bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoietin and Vascular Endothelial Growth Factor induce activation of Matrix Metalloproteinase‐2 via a common pathway
Authors: Ispanovic Eric , Haas Tara L
Published: June 2021

Angiogenesis is the formation of new capillaries from pre‐existing ones. Proteolysis of the basement membrane and interstitial matrix is mediated by the matrix metalloproteinases (MMPs) and is critical to the angiogenic process. Recent research has found that a number of cell types outside of the bone marrow, including endothelial cells, express the erythropoietin (EPO) receptor and respond to EPO. The angiogenic potential of EPO has been shown to be on par with that of vascular endothelial growth factor (VEGF). Little is known about how EPO induces angiogenesis. EPO treatment (2.5 nM, 2 hrs) induced cortical actin polymerization and increased the amount of MT1‐MMP on the cell surface similarly to the response seen with VEGF treatment (25 ng/mL, 2 hrs). The increase in cell surface localization correlated with an increase in MMP‐2 activation in response to both EPO and VEGF treatment. Both EPO and VEGF activated the PI3K/P‐AKT pathway and required PI3K activity for MMP‐2 activation. Together, these data provide new insight into the mechanisms by which EPO may induce angiogenesis, thus improving our understanding of the physiological roles of EPO. Funded by NSERC.
eNOS Internalization Mediated by Caveolar Endocytosis Regulates Microvascular Permeability.
Authors: Sanchez Fabiola Alejandra , Kim David D , Duran Ricardo G , Meininger Cynthia J , Duran Walter N
Published: June 2021

Endothelial nitric oxide synthase (eNOS) is highly regulated through phosphorylation, protein interactions and subcellular location. We demonstrated that platelet‐activating factor (PAF) activates eNOS and induces enzyme internalization in association with hyperpermeability. Here, we tested two hypotheses A) eNOS endocytosis regulates endothelial hyperpermeability to macromolecules, and B) eNOS internalization is mediated by caveolar endocytosis. We used bovine coronary postcapillary venular endothelial cells (CVEC). PAF increased NO production from 4.3 ± 3.8 to 467 ± 22.6 nmol/L and permeability to FITC‐dextran‐70 from 2.0 ± 0.23 × 10−6 cm/s to 6.0 ± 1.0 × 10−6 cm/s. PAF translocated eNOS from plasma membrane to cytosol. Application of 5 mM cyclodextrin (CD) and transfection of CVEC with dyn2K44A (a dynamin dominant negative mutant) blocked NO production and inhibited permeability responses to PAF. We used biotinylation of ECV304‐CD8eNOS‐GFP cells and Western blotting to show that eNOS is internalized via caveolae in response to PAF. Our data demonstrate that eNOS is internalized via caveolar endocytosis, and that this process is involved regulating endothelial permeability in response to inflammatory agonists. (Supported by NIH grant 5RO1 HL706434‐05).
Podocyte specific over‐expression of VEGF‐A165b, unlike VEGF‐A165, does not cause collapsing glomerulopathy
Authors: Ferguson Joanne Kathryn , Qiu Yan , Neal Chris R , Sage Leslie , Bates David O , Salmon Andrew H J , Harper Steve
Published: June 2021

