bluuebunny/crossref_metadata_2025_split huggingface dataset:
Telemetry‐based recording of renal cortical oxygenation in conscious rats with chronic kidney disease (890.4)
Authors: Koeners Maarten , Emans Tonja , Ow Connie , Evans Roger , Malpas Simon 
Published: June 2021

Multiple forms of acute and chronic kidney disease are associated with renal tissue hypoxia. This has led to the proposition that renal hypoxia is not just a consequence of kidney disease, but rather a primary pathogenic event. Using telemetric technology to chronically measure the partial pressure of oxygen (PO2), we recorded cortical PO2 during the early stages of development of chronic kidney disease (CKD) in conscious rats. Male Wistar rats underwent 5/6 subtotal nephrectomy (n = 4) or sham (n = 4) surgery. After an initial increase, cortical PO2 values decreased in the first 2 weeks after induction of CKD and thereafter remained stable for the subsequent 6 weeks with an average reduction of 42 ± 12% compared to baseline. Sham operation had little effect on cortical PO2. The decrease in tissue PO2 in the remnant kidney occurred before proteinuria and uremia suggesting that hypoxia precedes the symptoms of CKD and therefore could be a major driver of the subsequent pathology. This is consistent with the hypothesis that kidney hypoxia is central in the pathogenesis of CKD.Grant Funding Source: EU, FP7, Marie Curie Actions, International Outgoing Fellowship
Dietary NaCl modulates the urinary organic metabolite profile: influence of the organic anion transporter OAT3 (892.34)
Authors: Vallon Volker , Fu Yiling , Kuczkowski Alexander , Anzai Naohiko , Nigam Sanjay , Eraly Satish , Gerasimova Maria , Ramachandra Rao Satish 
Published: June 2021

The organic anion transporter 3 (OAT3) mediates uptake of organic anions across the basolateral membrane of the proximal tubule in exchange for α‐ketoglutarate. Mice lacking OAT3 have reduced blood pressure, but the underlying mechanism is unclear. Renal metabolism is closely linked to transport function, but little is known about the metabolic response of the kidney to changes in NaCl diet. Here we show that a low NaCl diet (0.01%) increases renal OAT3 protein expression by 50‐60% vs. high NaCl diet (4%) and that NaCl intake has distinct effects on the urinary metabolite pattern in wild‐type mice (WT): α‐ketoisocaproate, α‐ketoisovalerate, α‐keto‐3methyl valerate, and α‐ketoglutarate were not detectable in urine under high NaCl diet, whereas a low NaCl diet was associated with mean urinary concentrations of 50‐300 ng/µmol creatinine (each P<10‐6). Low NaCl diet was also associated with 10‐20 fold higher urinary excretion of pyruvate (P<10‐3). In contrast, urinary excretion of benzoate, 3‐OH‐butyrate, malonate, and succinate were lower under low vs. high NaCl diet (each P<10‐3), the latter being also an activator of GPR91, which stimulates renin release. Similar differences were observed in Oat3‐/‐ mice, except that a consistent and significant stimulatory effect of low NaCl diet on urinary α‐ketoglutarate was not detected. We conclude that dietary NaCl intake has a distinct influence on the urinary organic metabolite pattern, including substrates and components of the citric acid cycle and other molecules used as energy source. This pattern is mainly independent of OAT3, even though dietary NaCl regulates renal expression of OAT3. Grant Funding Source: Supported by NIH grants R01HL94728, R01DK56248, P30DK079337, and the Department of Veterans Affairs
A druggable binding site in the emerging diuretic target ROMK (893.15)
Authors: Swale Daniel , Sheehan Jonathan , Meiler Jens , Denton Jerod 
Published: June 2021

An emerging body of evidence suggests the Renal Outer Medullary potassium channel (ROMK, Kir1.1) represents a novel diuretic target for the treatment of hypertension. We recently employed high‐throughput screening and medicinal chemistry to develop the first highly selective small‐molecule ROMK inhibitor, VU591 (Bhave et al., 2011). Its clean ancillary pharmacology raises important questions about how drug selectivity can be achieved in a structurally simple K+ channel. The voltage‐dependence of VU591 block suggests the binding site is located in the membrane‐spanning pore, prompting us to perform scanning mutagenesis analysis of this region of ROMK. Most mutations had no effect on VU591 sensitivity; however, mutation of Val168 and Asp171, which are unique to ROMK among other inward rectifier K+ channels, led to side chain‐specific changes in channel block. In support of our functional data, comparative molecular modeling and in silico docking analysis revealed an energetically favorable interaction between VU591 and the side chains of V168 and N171. This study reveals key structural elements required for selective inhibition of this emerging diuretic target.Grant Funding Source: Supported by NIH 1RO1DK082884 to Jerod S Denton
Modulation of KCa3.1 by ubiquitylation in a polarized epithelium (893.20)
Authors: Lee Shih‐Liang , Devor Daniel , Hamilton Kirk 
Published: June 2021

