Identification of hub anoikis-associated genes and risk signature in cutaneous melanoma.

OBJECTIVE: As one of the most lethal and aggressive cutaneous malignancies, cutaneous melanoma (CM) greatly threatens human health and has long challenged clinicians because of its poor therapeutic response. Anoikis is a newly discovered form of apoptosis that was originally identified in the extracellular matrix (ECM). Recent studies have reported that anoikis is central to cancer metastasis. The aim of this study is to explore the role of anoikis-associated genes in CM. MATERIALS AND METHODS: We identified hub anoikis-associated genes in CM and constructed a risk signature for patients with CM. Gene expression from The Cancer Genome Atlas (TCGA) database was used to screen hub anoikis-associated genes connected with CM, and the Gene Expression Omnibus (GEO) dataset was applied to externally validate the identified genes. Weighted gene co-expression network analysis (WGCNA), differential expression, univariate Cox regression, and least absolute shrinkage and selection operator (LASSO) analyses were used to identify hub genes. Immune cell infiltration in CM was also evaluated to explore the association between hub genes and immune heterogeneity. Finally, an anoikis-associated prognostic model was constructed. RESULTS: Following complex analysis, FASLG, SOD2, BST2, PIK3R2, IKZF3, CDK2, and RAC3 were identified as hub anoikis-associated genes. Indeed, Kaplan-Meier and receiver operating characteristic analyses suggested that the expression patterns of hub genes can be used as prognostic factors for CM survival. The expression and survival trends of hub genes were verified in the validation cohort. Immune cell infiltration analysis showed that the number of immune cells varied among patients with CM and identified seven genes. Furthermore, functional analyses indicated that the constructed risk signature was significantly associated with patient survival, age, and tumor growth and could also serve as an independent prognostic factor for patients with CM. CONCLUSIONS: We suggest that the hub genes FASLG, SOD2, BST2, PIK3R2, IKZF3, CDK2, and RAC3 are involved in the anoikis-associated signature. The pattern of hub anoikis-associated genes may have a prognostic potential for CM progression and overall patient survival.

Kidney tubular transcription co-activator, Yes-associated protein 1 (YAP), controls the expression of collecting duct aquaporins and water homeostasis.

Final urine volume and concentration are defined by water reabsorption through the water channel proteins aquaporin (AQP)-2, -3 and -4 in the collecting duct. However, the transcriptional regulation of these AQPs is not well understood. The Hippo/Yes-associated protein 1 (YAP) pathway plays an important role in organ size control and tissue homeostasis. When the Hippo pathway including the Mst1/Mst2 kinases is inhibited, YAP is activated and functions as a transcription co-activator. Our previous work revealed a pathological role of tubular YAP activation in chronic kidney disease, but the physiological role of YAP in the kidney remains to be established. Here, we found that tubule-specific Yap knockout mice showed increased urine output and decreased urinary osmolality. Decreases in Aqp2, -3 and -4 mRNA and protein abundance in the kidney were evident in Yap knockout mice. Analysis of Mst1/Mst2 double knockout and Mst1/Mst2/Yap triple knockout mice showed that expression of Aqp2 and Aqp4 but not Aqp3 was dependent on YAP. Furthermore, YAP was recruited to the promoters of the Aqp2 and Aqp4 genes and stimulated their transcription. Interestingly, YAP was found to interact with transcription factors GATA2, GATA3 and NFATc1. These three factors promoted Aqp2 transcription in a YAP dependent manner in collecting duct cells. These three factors also promoted Aqp4 transcription whereas only GATA2 and GATA3 enhanced Aqp3 transcription. Thus, our results suggest that YAP promotes Aqp2 and Aqp4 transcription, interacts with GATA2, GATA3 and NFATc1 to control Aqp2 expression, while Aqp-2, -3 and -4 exploit overlapping mechanisms for their baseline transcriptional regulation.

Does race affect the long-term survival benefit of systemic therapy in pancreatic adenocarcinoma?