Regulation of podocyte expression of vascular endothelial growth factor A (VEGF‐A) is critical for the establishment and maintenance of the glomerular filtration barrier (GFB). Podocyte‐specific VEGF‐A165 overexpression during development causes collapsing glomerulopathy (Eremina et al, 2003). VEGF‐A165 contributes to the high water permeability of glomeruli (Salmon et al 2006). Podocytes express both VEGF‐A isoform families: VEGF‐Axxx and VEGF‐Axxxb. We sought to determine the role of VEGF‐A165b in the glomerulus, using heterozygous transgenic mice whose podocytes overexpress VEGF‐A165b under nephrin promoter control throughout development and adulthood. Glomerular LpA and albumin reflection coefficient (σ) were measured with an oncometric technique. (Salmon et al 2006; Sage et al 2007). Mice appeared healthy until at least 18 months old, with no overt renal phenoytpe. LpA was reduced by 40% compared with glomeruli form wild‐type littermates, but glomerular σ was unchanged. Glomeruli appeared normal by light microscopy, with no evidence of glomerular collapse. Resin embedded Toluidine Blue stained sections showed a disrupted filtration barrier with absence of sub podocyte space. Our results indicate that VEGF‐A165b plays a strikingly different role to VEGF‐A165 in glomerular development and physiology.
Rnd3 inhibits thrombin‐induced endothelial barrier dysfunction
Authors: Breslin Jerome W , Daines Dayle A , Wu Mack H , Yuan Sarah Y
Published: June 2021

The small GTPase Rho has previously been shown to be involved in elevated microvascular permeability. Recently, a new member of the Rho family, Rnd3, was described as an endogenous inhibitor of Rho signaling. We hypothesized that Rnd3 promotes endothelial barrier integrity. To test this hypothesis, we evaluated barrier function of cultured human umbilical vein endothelial cells (HUVEC) under basal conditions and after stimulation with 1 U/ml thrombin. To test the role of Rnd3, we either overexpressed a MAT‐FLAG‐Rnd3 fusion protein or selectively knocked down expression of endogenous Rnd3 with siRNA. Expression levels were confirmed by Western blotting. Endothelial barrier function was evaluated by measuring transendothelial electrical resistance or by determining the permeability to FITC‐albumin in cultured HUVEC monolayers. We also evaluated whether thrombin alters Rnd3 localization using immunofluorescence microscopy, as well as Ser and Tyr phosphorylation of Rnd3, with immunoprecipitation and Western blotting. The results show that overexpression of Rnd3 in HUVEC attenuates thrombin‐induced endothelial hyperpermeability, whereas selective knockdown of Rnd3 extended the time‐course. In addition, we observed that Rnd3 mainly localizes in the perinuclear region of unstimulated endothelial cells but distributed throughout the cytoplasm and in some cases at the plasma membrane in thrombin‐treated cells. Thrombin also increased the degree of Ser and Tyr phosphorylation of Rnd3. These findings suggest that Rnd3 promotes endothelial barrier integrity, and that thrombin may affect endothelial permeability via posttranslational modification of Rnd3 in endothelial cells. Supported by NIH grants HL61507, HL70752, and HL73324.
Calibration of a fluorescent membrane for use in mapping microvascular nitric oxide (NO) gradients
Authors: Nugent William , Golub Aleksander , Pittman Roland
Published: June 2021

The concentration of NO, a key mediator of microvascular oxygen (O2) delivery both through its influence on vascular tone and mitochondrial O2 consumption, has been measured locally around microvessels using microelectrodes. To extend these measurements beyond in vivo point‐sampling, we have developed a wide‐field, spatio‐temporal sensor using ~3 μm silica gel microspheres that are coated with an NO‐sensitive fluorescent indicator (DAF‐2) and embedded in a 10 μm thick layer of Nafion. To provide for accurate quantification, the membrane is undergoing in vitro calibration tests using a commercially available flow chamber with either bolus or continuous (20 μl/s) perfusion of solutions containing known [NO]'s. Upon taking a baseline series of images using low‐light epi‐illumination—to minimize photobleaching—along with image intensification, the NO perfusate is added without image time‐course interruption. DAF‐2's conversion to a highly fluorescent form is dependent on both [NO] & [O2], and thus with [O2] accounted for, the rate of rise of fluorescence can be linked to a specific [NO]. Over time, the bead field is expected to develop like a photograph when exposed to the heterogeneous distribution of [NO] in vivo, yielding quantifiable information regarding NO dynamics in the microcirculation. Supported by NIH Grants HLI8292&HL79087
Ischemia in the Pulmonary Vasculature: Redox Signaling for Angiogenesis
Authors: Chatterjee Shampa , Hong Nankang , Fisher Aron B.
Published: June 2021