In this study, we examined the role of ubiquitylation in the retrograde pathway of KCa3.1, for the first time in a polarized epithelium. Previously, we have shown that UBEI‐41, an inhibitor of ubiquitinin‐activating enzyme, reduced ubiquitylation and internalization of KCa3.1 (Balut et al. FASEB J 25: 2938‐48, 2011) in a non‐polarized epithelium. We asked the question ‘Is KCa3.1 ubiquitylated at the basolateral membrane?’ In order to test this, we used our Fischer Thyroid epithelial cell line stably expressing KCa3.1‐BLAP along with Bir‐A‐KDEL (cells grown on filters) that allows the biotinylation of channels prior to leaving the Golgi. Initially, we determined the time course of KCa3.1 by labeling (at 4C) the surface channels with streptavidin and then incubating cells at 37C for 0, 1, 3, 5, 8 and 12 hours. The half‐life of KCa3.1 was 3 hrs (n=6). To confirm that KCa3.1 was ubiquityated in a polarized epithelium, we conducted a similar experiment, as above, in which the cells were incubated with UBEI‐41 (50 µM). UBEI‐41 increased the half‐life of KCa3.1 to 12 hrs (n=4). If, channels were ubiquitylated at the membrane, in the presence of UBE‐41, then we hypothesized that there would be an increase number of channels at the membrane. To test this hypothesis, we used the Ussing chamber to examine an increase in function of KCa3.1 in the presence of UBEI‐41. Indeed, after 1 hr, K current in the presence of UBEI‐41 was increased compared to the K current of cells that were not treated with UBEI‐41. These data suggest that KCa3.1 is ubiquitylated at the membrane prior to being internalized.Grant Funding Source: This work was supported by the Department of Physiology, Univ. of Otago (KLH) and NIH (DCD).
Identification of two extracellular sites in the transmembrane region of SLC26A9 chloride channel ő Cl/HCO3 exchanger (893.27)
Authors: Chen An‐Ping , Chang Min‐Hwang , Romero Michael 
Published: June 2021

The SLC26A9 protein mediates Cl‐ transport as a Cl‐ channel and a Cl‐/HCO3 exchanger in the several epithelial cells, e.g., gut, lung, kidney. Topology analysis from BicA, a member of the SulP/SLC26A family, suggests that Slc26 proteins consists of 12 predicted transmembrane regions. However, the topology model of human SLC26 transporters has not yet been directly investigated. In this study, 12 HA‐inserted SLC26A9 mutants were constructed and expressed in Xenopus oocytes. Chemiluminescence was used to determine which HA‐tags were accessible from the extracellular surface. 157‐160 and 352‐353 sites of human SLC26A9 were identified as extracellular sites. Interestingly, the SLC26A9 HA (159‐160) mutant has ~40% Cl‐ transport activity, but this decreased function could be rescued by coexpression with another low function cSNP (T127N‐SLC26A9). These results revealed that the topology of TM region of SLC26A9 may different from that of BicA and also indicate that human SLC26A9 functions as dimer.Grant Funding Source: Supported by NIH EY 017732 (MFR)
Na+/H+ exchanger NHE9 affects tumor progression of human glioblastomas by altering endosomal pH (893.34)
Authors: Llongueras Jose , Kondapalli Kalyan , Hack Anniesha , Capilla‐González Vivian , Smith Chris , Guerrero‐Cazares Hugo , Quiñones‐Hinojosa Alfredo , Rao Rajini 
Published: June 2021

