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.

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.

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.

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.

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.

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.

Deletion of ß1-integrin in collecting duct principal cells leads to tubular injury and renal medullary fibrosis.

The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of ß1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of ß1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of ß1-integrin in CD cells, specifically in the PCs. We conditionally deleted ß1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, ß-1f/fAQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-ß (TGF-ß)-induced protein, fibronectin, and TGF-ß receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-ß signaling pathway. Therefore, our data reveal that normal expression of ß1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of ß1-integrin in the development and/or maintenance of the CD structure and function.

EGF Receptor Inhibition by Erlotinib Increases Aquaporin 2-Mediated Renal Water Reabsorption.

Nephrogenic diabetes insipidus (NDI) is caused by impairment of vasopressin (VP) receptor type 2 signaling. Because potential therapies for NDI that target the canonical VP/cAMP/protein kinase A pathway have so far proven ineffective, alternative strategies for modulating aquaporin 2 (AQP2) trafficking have been sought. Successful identification of compounds by our high-throughput chemical screening assay prompted us to determine whether EGF receptor (EGFR) inhibitors stimulate AQP2 trafficking and reduce urine output. Erlotinib, a selective EGFR inhibitor, enhanced AQP2 apical membrane expression in collecting duct principal cells and reduced urine volume by 45% after 5 days of treatment in mice with lithium-induced NDI. Similar to VP, erlotinib increased exocytosis and decreased endocytosis in LLC-PK1 cells, resulting in a significant increase in AQP2 membrane accumulation. Erlotinib increased phosphorylation of AQP2 at Ser-256 and Ser-269 and decreased phosphorylation at Ser-261 in a dose-dependent manner. However, unlike VP, the effect of erlotinib was independent of cAMP, cGMP, and protein kinase A. Conversely, EGF reduced VP-induced AQP2 Ser-256 phosphorylation, suggesting crosstalk between VP and EGF in AQP2 trafficking and a role of EGF in water homeostasis. These results reveal a novel pathway that contributes to the regulation of AQP2-mediated water reabsorption and suggest new potential therapeutic strategies for NDI treatment.

High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentration.

A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, ß-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and ß-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2-4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.

Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

Vasopressin induces apical expression of caveolin in rat kidney collecting duct principal cells.

Caveolin (Cav)1 is expressed in the basolateral membrane domain of renal collecting duct (CD) principal cells (PCs), where it is associated with caveolae. To reveal any potential involvement of Cav1 in vasopressin signaling, we used specific monoclonal and polyclonal antibodies to examine its localization in CD PCs of Brattleboro (BB) rats treated with vasopressin (DDAVP). Compared with controls, immunofluorescence revealed a time-dependent increase in Cav1 expression in the apical membrane domain of PCs, where it overlapped with aquaporin-2 (AQP2). After 24 h of DDAVP treatment, Cav1 was visible as an increased number of small apical spots. The staining gradually became more extensive, and, after 2 wk of DDAVP, it occupied the majority of the apical membrane domain of many PCs. Cav1 also assumed an apical localization in PCs of DDAVP-treated Sprague-Dawley and Long-Evans rats. Similarly, Cav2 appeared at the apical pole of PCs after DDAVP treatment of BB, Sprague-Dawley, and Long-Evans rats. Immunogold electron microscopy confirmed bipolar Cav1 membrane expression in DDAVP-treated BB rats, whereas caveolae were only detected on the basolateral membrane. Immunoblot analysis of BB rat whole kidney homogenates revealed no significant increase in Cav1 levels in DDAVP-treated rats, suggesting that DDAVP induces Cav1 relocalization or modifies its targeting. We conclude that Cav1 and Cav2 trafficking and membrane localization are dramatically altered by the action of DDAVP. Importantly, the absence of apical caveolae indicates that while Cavs may have an as yet undetermined role in vasopressin-regulated signaling processes, this is probably unrelated to AQP2 internalization by caveolae.

A zebrafish model of conditional targeted podocyte ablation and regeneration.

Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture, and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion, and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

Low levels of a natural IgM antibody are associated with vein graft stenosis and failure.