We have reported earlier that ischemia in the pulmonary vasculature results in reactive oxygen species (ROS) generation. In in vitro experiments using pulmonary microvascular endothelial cells (PMVEC), this post ischemic ROS was found to cause increased endothelial cell proliferation. We hypothesized that this proliferation is an angiogenic response of the endothelium. Here we investigated the angiogenic potential of flow adapted ischemic PMVEC (72 h flow adaptation, 1 h ischemia) as compared to static cells using in vitro and in vivo angiogenic assays. In matrigel dishes, flow adapted ischemic PMVEC showed ~4 fold higher tube formation than static cells. In the matrigel plug assay, where cells mixed in matrigel were implanted as a plug subcutaneously into nude mice, ischemic cells showed more extensive appearance of new vessels and endothelial markers as compared to static cells. Hind limb ischemia in wild type mice and mice with knockout of gp91phox (the membrane component of the NADPH oxidase) was used to study angiogenesis in vivo. Five days post ischemia, the growth of new vessels as ascertained by microscopic evaluation of the ischemic tissue was lower in gp91phox knockout mice where ROS generation was compromised. We thus conclude that the ROS‐dependent proliferation with ischemia is an angiogenic signal and represents an attempt at neovascularization to restore the impeded blood flow.
Role of PI3Kinase/Akt and Rac in activation of the endothelial NADPH oxidase with Ischemia
Authors: liu Weidong , Chatterjee Shampa , Fisher Aron B
Published: June 2021

We have shown previously that ischemia or abrupt cessation of flow in the lung endothelia in situ and in pulmonary microvascular endothelial cells (PMVEC) in vitro causes ROS production. This occured via NADPH oxidase as knockout of gp91phox prevented the response. We also reported that the trigger for NADPH oxidase activation is depolarization of the endothelial cell plasma membrane (via KATP channels) as preventing the depolarization with a channel agonist prevented the ROS generation. In phagocytes, the small G protein, rac (Rac1) is known to participate in NADPH assembly and activation. To investigate if Rac plays a similar role in endothelial NADPH oxidase assembly, we examined the link between endothelial cell membrane depolarization, Rac activation and NADPH oxidase assembly. Using PMVECs transfected with RacGFP, Rac translocation was monitored with membrane depolarization (achieved either by high external K+ or by ischemia). The significant increase in Rac translocation that was observed with depolarization was attenuated by C difficile Toxin B (Rac inhibitor) as also by wortmannin and LY294002 (PI3K inhibitors). PMVECs infected with dominant negative PI3K (mutant PI3K p85 subunit) showed no translocation of Rac to plasma membrane upon depolarization of the cells. These results indicate that PI3K/Akt may be upstream of Rac and that Rac 1 translocation is required for NADPH oxidase activation.
Extracellular calcium regulates embryonic mouse lung function through the calcium‐sensing receptor
Authors: Finney Brenda , del Moral Pierre M. , Wilkinson William W. , Cole Martin , Martin David , Warburton David , Kemp Paul J. , Riccardi Daniela
Published: June 2021