BACKGROUND: Systemic therapy is a key management component of pancreatic ductal adenocarcinoma(PDAC). Racial disparities exist in PDAC, often linked to socioeconomic variables. We investigated the impact of race in PDAC patients who had undergone systemic therapy and surgical resection. METHODS: A retrospective analysis was performed for all patients who underwent surgical resection for PDAC from 2010 to 2018. RESULTS: 234 patients (78.2% White; 21.8% Black) were included. Black patients presented at a younger age with larger tumors. White patients benefited from systemic therapy with longer overall survival (35vs20 months, p = 0.002). This survival advantage was not present in Black patients (21vs15 months, p = 0.15). Black patients receiving systemic therapy had similar survival as White patients who did not (p = 0.81). CONCLUSION: Black PDAC patients present at younger ages and with larger initial tumors. In our population, White patients had a longer overall survival after both surgical and systemic therapy. These findings may indicate differences in tumor biology. Further prospective studies are needed.

HPLC-UV assay of tramadol and O-desmethyltramadol in human plasma containing other drugs potentially co-administered to participants in a paediatric population pharmacokinetic study.

Multimodal analgesia is employed in paediatric pain management to maximise analgesia and minimise side effects. Tramadol is dosed at 1-1.5 mg/kg to treat severe pain in children but the assay for tramadol in plasma samples for pharmacokinetic and toxicology studies does not often consider concurrently administered medications. In this study we developed and validated an HPLC-UV method to quantify tramadol and its main metabolite (O-desmethyltramadol) in human plasma in the presence of seven potentially interfering drugs. Sample preparation method was developed by combining liquid-liquid extraction and protein precipitation. Chromatographic separation was achieved on a BDS-Hypersil-C18 column (5 µm, 250 × 4.6 mm) using a double gradient method. The limit of quantification was 6.7 ng/ml for both tramadol and ODT. The precision and accuracy were in compliance with ICH guidelines. This method was successfully employed to analyse the blood samples of 137 paediatric participants in a tramadol pharmacokinetic trial.

Inhibiting Focal Adhesion Kinase Ameliorates Cyst Development in Polycystin-1-Deficient Polycystic Kidney Disease in Animal Model.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by numerous cysts originating from renal tubules and is associated with significant tubular epithelial cell proliferation. Focal adhesion kinase (FAK) promotes tumor growth by regulating multiple proliferative pathways. METHODS: We established the forskolin (FSK)-induced three-dimensional (3D) Madin-Darby Canine Kidney cystogenesis model and 8-bromoadenosine-3`,5`-cyclic monophosphate-stimulated cyst formation in ex vivo embryonic kidney culture. Cultured human renal cyst-lining cells (OX-161) and normal tubular epithelial cells were treated with FAK inhibitors or transfected with green fluorescent protein-tagged FAK mutant plasmids for proliferation study. Furthermore, we examined the role of FAK in two transgenic ADPKD animal models, the kidney-specific Pkd1 knockout and the collecting duct-specific Pkd1 knockout mouse models. RESULTS: FAK activity was significantly elevated in OX-161 cells and in two ADPKD mouse models. Inhibiting FAK activity reduced cell proliferation in OX-161 cells and prevented cyst growth in ex vivo and 3D cyst models. In tissue-specific Pkd1 knockout mouse models, FAK inhibitors retarded cyst development and mitigated renal function decline. Mechanically, FSK stimulated FAK activation in tubular epithelial cells, which was blocked by a protein kinase A (PKA) inhibitor. Inhibition of FAK activation by inhibitors or transfected cells with mutant FAK constructs interrupted FSK-mediated Src activation and upregulation of ERK and mTOR pathways. CONCLUSIONS: Our study demonstrates the critical involvement of FAK in renal cyst development, suggests that FAK is a potential therapeutic target in treating patients with ADPKD, and highlights the role of FAK in cAMP-PKA-regulated proliferation.

Efficient passivation of n-type and p-type silicon surface defects by hydrogen sulfide gas reaction.