Na+(K+)/H+ Exchangers (NHEs) are a large family of transport proteins that conduct electroneutral countertransport of cations with protons across lipid bilayers. A newly discovered Na+/H+ Exchanger, NHE9 (SLC9A9), has recently been shown to localize to a perinuclear region corresponding to recycling endosomes, where it is thought to regulate luminal pH and control cargo trafficking and degradation. Given the mounting evidence implicating NHE9 in neurological diseases including autism and ADHD, we explored the functional consequence of altered NHE9 expression levels in brain cancer.Glioblastoma Multiforme (GBM) is the most aggressive form of malignant glioma that accounts for over 50% of all gliomas and is frequently characterized by the amplification and membrane persistence of epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosomal‐mediated degradation, and we postulated that NHE9 might alter this pathway. Here, we have found that levels of NHE9 are amplified in a large subset of GBMs. Using primary GBM cell cultures with naturally high or low levels of NHE9, we have found that altering those levels regulates tumorigencity and migratory capacity in vitro and in nude mouse xenografts. Furthermore, increased NHE9 levels alkalinizes the luminal pH of endosomes, which inhibits EGFR degradation and promotes oncogenic signaling downstream of MAPK and Akt. Finally, we show that inhibiting NHE9 with amiloride‐derived inhibitor EIPA increases EGFR responsiveness to Erlotinib. Our findings demonstrate that NHE9 is a target gene that should be explored as a treatment for a subset of GBMs.Grant Funding Source: Supported by NIH grant R01 DK054214
Conserved polarization of Na,K‐ATPase in vertebrate skin epithelia (895.1)
Authors: Modyanov NIikolai , Korneenko Tatyana , Shakhparonov Mikhail , Pestov Nikolay 
Published: June 2021

Development of epidermis creates stratified epithelium with different sets of ion‐transporting enzymes in its layers. We have characterized expression of Na,K‐ and H,K‐ATPases (X=Na or H) in rat skin including all α and β subunits and six FXYD isoforms. Na,K‐ATPase is predominantly represented by α1 and β3 isoforms. Among H,K‐ATPases, only the α‐subunit of non‐gastric H,K‐ATPase (αng) was detected. Expression of ATP1A1, ATP1B4 and FXYD6 genes are down‐regulated in adult rat skin as compared to the newborn one, whereas the level of αng and FXYD4/CHIF are higher in adult skin. Immunohistochemistry with the use of monoclonal antibodies indicates that αng and α1 are differentially expressed during keratinization: α1 is gradually decreased whereas αng is enriched in the outer live cell layer, stratum granulosum. Na,K‐ATPase α1 is abundant in cells of the innermost layer, stratum basale, and, unexpectedly, is completely undetectable in their basal membranes. Epidermis of a frog species, Xenopus laevis, also show lateroapical location of Na,K‐ATPase, as well as in the chicken epidermis. These findings indicate that Na,K‐ATPase in epidermis is not basolateral as it is in most epithelial cells. On the other hand, Na,K‐ATPase is basolateral in epithelium of the cutaneous secretory glands. These features appear to be conserved in tetrapod vertebrates.Supported by RFBR grant 13‐04‐01413.Grant Funding Source: RFBR grant 13‐04‐01413
High saturated fat diet alters skeletal muscle phospholipid composition and increases SERCA activity (895.2)
Authors: Trojanowski Natalie , Castelli Laura , Miotto Paula , Ward Wendy , LeBlanc Paul 
Published: June 2021

Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) is a transmembrane protein whose activity is, in part, dependent on its lipid environment. Past studies using synthetic membranes have demonstrated that 20‐22 carbon long monounsaturated fatty acids are associated with maximal SERCA activity. However, this association has not been examined in biological membranes. Thus, the objective of this study was to examine this association in a biological system and the response to high saturated fat (HSF) diet‐mediated membrane alterations in skeletal muscle. Female Wistar rats (28 days old) were fed control (CON; AIN93G, 7% soybean oil by weight) or HSF (AIN93G, 20% lard by weight) diet for 16 weeks. Mixed hind limb muscle phospholipids from rats fed a HSF diet had lower percent mole fraction of polyunsaturated fatty acids, unsaturation index (UI), and average chain length (ACL); and higher saturated fatty acids and SERCA activity compared to CON (p<0.05). Correlation analysis demonstrated ACL (R2= 0.83, p < 0.05) and UI (R2=0.53, p = 0.062) were negatively correlated with SERCA activity. These results suggest that SERCA activity may be influenced by phospholipid composition in biological membranes differently than in synthetic membranes. Future studies will examine the lipid annulus closely associated with SERCA.Grant Funding Source: Supported by Natural Sciences and Engineering Research Council (NSERC)
Amino acids involved in lubiprostone activation of human ClC‐2 (896.11)
Authors: Cuppoletti John , Malinowska Danuta , Chakrabarti Jayati , Tewari Kirti , Ueno Ryuji 
Published: June 2021