The widespread distribution of the extracellular calcium‐sensing receptor (CaR) and its sensitivity to acute physiologic changes in extracellular calcium concentration ([Ca2+]o) make it ideally suited to be a key regulator of Ca2+o‐dependent cell fate during development. We investigated whether [Ca2+]o, acting through the CaR, regulates murine lung development using embryonic day 12.5 lungs in whole organ culture. Ontogeny of CaR expression, determined using qPCR and immunohistochemistry, supported the use of this time point for experimentation. Branching morphogenesis and trans‐epithelial potential difference were exquisitely sensitive to changes in [Ca2+]o. Branching morphogenesis was maximal between 1 and 1.2mM (adult level) and was maximally inhibited at 1.7mM [Ca2+]o (fetal level). To probe the involvement of CaR in mediating the inhibitory effects of high [Ca2+]o, we co‐treated cultured lungs with the CaR specific calcimimetic, AMG R‐568 (10nM) in the presence of 1.2mM [Ca2+]o; this suppressed lung branching rate to a level not significantly different from that at 1.7mM [Ca2+]o. Together, our data suggest that fetal environmental Ca2+ signals are important extrinsic factors that modulate the intrinsic lung developmental programme, and they strongly support a primary role for the CaR in preventing hyperplastic lung disease in utero. This project is supported by the BBSRC.
Faster aqueous than phospholipid exocytosis during alveolar surfactant secretion
Authors: Islam Mohammad Naimul , Das Shonit , Perlman Carrie E , Lindert Jens , Bhattacharya Jahar
Published: June 2021

Lamellar bodies (LB) of alveolar type 2 cells consist of an aqueous phase that surrounds a phase containing surfactant protein B (SPB) in complex with surfactant phospholipids. Surfactant secretion entails exocytosis of the aqueous and the SPB‐phospholipid phases. To determine the extent to which these phases are secreted differently, we imaged by live confocal microscopy, single alveoli of isolated, blood‐perfused rat lungs held at alveolar pressure (Palv) of 5 cmH2O. Then, by alveolar micropuncture, we co‐loaded type 2 cells with purified, fluorescent SPB (BODIPY‐SPB) and the LB‐localizing marker, LysoTracker Red (LTR) to mark respectively, the phospholipid and the aqueous phases in LB. In single alveoli, type 2 cells were revealed by the high fluorescence of both BODIPY‐SPB and LTR. Thirty minutes after a single 15‐s inflation to Palv of 15 cmH2O, type 2 cell fluorescence decreased for BODIPY‐SPB by 46.6 ± 2.9% of initial (mean ± SE; n=4), but markedly more for LTR, namely by 72.5 ± 4.5% of initial (p<0.05). Thus, the fluorescence decay rate for BODIPY‐SPB was half that for LTR (P<.05). We interpret that during surfactant secretion, phospholipid versus aqueous secretion rates differ markedly. The difference might be attributable to differences in phase mobility resulting from the geometry of the LB exocytosis pathway (support: HL64896).
Co‐culture of alveolar epithelial and endothelial cells blunts failure of alveolar barrier function in hypoxia
Authors: Zuegel Stefanie , Bartsch Peter , Mairbaurl Heimo
Published: June 2021

Alveolar hypoxia increases alveolar barrier permeability and inhibits reabsorption favoring formation of pulmonary edema. Monocultures of primary rat alveolar type II cells (ATII) and rat lung microvascular endothelial cells (EC) are commonly used models. We designed a co‐culture (CC) system of EC and ATII on nucleopore filters to study mechanisms of hypoxia (1.5% O2, 24 h) induced failure of alveolar barrier function. CC showed a 40% higher resistance and a higher short‐circuit current (ISC) in normoxia and hypoxia than ATII monolayers. In normoxia, Na/K‐ATPase capacity was higher in CC than in ATII, but ENaC activity was comparable. Hypoxia inhibited ISC (−40%), Na/K‐ATPase (−25%) and ENaC (−25%) only in ATII but not in CC. In normoxia, Na‐fluorescein permeability was lowest in CC. It was 9x higher in ATII and 120x higher in EC than in CC. Hypoxia increased fluorescein‐permeability in ATII (+55%) and in CC (+400%) but not in EC alone. FITC‐albumin‐permeability in normoxia was 35x higher in EC than in ATII and in CC. Hypoxia increased albumin‐permeability only in ATII (+35%). These findings indicate that the interaction between alveolar epithelial and endothelial cells tightens the alveolar barrier and increases trans‐alveolar Na‐transport activity. Hypoxia‐effects on permeability and ion transport are less pronounced in the co‐culture than in monocultures of alveolar epithelial cells.
Mathematical model of lacrimal acinar cell pH and volume regulation
Authors: Huang Wei Chieh , Clausen Chris , Brink Peter , Walcott Benjamin , Moore Leon C
Published: June 2021