An efficient surface defect passivation is observed by reacting clean Si in a dilute hydrogen sulfide-argon gas mixture (<5% H2S in Ar) for both n-type and p-type Si wafers with planar and textured surfaces. Surface recombination velocities of 1.5 and 8 cm s-1are achieved on n-type and p-type Si wafers, respectively, at an optimum reaction temperature of 550 °C that are comparable to the best surface passivation quality used in high efficiency Si solar cells. Surface chemical analysis using x-ray photoelectron spectroscopy shows that sulfur is primarily bonded in a sulfide environment, and synchrotron-based soft x-ray emission spectroscopy of the adsorbed sulfur atoms suggests the formation of S-Si bonds. The sulfur surface passivation layer is unstable in air, attributed to surface oxide formation and a simultaneous decrease of sulfide bonds. However, the passivation can be stabilized by a low-temperature (300 °C) deposited amorphous silicon nitride (a-Si:NX:H) capping layer.

Pdcd10-Stk24/25 complex controls kidney water reabsorption by regulating Aqp2 membrane targeting.

PDCD10, also known as CCM3, is a gene found to be associated with the human disease cerebral cavernous malformations (CCMs). PDCD10 forms a complex with GCKIII kinases including STK24, STK25, and MST4. Studies in C. elegans and Drosophila have shown a pivotal role of the PDCD10-GCKIII complex in maintaining epithelial integrity. Here, we found that mice deficient of Pdcd10 or Stk24/25 in the kidney tubules developed polyuria and displayed increased water consumption. Although the expression levels of aquaporin genes were not decreased, the levels of total and phosphorylated aquaporin 2 (Aqp2) protein in the apical membrane of tubular epithelial cells were decreased in Pdcd10- and Stk24/25-deficient mice. This loss of Aqp2 was associated with increased expression and membrane targeting of Ezrin and phosphorylated Ezrin, Radixin, Moesin (p-ERM) proteins and impaired intracellular vesicle trafficking. Treatment with Erlotinib, a tyrosine kinase inhibitor promoting exocytosis and inhibiting endocytosis, normalized the expression level and membrane abundance of Aqp2 protein, and partially rescued the water reabsorption defect observed in the Pdcd10-deficient mice. Our current study identified the PDCD10-STK-ERM signaling pathway as a potentially novel pathway required for water balance control by regulating vesicle trafficking and protein abundance of AQP2 in the kidneys.

An evaluation of social media utilization by general surgery programs in the COVID-19 era.

BACKGROUND: Our study investigates how general surgery residency programs utilized social media to adapt to the challenges of COVID-19. METHODS: 319 participating general surgery residency programs provided by the Electronic Residency Application Service were analyzed in this study. Associated Twitter, Instagram, and Facebook accounts were assessed to find virtual open houses and externships. RESULTS: Of the 319 program, 188 (59%) were found to have a social media presence. A total of 348 social media accounts were found, as some of the programs had separate residency and department accounts. Of all the social media accounts, 112 (32%) of the accounts were created after March 1, 2020. Virtual open houses opportunities were found to be advertised across all platforms. CONCLUSION: Many general surgery programs responded to the physical limitations of COVID-19 pandemic by increasingly utilizing social media during the COVID-19 pandemic. Virtual opportunities should be considered as a novel approach for future outreach and recruitment.

Evaluating the Highest- and Lowest-cited Research Articles in the Cardiothoracic Surgery Literature.

BACKGROUND: Understanding the differences between articles that amass a high number of citations and those that receive very few allows investigators to write journal articles that maximize the impact of their research. There are minimal data regarding these two cohorts in the cardiothoracic surgery literature. METHODS: We identified all primary research articles from 1998 to 2008 from The Journal of Thoracic and Cardiovascular Surgery, The Journal of Cardiac Surgery, The Annals of Thoracic Surgery, and The European Journal of Cardio-Thoracic Surgery (n = 4276). Eighty-seven of these articles accrued 0 or only 1 citation within 10 y of publication. We compared this "low citation" cohort to the "high citation" cohort made up of the 87 highest-cited articles from the same journals over the same time period. RESULTS: When compared with the low-citation articles, high-citation articles were significantly more likely to be clinical in nature (P < 0.0001), have observational study design (P < 0.0001), involve multidisciplinary authorship (P < 0.0001), and have more funding reported (P = 0.0039). With regard to technical aspects of the article, the high-citation articles were likely to have longer titles (P = 0.0086), punctuation in the title (P = 0.0027), longer abstracts (P = 0.0007), more words in the manuscript (P < 0.0001), more authors (P < 0.0001), more declared conflict of interests (P = 0.0167), more references (P < 0.0001), more tables (P < 0.0001), more figures (P = 0.0024), and more pages (P < 0.0001). There was no significant difference in the year of publication among both cohorts. CONCLUSIONS: This review suggests that there are several important distinguishing characteristics that should be considered by investigators when designing and implementing cardiothoracic research studies to maximize the impact of their published research.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