Objective. The study’s objective was to determine amino acids important for activation of human ClC‐2 (hClC‐2) by lubiprostone. NMR studies of ClC‐0 by Alioth et al (J Mol Biol 369:1163‐9, 2007) identified an α‐helical region that interacts with ClC‐0 CBS domains, important to channel regulation, which contains one of two PKA phosphorylation sites unique to hClC‐2 (RGET691).Methods. Whole cell patch clamp of wild type and mutant hClC‐2 expressed in EBNA293 cells were carried out as described (Cuppoletti et al, Cell Biochem Biophys 66: 53‐63, 2013). Wild type rabbit and mouse ClC‐2 were also studied.Results. Single amino acid mutagenesis of the PKA phosphorylation site RGET691‐>A shifted the EC50 for lubiprostone stimulation of wild type hClC‐2 from 27.6 ± 4.5 nM (4) to 445.8 ± 2.1 nM (3). The triple mutant RGET691‐>AGAA lost all lubiprostone stimulation. Both mutants were still stimulated by forskolin/IBMX. Both rabbit and mouse ClC‐2 were also stimulated by lubiprostone with EC50s of 350 ± 37.9 nM (3) and 64.3 ± 3.7 nM (4), respectively.Conclusions. RGET691 plays an essential role in lubiprostone stimulation of human ClC‐2. In light of NMR evidence that this region may interact with CBS domains and contains the RGET691 phosphorylation site in hClC‐2, binding of lubiprostone to this region in human, rabbit and mouse ClC‐2 may alter the interaction of this region with CBS domains, leading to direct modulation of ClC‐2.Grant Funding Source: Supported by Sucampo AG
Cdc42 plays a requisite role in dopamine transporter endocytic trafficking (896.6)
Authors: Wu Sijia , Melikian Haley 
Published: June 2021

Dopaminergic neurotransmission is essential for voluntary movement, reward and cognition, and dopamine (DA) signaling is implicated in Parkinson’s disease, addiction and schizophrenia. The presynaptic Na+/Cl‐‐dependent DA transporter (DAT) recaptures released DA, thereby temporally and spatially sculpting DAergic neurotransmission. DAT is potently inhibited by addictive (cocaine, amphetamine [AMPH]) and therapeutic (methylphenidate) psychostimulants. DAT loss‐of‐function mutations lead to infantile Parkinsonism and several DAT coding variants are associated with ADHD. Thus, intrinsic cellular mechanisms modulating DAT availability are likewise predicted to significantly impact DAergic neurotransmission. PKC activation and AMPH exposure acutely decrease DAT surface expression via robust endocytic trafficking. However, the mechanisms governing DAT membrane trafficking are not clearly defined. We recently demonstrated that 1) Rin GTPase activity is required for PKC‐stimulated DAT internalization and 2) DAT endocytic recycling relies on a dynamin/actin‐dependent mechanism. Both Rin and the actin cytoskeleton are intimately coupled to cdc42, suggesting that cdc42 activity may impart multimodal control over DAT endocytic trafficking. Here, we used small molecule cdc42 inhibitors and cdc42 mutants to test this possibility in dopaminergic cells lines and acute mouse striatal slices. Cdc42 inhibitors casin and pirl1 significantly decreased DA uptake in SK‐N‐MC cells. DAT functional losses were accompanied by decreased DAT surface expression in SK‐N‐MC cells and in mouse striatal slices. Studies using shRNA and cdc42 GTPase mutants will further define the role of cdc42 in DAT membrane trafficking.Grant Funding Source: DA035244
Investigation of MCT1 transporter protein in the human gastrointestinal tract (897.1)
Authors: Al‐mousawi Hashmiyah , Courtney Danielle , McGrane Alison , Winter Desmond , Baird Alan , Stewart Gavin 
Published: June 2021