When stimulated, lacrimal acinar cells (LAC) secrete K+, Cl− and water. This is associated with cell shrinkage, and activation of the Na+‐K+‐2Cl− cotransporter (NKCC1), and of the Na+‐H+ (NHE1) and Cl−‐HCO3− (AE) exchangers. These transporters pump Na+ and Cl− into the cell to maintain tear secretion. However, experiments have shown that LAC ion transport is relatively insensitive to inhibition of either the NHE1 or NKCC1 transporters. In this study, we used a mathematical model of LAC ion and water transport to examine interactions between tear secretion, cell volume (CV) regulation and pH homeostasis. The model is based on mass conservation, electroneutrality constraints, and includes kinetic models of Na+‐K+‐ATPase, NKCC1, the K+‐Cl− cotransporter (KCC), NHE1, AE, CO2 hydration, and passive fluxes of ions, non‐electrolytes and water. The effects of muscarinic stimulation to enhance apical Cl− and K+ permeabilities and NHE1 activity are also included, as are the effects of CV changes on the NKCC1 and KCC transporters. During secretion, the model predicts increased NHE1 and AE ion transport when NKCC1 is inhibited, and higher NKCC1 uptake when NHE1 in inhibited. In both cases, the compensatory changes are driven by reduced cell [Cl−]. The results suggest that interactions of the transporters in tear secretion, CV and pH control are in part mediated by altered cytosolic Cl− levels. Supported by NIH grant EY14604.
Slo2.1 (Slick), but not Slo2.2 (Slack), is regulated by changes in cell volume
Authors: Stolpe Kathleen , Poulsen Asser Nyander , Søe Rikke , Meinild‐Lundby Anne‐Kristine , Klærke Dan
Published: June 2021

A number of physiological processes, such as nerve activity, epithelial transport and apoptosis, involve changes in cell volume. The KCa1.1 ‐ (Slo, BK, MaxiK) ‐ related potassium channels Slo2.1 (Slick) and Slo2.2 (Slack) are activated by intracellular Na+ and Cl– and inhibited by intracellular ATP. Both channels are widely expressed in different tissues, including dura mater, middle cerebral artery and basilar artery confirmed by RT‐PCR. In this study we examined the possible regulation of Slo2.1 and Slo2.2 by small, fast changes in cell volume in Xenopus oocytes after co‐expression with aquaporins (AQP1) to ensure high water permeability. Our results show that Slo2.1 is dramatically stimulated (to 196% of control) by cell swelling of approximately 5%, and inhibited (to 44% of control) by a similar decrease in cell volume. In contrast, changes in cell volume did not affect the Slo2.2 channels. Therefore it is suggested that cell volume is a significant regulatory parameter for Slo2.1 and that these channels may play an important role in regulating intracellular potassium concentration during changes in cell volume.
Renal dysfunction and water channels in fresh water unadapted European sea‐bass (Dicentrarchus labrax)
Authors: Giffard‐Mena Ivone , Boulo Viviane , Lorin‐Nebel Catherine , Castille René , Charmantier Guy
Published: June 2021

