reduction in malondialdehyde accumulation in Si and SiNPs treated plants was positively related to their membrane stability index. The reverse transcription PCR analysis showed that Si treatment but not SiNP treatment stimulated the overexpressions of both Triticum aestivum plasma membrane intrinsic protein (TaPIP1) and Triticum aestivum nodulin 26-like intrinsic protein (TaNIP2) aquaporin genes parallelly with an improvement in the relative water content. This investigation reveals that Si was more effective than SiNPs in restoring the heat stress injuries. To the best of our knowledge, this is the first investigation exploring the effects of Si and SiNPs in improving thermotolerance of wheat seedlings.

Effect of aquaporin 4 protein overexpression in nigrostriatal system on development of Parkinson’s disease

Objects Recent studies indicated that aquaporin 4 (AQP4), as the main water channel in the central nervous system (CNS), participated in the onset and progression of Parkinson’s disease (PD). But how the AQP4 influenced the exacerbation of PD has not been described in detail. In this study, the effect of the AQP4 protein overexpression in nigrostriatal system that include substantia nigra (SN) and striatum (CPu) on the development of PD was investigated. Methods Forty male Sprague Dawley rats were equally divided into two groups at random: PD group and control group, PD group undergoing surgery and receiving 6-hydroxydopamine (6-OHDA). Using MRI tracer-based method, extracellular space (ECS) diffusion parameters of nigrostriatal system for all rats were measured, including the clearance coefficient (k’) and the half-life (t1/2). Immunohistochemistry of AQP4 was performed for 20 rats. Results The area of dark-stained AQP4 immunoreactivity increased markedly in SN of PD rats, there were significant differences between two groups (SN: t = 5.809, p < 0.0001; CPu: t = 5.943, p < 0.0001). And the diffusion parameters were significantly greater in PD group than that of control group, including k′ (SN: t = 5.519, p < 0.0001; CPu: t = 2.149, p = 0.045) and t1/2 (SN: t = 6.131, p < 0.0001; CPu: t = 6.708, p < 0.0001). There was a significant positive correlation between the AQP4 expression level and the k’ values (SN: r = 0.827, p = 0.0031; CPu: r = 0.641, p = 0.0046), and a significant negative correlation between AQP4 and the t1/2 values (SN: r=−0.654, p = 0.0403; CPu: r=-0.664, p = 0.0362). Conclusions The results indicated that AQP4 expression was increased in nigrostriatal system of PD rats, therefore, the overexpression of AQP4 led to acceleration of the diffusion and drainage process of drugs in ECS, reduced the effect of drugs for the treatment of PD, inhibited the development of PD.

Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct.

Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates with a retromer complex, which regulates intracellular trafficking and the abundance of membrane proteins. Since the carboxyl terminus of aquaporin-2 (AQP2c) has a class I PDZ-interacting motif (X-T/S-X-Φ), the role of SNX27 in the regulation of AQP2 was studied. Co-immunoprecipitation assay of the rat kidney demonstrated an interaction of SNX27 with AQP2. Glutathione S-transferase (GST) pull-down assays revealed an interaction of the PDZ domain of SNX27 with AQP2c. Immunocytochemistry of HeLa cells co-transfected with FLAG-SNX27 and hemagglutinin (HA)-AQP2 also revealed co-localization throughout the cytoplasm. When the PDZ domain was deleted, punctate HA-AQP2 labeling was localized in the perinuclear region. The labeling was intensively overlaid by Lysotracker staining but not by GM130 labeling, a cis-Golgi marker. In rat kidneys and primary cultured inner medullary collecting duct cells, the subcellular redistribution of SNX27 was similar to AQP2 under 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Cell surface biotinylation assay showed that dDAVP-induced AQP2 translocation to the apical plasma membrane was unaffected after SNX27 knockdown in mpkCCD cells. In contrast, the dDAVP-induced AQP2 protein abundance was significantly attenuated without changes in AQP2 mRNA expression. Moreover, the AQP2 protein abundance was markedly declined during the dDAVP withdrawal period after stimulation under SNX27 knockdown, which was inhibited by lysosome inhibitors. Autophagy was induced after SNX27 knockdown in mpkCCD cells. Lithium-induced nephrogenic diabetes insipidus in rats revealed a significant downregulation of SNX27 in the kidney inner medulla. Taken together, the PDZ domain-containing SNX27 interacts with AQP2 and depletion of SNX27 contributes to the autophagy-lysosomal degradation of AQP2.