BACKGROUND: Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. METHODS: Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. RESULTS: Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. CONCLUSIONS: A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Pathological cardiac remodeling occurs early in CKD mice from unilateral urinary obstruction, and is attenuated by Enalapril.

Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD) and end-stage renal disease. Despite increasing recognition of a close interplay between kidney dysfunction and cardiovascular disease, termed cardiorenal syndrome (CRS), the underlying mechanisms of CRS remain poorly understood. Here we report the development of pathological cardiac hypertrophy and fibrosis in early stage non-uremic CKD. Moderate kidney failure was induced three weeks after unilateral urinary obstruction (UUO) in mice. We observed pathological cardiac hypertrophy and increased fibrosis in UUO-induced CKD (UUO/CKD) animals. Further analysis indicated that this cardiac fibrosis was associated with increased expression of transforming growth factor ß (TGF-ß) along with significant upregulation of Smad 2/3 signaling in the heart. Moreover early treatment of UUO/CKD animals with an angiotensin-converting-enzyme inhibitor (ACE I), Enalapril, significantly attenuated cardiac fibrosis. Enalapril antagonized activation of the TGF-ß signaling pathway in the UUO/CKD heart. In summary our study demonstrates the presence of pathological cardiac hypertrophy and fibrosis in mice early in UUO-induced CKD, in association with early activation of the TGF-ß/Smad signaling pathway. We also demonstrate the beneficial effect of ACE I in alleviating this early fibrogenic process in the heart in UUO/CKD animals.

A measurement of the attenuation of radiation from F-18 by a PET/MR scanner.

The attenuation of 511 keV photons by the structure of a PET/MR scanner was measured prior to energizing the magnet. The exposure rate from a source of fluorine-18 was measured in air and, with the source placed at the isocenter of the instrument, at various points outside of the scanner. In an arc from 45 to 135 degrees relative to the long axis of the scanner and at a distance of 1.5 m from the isocenter, the attenuation by the scanner is at least 5.6 half-value layers from the MR component alone and at least 6.6 half-value layers with the PET insert installed. This information could inform better design of the radiation shielding for PET/MR scanners.

HU deviation in lung and bone tissues: Characterization and a corrective strategy.

INTRODUCTION: In the era of precision medicine, quantitative applications of x-ray Computed Tomography (CT) are on the rise. These require accurate measurement of the CT number, also known as the Hounsfield Unit. In this study, we evaluated the effect of patient attenuation-induced beam hardening of the x-ray spectrum on the accuracy of the HU values and a strategy to correct for the resulting deviations in the measured HU values. MATERIALS AND METHODS: A CIRS electron density phantom was scanned on a Siemens Biograph mCT Flow CT scanner and a GE Discovery 710 CT scanner using standard techniques that are employed in the clinic to assess the HU deviation caused by beam hardening in different tissue types. In addition, an anthropomorphic ATOM adult male upper torso phantom was scanned on the GE Discovery 710 scanner. Various amounts of Superflab bolus material were wrapped around the phantoms to simulate different patient sizes. The mean HU values that were measured in the phantoms were evaluated as a function of the water-equivalent area (Aw ), a parameter that is described in the report of AAPM Task Group 220. A strategy by which to correct the HU values was developed and tested. The variation in the HU values in the anthropomorphic ATOM phantom under different simulated body sizes, both before and after correction, were compared, with a focus on the lung and bone tissues. RESULTS: Significant HU deviations that depended on the simulated patient size were observed. A positive correlation between HU and Aw was observed for tissue types that have an HU of less than zero, while a negative correlation was observed for tissue types with HU values that are greater than zero. The magnitude of the difference increases as the underlying attenuation property deviates further away from that of water. In the electron density phantom study, the maximum observed HU differences between the measured and reference values in the cortical bone and lung materials were 426 and 94 HU, respectively. In the anthropomorphic phantom study, the HU difference was as much as -136.7 ± 8.2 HU (or -7.6% ± 0.5% of the attenuation coefficient, AC) in the spine region, and up to 37.6 ± 1.6 HU (or 17.3% ± 0.8% of AC) in the lung region between scenarios that simulated normal and obese patients. Our HU correction method reduced the HU deviations to 8.5 ± 9.1 HU (or 0.5% ± 0.5%) for bone and to -6.4 ± 1.7 HU (or -3.0% ± 0.8%) for lung. The HU differences in the soft tissue materials before and after the correction were insignificant. Visual improvement of the tissue contrast was also achieved in the data of the simulated obese patient. CONCLUSIONS: The effect of a patient's size on the HU values of lung and bone tissues can be significant. The accuracy of those HU values was substantially improved by the correction method that was developed for and employed in this study.