The euryhalin teleost D. labrax is able to live within a wide salinity range between fresh water (FW) and sea water. However some juvenile individuals are unable to adapt to FW (FWU‐fish). The expression of aquaporins (AQP1 & AQP3) and Na+/K+‐ATPase expression was evaluated in osmoregulatory organs. The structure of gills, gut and kidney were analysed by histology. Overexpression of AQP1 in gut and of AQP3 in gills in FWU‐fish compared to FW‐adapted fish suggest an increase in water permeability of these organs. The presence of renal bodies in distended collecting duct and urinary bladder of FWU‐fish, as well as a lower Na+/K+‐ATPase activity in FWU kidney indicate a renal failure as the origin of lower osmotic blood pressure which leads to fish die. Fish dyes 48 h after the occurrence of abnormal behavior in the shoal. FWU‐fish survive if they are transferred to 10 ppt (340 mOms.kg−1)or more.
Distribution and Regulation of Aquaporins in Intestinal Epithelial Cells
Authors: Chow Elaine , MacNaughton Wallace
Published: June 2021

Sodium absorption and chloride secretion create osmotic gradients that drive water transport across intestinal epithelial cells. Aquaporins (AQP) are tetrameric water‐selective channels that may play an important role in facilitating transcellular water movement and maintaining physiological electrolyte balance. In the kidney, key AQPs are regulated by intracellular cAMP. We hypothesized that the cellular distribution of AQPs in the intestinal epithelium is regulated by cAMP. RT‐PCR of three intestinal epithelial cell lines demonstrated mRNA expression of: AQP1, 2, 3, and 5 in SCBN; AQP1, 2, 3, 5, 7, 8, and 9 in T84; and AQP1, 2, 3, 4, and 5 in CMT. Confocal imaging showed immunoreactivity for AQP1, 2, 3, 4, and 7 in SCBN and AQP1, 4, 6, and 9 in T84. Real time confocal live cell imaging tracked intracellular movement of GFP‐linked human AQP1 gene construct transfected into T84 cells. It suggested an increase in apical fluorescence of AQP1 within 20 min of forskolin (FSK) treatment. Cellular fractionation of CMT whole cell lysate by differential centrifugation showed a significant FSK‐dependent increase in AQP1 protein in the plasma membrane and no FSK‐induced change in the intracellular fraction. This study demonstrates that multiple AQPs are expressed in intestinal epithelial cell lines, and provides evidence for cAMP‐dependent regulation of AQP1 trafficking.

Supported by CCFC.
Inactivation of GATA3 in Principal Cells Leads to a Concentrating Defect
Authors: Riemondy Kent , Lucero Olivia , Baumgartner Brett , Mauch Teri Jo , Miller Lance , Nelson Raoul
Published: June 2021

GATA3, a member of the GATA zinc‐finger family of transcription factors, plays an important role in renal development; haploinsufficiency leads to Hypoparathyroidism, Deafness, and Renal agenesis. GATA3 has been shown to be expressed in the ureteric bud (UB) and collecting duct (CD) of the embryonic and adult kidneys, respectively. Its role in the developing UB is currently being explored; however, its role in the CD is completely unknown. The purpose of this study was to determine the role of GATA3 in renal principal cells of the adult mouse. We inactivated GATA3 using the AQP2‐Cre transgenic mouse (AQP2‐Cre) and the floxed GATA3 mouse (GATA3floxed). Inactivation of GATA3 in principal cells lead to a moderate concentrating defect, i.e., mice could only concentrate their urine ~60% of control; however, inactivation of GATA3 did not affect the ability to excrete an acute water load. Furthermore, other than the concentrating defect mice appear normal and healthy. We hypothesize that GATA3 transcriptionally regulates AQP2 or other collecting duct‐specific transporters involved in water reabsorption or maintaining the osmotic gradient. This study explores this hypothesis.
Differential Regulation of FXYD5, FXYD9 and FXYD11 Expression in Atlantic Salmon Gill During Parr‐Smolt Transformation and Sea Water Acclimation
Authors: Tipsmark Christian K , Madsen Steffen S
Published: June 2021