Acupoint Catgut Embedding Improves the Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Rats.

Background This study investigated the potential therapeutic effects of acupoint catgut embedding (ACE) at ST36 and BL13 on lipopolysaccharide- (LPS-) induced acute respiratory distress syndrome (ARDS) in rats. Materials and Methods Male Sprague-Dawley rats were randomized into the normal saline (NS group with a sham procedure), lipopolysaccharide (LPS group with a sham procedure), and LPS plus ACE (LPS+ACE with ACE at bilateral BL13 and ST36 acupoints one day before LPS injection) groups. After intratracheal instillation of normal saline or LPS (0.5 mg/kg), all rats were subjected to mechanical ventilation for 4 h. Their blood gas was analyzed before and after lung injury, and their lung pressure-volumes were measured longitudinally. The levels of TNF-α, IL-6, IL-10, and phosphatidylcholine (PC) and total proteins (TP) in bronchial alveolar lavage fluid (BALF) were assessed. Their wet to dry lung weight ratios, histology, myeloperoxidase (MPO), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were measured. Their lung aquaporin 1 (AQP1) and Occludin protein levels were analyzed. Results LPS administration significantly decreased the ratios of PaO2/FiO2 and pressure-volumes and induced lung inflammation and injury by increased concentrations of TNF-α, IL-6, IL-10, and TP in BALF and MPO and MDA in the lung but decreased PC in BALF and SOD activity in the lungs. LPS also reduced AQP1 and Occludin protein levels in the lung of rats. In contrast, ACE significantly mitigated the LPS-induced lung injury, inflammation, and oxidative stress and preserved the AQP1 and Occludin contents in the lung of rats. Conclusions ACE significantly improved respiratory function by mitigating inflammation and oxidative stress and preserving AQP1 and Occludin expression in the lung in a rat model of LPS-induced ARDS.

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema

SummarySwelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Concomitant enlargement of perivascular spaces and decrease in glymphatic transport in an animal model of cerebral small vessel disease

ObjectivesTo observe glymphatic transport and evaluate enlarged perivascular spaces (PVSs) in spontaneously hypertensive rats (SHRs). MethodsSHRs were used as an animal model of cerebral small vessel disease, and Wistar Kyoto (WKY) rats were used as the control group. Histopathology was used to evaluate the enlargement of PVSs. A fluorescent tracer was infused into the cisterna magna of rats, and the proportion of the brain parenchyma area exposed to the fluorescent tracer was later quantified to evaluate the influx and efflux function of the glymphatic system. The global and polarized expression of aquaporin protein 4 (AQP4) was analyzed by immunofluorescence. ResultsCompared with WKY rats, SHRs exhibited obviously enlarged PVSs and significantly decreased influx and efflux function of the glymphatic system. The results showed a significant decrease in AQP4 polarity in SHRs, but a difference in global AQP4 expression was not observed between SHRs and WKY rats. ConclusionsImpaired glymphatic transport may be involved in the pathogenesis of arteriolosclerotic cerebral small vessel disease, and enlarged PVSs may be a manifestation of the impaired glymphatic system.

Lithium Chloride and GSK3 Inhibition Reduce Aquaporin-2 Expression in Primary Cultured Inner Medullary Collecting Duct Cells Due to Independent Mechanisms.

Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism.

Computational Modeling on Aquaporin-3 as Skin Cancer Target: A Virtual Screening Study.

Aquaporin-3 (AQP3) is one of the aquaglyceroporins, which is expressed in the basolateral layer of the skin membrane. Studies have reported that human skin squamous cell carcinoma overexpresses AQP3 and inhibition of its function may alleviate skin tumorigenesis. In the present study, we have applied a virtual screening method that encompasses filters for physicochemical properties and molecular docking to select potential hit compounds that bind to the Aquaporin-3 protein. Based on molecular docking results, the top 20 hit compounds were analyzed for stability in the binding pocket using unconstrained molecular dynamics simulations and further evaluated for binding free energy. Furthermore, examined the ligand-unbinding pathway of the inhibitor from its bound form to explore possible routes for inhibitor approach to the ligand-binding site. With a good docking score, stability in the binding pocket, and free energy of binding, these hit compounds can be developed as Aquaporin-3 inhibitors in the near future.