Bacterially‐derived short chain fatty acids, such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the uptake of butyrate into colonic epithelial cells, via MCT1 transporters, which regulates cell differentiation (Cuff et al., 2005). Although previously studied, the exact localisation of MCT1 protein along the human gastrointestinal tract remains controversial (Gill et al., 2006; Iwanaga et al., 2006). The aim of this study was therefore to re‐investigate MCT1 protein abundance in various human gastrointestinal tissues. Initial RT‐PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, Western analysis utilising the MCT12A antibody (Source Bioscience, UK) detected higher abundance of a 45 kDa MCT1 protein in colon compared to ileum (P<0.05, N = 4, unpaired t‐test). Importantly, MCT1 abundance was found to be significantly higher in ascending and descending colon compared to sigmoid colon (P<0.01, N = 11, ANOVA). In contrast, ileum samples obtained from ileostomy reversal surgeries showed a high MCT1 abundance, with no significant difference between proximal and distal ileum (NS, N = 7, paired t‐test). These data appear to confirm that MCT1 protein abundance is correlated to levels of bacterially‐derived short‐chain fatty acids along the gastrointestinal tract. In addition, they highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states.Grant Funding Source: Supported by Science Foundation Ireland and the Kuwaiti government
MCT‐1 and SMCT‐1 regulation by miR‐29a, b and c in intestinal epithelial cells (908.8)
Authors: Anbazhagan Arivarasu , Borthakur Alip , Alrefai Waddah , Dudeja Pradeep 
Published: June 2021

Monocarboxylate transporter‐1 (MCT1) and sodium‐coupled monocarboxylate transporter‐1(SMCT‐1) are the major proteins involved in the absorption of short chain fatty acids (SCFA) in the colon. SCFAs exert myriad of beneficial effects including the amelioration of inflammation. Therefore, it is important to understand the mechanisms of regulation of MCT1 and SMCT‐1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In this regard, miR‐29 a, b have been recently shown to alter MCT1 expression in pancreatic ‐cells. Whether these miRs modulate colonic MCT1 and SMCT1expression is not known. The 3`‐untranslated regions (UTR) of MCT1 and SMCT‐1 genes were cloned into pmirGLO vector upstream of luciferase and transienstly transfected with miR29a,b and c mimics into colonic Caco‐2 and T‐84 cells. Luciferase activity and MCT1 and SMCT‐1 expression were determined. The presence of UTR of these genes significantly decreased luciferase activity (~40%) as compared to pmiRGLO empty vector. Co‐transfection with miR‐29 a, b or c resulted in further decrease in 3`‐UTR activity of MCT1 and SMCT1 luciferase constructs. MCT1 and SMCT‐1 mRNA expression was not altered in response to mimic transfections, whereas the protein expression was significantly decreased (~30%). Further, the expression of miR 29 a and c was significantly lower in mouse colon as compared to small intestine, consistent with higher levels of MCT1 and SMCT1 protein in the colon. Our studies demonstrated that miR 29 a, b and c down‐regulate MCT1 and SMCT‐1 expression in colonic epithelial cells and may partly explain the differential expression of these transpoters along the length of the GI tract.(Supported by NIDDK and Dept of Veteran Affairs)Grant Funding Source: NIDDK and Dept of Veteran Affairs
Specialize or disappear: anisomerism, empirical evidence of the differentiation or loss of polyisomere structures during gnathostome evolution based on a comparative study of head and appendicular muscles (918.27)
Authors: Ziermann Janine , Diogo Rui 
Published: June 2021

Some authors have defended the notion of anisomerism, i.e. that there is an evolutionary tendency for the original polyisomeres of an organism (e.g., branchial arches, digits) to become differentiated so that phylogenetically derived organisms do not consist of a linear series of similar parts. This notion has been often related with the notion of functional uncouplings, i.e. that the differentiation of a polyisomere into an anatomically different structure allows this structure to not only have a new function but also to be liberated from its original functional complex and therefore to become functionally associated with other structures of the body. We provide here the first detailed empirical analysis of this issue based on data obtained from a comparison of the head and appendicular muscles of all major groups of gnathostomes. Our results support the idea that there is effectively a trend for the muscles associated for instance with the branchial arches or with the digits to specialize or, instead, to become lost in evolution. One example concerns our own species, Homo sapiens, which almost completely lacks any of the original similar polyisomere head muscles found in taxa such as sharks (e.g., branchial adductors), and that lacks many of the original similar polyisomere autopodial (hand/foot) muscles found in tetrapods such as salamanders or lizards (e.g., the flexores breves superficiales).Grant Funding Source: Faculty Start‐Up Package, Howard Univ. Coll. Medicine