The Na,K‐ATPase is the major driving force for ion secretion in teleost gill chloride cells. Accordingly, gill enzyme abundance increases and isoform composition changes in response to increased salinity and during the salmon parr‐smolt transformation (PST) prior to seaward migration in the spring. We identified 3 isoforms of the FXYD protein family (FXYD5, FXYD9 and FXYD11) in the salmon gill, which may assist in regulating the catalytic rate of the Na,K‐ATPase in these situations. FXYD proteins are auxiliary subunits which may associate with the alpha‐subunit of the Na,K‐ATPase. Expression in the gill of the 3 isoforms was investigated during PST and acclimation of freshwater (FW) salmon to sea water (SW). During the PST, FXYD5 mRNA levels were unaltered, whereas the expression of FXYD9 and FXYD11 peaked in May, coinciding with optimal SW‐tolerance and peak levels of Na,K‐ATPase of the FW dwelling salmon. Abrupt SW‐transfer induced an increase in Na,K‐ATPase abundance and FXYD5 expression but no overall changes in FXYD9 and FXYD11. The parallel increase in FXYD9 and FXYD11 levels with Na,K‐ATPase abundance during PST but not during SW‐acclimation suggests that these auxillary proteins play a particular role in controlling catalytic rate in situ when the FW fish prepares for SW entry.

The research was supported by the Carlsberg Foundation (2005‐1‐311) and the Danish Natural Research Council (272‐06‐0526).
Molecular dissection of tyraminergic communication in the Drosophila Malpighian tubule
Authors: Blumenthal Edward Michael
Published: June 2021

The biogenic amine tyramine (TA) acts as a diuretic agent in the Drosophila Malpighian tubule (MT), increasing urine secretion by activating a transepithelial chloride conductance. The MT can also synthesize TA when its precursor, L‐tyrosine, is present in the peritubular fluid. The goal of this study was to identify two of the gene products necessary for TA signaling: tyrosine decarboxylase (tdc), the enzyme that converts tyrosine to TA, and the TA receptor (TAR). The Drosophila genome contains two tdc genes: tdc1 and tdc2 (Cole et al., 2005). The tdc1 gene is highly expressed in the MT while tdc2 is not. MTs from tdc1 mutant flies show no electrophysiological or diuretic responses to tyrosine but normal responses to TA; in contrast, mutation of tdc2 does not alter tyrosine sensitivity. Overexpressing tdc1 in the principal cells, but not the stellate cells, of tdc1 mutants, rescues tyrosine sensitivity, suggesting that TA synthesis occurs in the principal cells. The TAR in the MT is unlikely to be encoded by the canonical TAR gene tyrR, which is expressed in the MT at very low levels. In contrast, the recently identified TAR gene CG7431 (Cazzamali et al., 2005) is abundantly expressed in the MT. Mutation of CG7431 results in complete insensitivity to TA, suggesting that CG7431 encodes the TAR in the MT. Rescue experiments are underway to determine the cell type in which this receptor functions. Supported by Marquette University
Phorbol ester stimulation enhances endocytosis and ubiquitination of the sodium chloride co‐transporter
Authors: Ko Benjamin , Cooke Leslie L , Hoover Robert S
Published: June 2021

The sodium chloride co‐transporter (NCC) is the principal salt‐absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters stimulate Ras guanyl‐releasing protein 1, triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression. Surface expression may decrease either by inhibition of forward trafficking or enhanced endocytosis. To investigate the mechanism of altered NCC surface expression in this system, mouse distal convoluted tubule (mDCT) cells were biotinylated at 4ºC, then re‐warmed to 37ºC and treated for 20 minutes with either vehicle (DMSO) or 100nM TPA (phorbol ester). Cells were washed with MesNa to remove any biotin remaining at the cell surface, then lysed. Strepavidin pulldown and immunoblotting showed that after 20 minutes of TPA treatment, the amount of internalized NCC increased by 459±25% compared to control (p<0.01). The ubiquitination of NCC may mediate this process, as TPA stimulation resulted in a 485±33% increase in ubiquitinated NCC as compared to control (p<0.05), measured by immunoprecipitation of NCC followed by immunoblotting for ubiquitin in mDCT cell lysates treated with TPA or vehicle. These data indicate that phorbol ester stimulation of mDCT cells triggers enhanced endocytosis and increased ubiquitination of NCC.