BACKGROUND: All humans have natural, protective antibodies directed against phosphorylcholine (PC) epitopes, a common inflammatory danger signal appearing at sites of cell injury, oxidative stress, and on bacterial capsules. In large human cohorts, low levels of anti-PC IgM were associated with a significantly increased risk of stroke or myocardial infarction. However, it is not known if these antibodies protect against the premature closure of arterial reconstructions. METHODS: A prospective, observational study of patients undergoing elective, infrainguinal, autogenous vein bypasses for atherosclerotic occlusive disease of the legs was conducted. Clinical data were recorded prospectively, and preoperative levels of anti-PC IgM measured with the CVDefine kit from Athera Biotechnologies (Solna, Sweden). The principal clinical end point was the loss of primary patency (loss of graft flow, or any intervention for stenosis). Patients were followed regularly by duplex ultrasound at 1, 3, 6, 12, 18 months, and yearly thereafter. RESULTS: Fifty-six patients were studied, for an average of 1.3 years. Indications for surgery were claudication (33.9%), ischemic rest pain (17.9%), and ischemia with ulceration or gangrene (48.2%). Seventeen (30.4%) patients experienced loss of primary patency (10 graft occlusions, seven surgical or endovascular revisions of graft stenoses). Kaplan-Meier survival analysis showed that the quartile of patients with the lowest anti-PC IgM levels had significantly worse primary graft patency (log-rank test, P = .0085). Uni- and multivariate Cox proportional hazards analysis revealed that the preoperative anti-PC IgM level was an important predictor of graft failure. Patients with IgM values in the lowest quartile had a 3.6-fold increased risk of graft failure (95% confidence interval: 1.1-12.1), even after accounting for other significant clinical or technical factors such as indication for surgery, site of distal anastomosis, or vein graft diameter. CONCLUSIONS: A naturally occurring IgM antibody directed against the proinflammatory epitope PC may be protective against vein graft stenosis and failure, through anti-inflammatory mechanisms. Measurement of this antibody may be a useful prognostic indicator, although larger studies of more diverse populations will be needed to confirm these results. The biological actions of anti-PC IgM suggest it may be useful in developing immunotherapies to improve bypass longevity.

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

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.

New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells.

Maintaining tight control over body fluid and acid-base homeostasis is essential for human health and is a major function of the kidney. The collecting duct is a mosaic of two cell populations that are highly specialized to perform these two distinct processes. The antidiuretic hormone vasopressin (VP) and its receptor, the V2R, play a central role in regulating the urinary concentrating mechanism by stimulating accumulation of the aquaporin 2 (AQP2) water channel in the apical membrane of collecting duct principal cells. This increases epithelial water permeability and allows osmotic water reabsorption to occur. An understanding of the basic cell biology/physiology of AQP2 regulation and trafficking has informed the development of new potential treatments for diseases such as nephrogenic diabetes insipidus, in which the VP/V2R/AQP2 signaling axis is defective. Tubule acidification due to the activation of intercalated cells is also critical to organ function, and defects lead to several pathological conditions in humans. Therefore, it is important to understand how these "professional" proton-secreting cells respond to environmental and cellular cues. Using epididymal proton-secreting cells as a model system, we identified the soluble adenylate cyclase (sAC) as a sensor that detects luminal bicarbonate and activates the vacuolar proton-pumping ATPase (V-ATPase) via cAMP to regulate tubular pH. Renal intercalated cells also express sAC and respond to cAMP by increasing proton secretion, supporting the hypothesis that sAC could function as a luminal sensor in renal tubules to regulate acid-base balance. This review summarizes recent advances in our understanding of these fundamental processes.

Calcitonin has a vasopressin-like effect on aquaporin-2 trafficking and urinary concentration.

The most common cause of hereditary nephrogenic diabetes insipidus is a nonfunctional vasopressin (VP) receptor type 2 (V2R). Calcitonin, another ligand of G-protein-coupled receptors, has a VP-like effect on electrolytes and water reabsorption, suggesting that it may affect AQP2 trafficking. Here, calcitonin increased intracellular cAMP and stimulated the membrane accumulation of AQP2 in LLC-PK1 cells. Pharmacologic inhibition of protein kinase A (PKA) and deficiency of a critical PKA phosphorylation site on AQP2 both prevented calcitonin-induced membrane accumulation of AQP2. Fluorescence assays showed that calcitonin led to a 70% increase in exocytosis and a 20% decrease in endocytosis of AQP2. Immunostaining of rat kidney slices demonstrated that calcitonin induced a significant redistribution of AQP2 to the apical membrane of principal cells in cortical collecting ducts and connecting segments but not in the inner stripe or inner medulla. Calcitonin-treated VP-deficient Brattleboro rats had a reduced urine flow and two-fold higher urine osmolality during the first 12 hours of treatment compared with control groups. Although this VP-like effect of calcitonin diminished over the following 72 hours, the tachyphylaxis was reversible. Taken together, these data show that calcitonin induces cAMP-dependent AQP2 trafficking in cortical collecting and connecting tubules in parallel with an increase in urine concentration. This suggests that calcitonin has a potential therapeutic use in nephrogenic diabetes insipidus.

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.