Manganese promotes intracellular accumulation of AQP2 via modulating F-actin polymerization and reduces urinary concentration in mice.

Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine-256, -264, and -269 residues and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization and uncovered its potent impact on water diuresis in vivo.

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Re-characterization of the Glomerulopathy in CD2AP Deficient Mice by High-Resolution Helium Ion Scanning Microscopy.

Helium ion scanning microscopy (HIM) is a novel technology that directly visualizes the cell surface ultrastructure without surface coating. Despite its very high resolution, it has not been applied extensively to study biological or pathology samples. Here we report the application of this powerful technology to examine the three-dimensional ultrastructural characteristics of proteinuric glomerulopathy in mice with CD2-associated protein (CD2AP) deficiency. HIM revealed the serial alteration of glomerular features including effacement and disorganization of the slit diaphragm, followed by foot process disappearance, flattening and fusion of major processes, and eventual transformation into a podocyte sheet as the disease progressed. The number and size of the filtration slit pores decreased. Strikingly, numerous "bleb" shaped microprojections were observed extending from podocyte processes and cell body, indicating significant membrane dynamics accompanying CD2AP deficiency. Visualizing the glomerular endothelium and podocyte-endothelium interface revealed the presence of endothelial damage, and disrupted podocyte and endothelial integrity in 6 week-old Cd2ap-KO mice. We used the HIM technology to investigate at nanometer scale resolution the ultrastructural alterations of the glomerular filtration apparatus in mice lacking the critical slit diaphragm-associated protein CD2AP, highlighting the great potential of HIM to provide new insights into the biology and (patho)physiology of glomerular diseases.

Ezrin directly interacts with AQP2 and promotes its endocytosis.

The water channel aquaporin-2 (AQP2) is a major regulator of water homeostasis in response to vasopressin (VP). Dynamic trafficking of AQP2 relies on its close interaction with trafficking machinery proteins and the actin cytoskeleton. Here, we report the identification of ezrin, an actin-binding protein from the ezrin/radixin/moesin (ERM) family as an AQP2-interacting protein. Ezrin was first detected in a co-immunoprecipitation (co-IP) complex using an anti-AQP2 antibody in a proteomic analysis. Immunofluorescence staining revealed the co-expression of ezrin and AQP2 in collecting duct principal cells, and VP treatment caused redistribution of both proteins to the apical membrane. The ezrin-AQP2 interaction was confirmed by co-IP experiments with an anti-ezrin antibody, and by pulldown assays using purified full-length and FERM domain-containing recombinant ezrin. By using purified recombinant proteins, we showed that ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain. Knocking down ezrin expression with shRNA resulted in increased membrane accumulation of AQP2 and reduced AQP2 endocytosis. Therefore, through direct interaction with AQP2, ezrin facilitates AQP2 endocytosis, thus linking the dynamic actin cytoskeleton network with AQP2 trafficking.