Pre-atherosclerotic flow and oncotically active solute transport across the arterial endothelium

Atherosclerosis starts with transmural (transwall) pressure-driven advective transport of blood-borne low-density lipoprotein (LDL) cholesterol across rare endothelial cell (EC) monolayer leaks into the arterial subendothelial intima (SI) wall layer where they can spread, bind to extracellular matrix and seed lesions. The local SI LDL concentration, which governs LDL’s binding kinetics, depends on the overall diluting transmural liquid flow. Transmural pressures typically compress the SI at physiological pressures, which keeps this flow low. Nguyen et al. (2015) showed that aortic ECs express the water channel protein aquaporin-1 (AQP1) and the transEC (δP) portion of the transmural (ΔP) pressure difference drives, in parallel, water across AQP1s and plasma across interEC junctions. Since the lumen is isotonic, selective AQP1-mediated water flow should quickly render the ECs’ lumen side hypertonic and the SI hypotonic; resulting transEC oncotic pressure differences, δπ, should oppose δP and quickly halt transEC flow. Yet Nguyen et al.’s (2015) transAQP1 flows persist for hours. To resolve this paradox, we extend our fluid filtration theory Joshi et al. (2015) to include mass transfer for oncotically active solutes like albumin. This addition nonlinearly couples mass transfer, fluid flow and wall mechanics. We simultaneously solve these problems at steady state. Surprisingly it finds that media layer filtration causes steady SI to exceed EC glycocalyx albumin concentration. Thus δπ reinforces rather than opposes δP, i.e., it sucks water from, rather than pushing water into the lumen from the SI. Endothelial AQP1s raise the overall driving force for flow across the EC above δP, most significantly at pressures too low to compress the SI, and they increase the ΔP needed for SI compression. This suggests the intriguing possibility that increasing EC AQP1 expression can raise this requisite compression pressure to physiological values. That is, increasing EC AQP1 may decompress the SI at physiological pressures, which would significantly increase SI thickness, flow and subsequently SI LDL dilution. This could retard LDL binding and delay preatherosclerotic lesion onset. The model also predicts that glycocalyx-degrading enzymes decrease overall transEC driving forces and thus lower, not raise, transmural water flux.

Chloroquine attenuates lithium-induced NDI and proliferation of renal collecting duct cells.

Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na+-K+-2Cl- cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and β-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Erlotinib Induces Dry Skin via Decreased in Aquaporin-3 Expression.

An adverse reaction of dry skin occurs frequently during treatment with anticancer epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). In this study, we conducted basic research to clarify the mechanism of EGFR-TKI-induced dry skin and propose new treatments or preventative measures. Dermal water content was significantly lower in the erlotinib-treated mice than in the control group. An assessment of the expression levels of functional genes in the skin revealed that only the expression of the water channel aquaporin-3 (AQP3) was significantly decreased in the erlotinib-treated group. When erlotinib was added to epidermal keratinocyte HaCaT cells, the expression levels of both AQP3 mRNA and protein decreased. Erlotinib treatment also significantly decreased the expression levels of phospho-EGFR and phospho-extracellular signal-regulated kinase (ERK), both in HaCaT cells and mouse skin. Dry skin due to erlotinib may be caused by the decreased expression of AQP3 in the skin, thereby limiting water transport from the vascular side to the corneum side. The decrease in AQP3 may also be attributable to ERK suppression via inhibition of EGFR activity by erlotinib. Therefore, substances that increase AQP3 expression may be effective for erlotinib-induced dry skin.

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A-Dependent Signaling in Kidney.

Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein aquaporin-2. Vasopressin signaling is initiated by binding to a G-protein-coupled receptor called V2R, which signals through heterotrimeric G-protein subunit Gs α, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteome-wide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2R-selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad-spectrum kinase inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide), and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic data sets have been made available online for modeling vasopressin signaling and signaling downstream from other G-protein-coupled receptors. SIGNIFICANCE STATEMENT: New developments in protein mass spectrometry are facilitating progress in identification of signaling networks. Using mass spectrometry, it is now possible to identify and quantify thousands of phosphorylation sites in a given cell type (phosphoproteomics). The authors describe the use of phosphoproteomics technology to identify signaling mechanisms downstream from a G-protein-coupled receptor, the vasopressin V2 subtype receptor, and its role of the regulation and dysregulation of water excretion in the kidney. Data from multiple phosphoproteomic data sets are provided as web-based resources.

Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA‐mediated control of the water channel aquaporin‐2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA‐Cα [Gene symbol: Prkaca]) and PKA catalytic β (PKA‐Cβ [Gene symbol: Prkacb]). To identify the relative roles of these two proteins, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA‐Cα (n=3) or PKA‐Cβ (n=3) was deleted using CRISPR‐Cas9‐based genome editing (generated in PMID 28973931). Cells were grown on permeable supports in the presence of the vasopressin analog dDAVP (0.1nM). Controls were cells carried through the genome editing procedure, but without deletion of PKA. Quantification used TMT mass tags in an Orbitrap Fusion Lumos ETD mass spectrometer. Preliminary measures of total protein amounts showed that PKA‐Cα deletion resulted in a near disappearance of AQP2 protein, while PKA‐Cβ deletion was associated with an increase in AQP2. A total of 4635 phosphopeptides were quantified (Figure 1). The red data points represent phosphopeptides that correspond to previously identified PKA target sites. Stringent statistical criteria (Pmod &lt; 0.01 and |log2(KO/control)| &gt; 0.503) revealed that 67 phosphopeptides were significantly altered with PKA‐Cα deletion, while 21 phosphopeptides were significantly altered with PKA‐Cβ deletion. Only four sites were changed in both. We used Gene Ontology (GO‐CC) Cellular Component terms to identify sets of target phosphoproteins localized in different regions of the cell. The target proteins identified in PKA‐Cα‐null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA‐Cβ‐null cells were largely associated with the actin cytoskeleton and cell junctions. In summary, PKA‐Cα and PKA‐Cβ appear to serve substantially different functions in renal collecting duct cells. The differences in the phosphorylation targets of PKA‐Cα and PKA‐Cβ suggest differences in the cellular localization of the two PKA subunits. These findings in addition to the dramatically different effect of PKA‐Cα and PKA‐Cβ deletion on AQP2 protein abundance support the conclusion that PKA‐Cα and PKA‐Cβ have non‐redundant functions in collecting duct cells.Support or Funding InformationK.S. was supported by the Biomedical Engineering Summer Internship Program at NIH and carried out all of the mass spectrometry experiments. Work was supported by NHLBI Project ZIA‐HL001285.Volcano plots showing effect of PKA‐Cα deletion (left) and PKA‐Cβ deletion (right) on phosphoproteome of mouse mpkCCD cells. Three biological replicates for each.Figure 1

AQP2 as a diagnostic immunohistochemical marker for pheochromocytoma and/or paraganglioma.

Aquaporin2 (AQP2) is water channel protein that is widely distributed among mammalian tissues and plays a major role in water homeostasis. However, little is known about the expression and significance of AQP2 in human adrenal tumors. Thus, we performed an immunohistochemical investigation of AQP2 in normal and neoplastic adrenal tissues. AQP2 protein expression was investigated in 190 adrenal tumor patients using immunohistochemistry. Correlation between protein expression and clinicopathological features was statistically analyzed. We demonstrated immunopositivity for AQP2 in all adrenal medulla-originating tumors, including 96 pheochromocytomas (PCC), 7 metastatic pheochromocytomas (MPCC), and 10 neuroblastic tumors (NT) and 13 extra-adrenal paragangliomas (EAPGL). Whereas, APQ2 was absent from the 52 adrenal cortical adenomas and 2 adrenal cortical carcinomas examined. The 10 metastatic carcinomas examined in adrenal tissue were also negative for AQP2. In 82 (85%) of the 96 samples from patients with PCC, we studied the relationship between clinicopathologic factors and AQP2 expression and our findings suggested that the tumors that exhibited diffuse expression pattern of AQP2 were larger in diameter than those exhibiting focal (P=0.007) or mediate expression pattern (P=0.001). AQP2 protein is significantly expressed in normal adrenal medullary tissues and medullary tumors (including PCC, MPCC and NT) as well as EAPGL. AQP2 expression may indicate the origin of normal adrenal tissues, and its expression in cancer tissue may reflect the maintenance of water metabolism via AQP2 during tumorigenesis. AQP2 may serve as a valuable marker for the differential diagnosis of adrenal tumors.