This work was supported by a NIH Grant K08 DK070668.
Vasopressin Activates Akt1 Through PI‐3K in Rat Inner Medullary Collecting Duct (IMCD)
Authors: Pisitkun Trairak , Jacob Vinitha , Yu Ming‐Jiun , Chou Chung‐Lin , Knepper Mark A
Published: June 2021

Regulation of osmotic water permeability in the rat IMCD depends on vasopressin‐mediated control of AQP2 trafficking. Although it has been established that this regulation requires both an increase in cAMP and in Ca‐calmodulin, full details of vasopressin signaling are lacking. We investigated activation of Akt1 by vasopressin analog dDAVP in suspensions of freshly isolated rat IMCD using immunoblotting to assess changes in Akt1 phosphorylation. dDAVP significantly increased Akt1 phosphorylation at T308 and S473, which are both associated with activation of kinase activity. Maximal activation was seen 5–10 minutes after dDAVP addition. The LY294002 (PI‐3K inhibitor) markedly reduced phosphorylation at both sites and prevented the dDAVP‐induced increase. Immunolocalization of rat tissue sections revealed that the p110γ subunit of PI‐3K is associated with the basolateral plasma membrane of IMCD, while Akt1 is distributed throughout the cells. Addition of CPT‐cAMP increased phosphorylation at S473 but not at T308. The intracellular Ca chelator BAPTA‐AM and the calmodulin‐inhibitor W‐7 decreased phosphorylation at both sites. Use of an Akt substrate specific antibody for 2‐D immunoblotting of rat IMCD revealed several spots that changed in abundance or shifted in pI in response to dDAVP. We conclude that PI‐3K dependent Akt1 activation is part of the overall vasopressin signaling response in the rat IMCD.
The Functional Roles of Cysteine Residues in the KCC4 K‐Cl Cotransporter
Authors: Mercado Adriana , Babilonia Elisa , Mount David
Published: June 2021

K+‐Cl− cotransporters (KCCs) are modulated by the cysteine‐reactive compound NEM. Reducing agents also inhibit oligomerization of KCC2, implicating cysteine‐dependent disulfide bridges. KCC4 contains a total of 23 cysteines, 18 conserved; we have examined the functional role of all 23 cysteines, using expression in Xenopus oocytes. Mutation to alanine (transmembrane) or serine (extra/intracellular orientation) yielded several non‐functional mutants. However, converting these to leucine and/or other residues we found that no single cysteine was essential for function. Single mutants of conserved cysteines within TM2 were only modestly functional, suggesting a critical functional role for these residues. Sequential mutation to generate a cysteine‐less protein has been partially successful, preserving function when up to 11 cysteines were modified. The apparent dimerization of KCC4 in immunoblots was not affected in compound mutants, suggesting that cysteines are not required for dimerization of this KCC paralog. In summary, no single cysteine in KCC4 is required for K+‐Cl− cotransport, nor are cysteines evidently required for dimerization. Progress has also been made in generating functional compound mutants that are profoundly cysteine‐depleted, suitable for defining cysteines targeted by activating/inhibiting stimuli and/or for other structure‐function studies.
Mechanisms of Human Hepatic Vitamin C Uptake: Studies of the hSCVT Systems
Authors: Reidling Jack C. , Subramanian Veedamali S. , Dahhan Tamara , Sadat Mohammed , Said Hamid M.
Published: June 2021

Ascorbic acid (AA), the reduced active form of vitamin C, is transported by two sodium‐ascorbate co‐transporters (SVCT1 & 2). Humans do not synthesize AA and the essential micronutrient is both an antioxidant and cofactor for several enzymes. The human liver participates in regulating and maintaining body AA homeostasis. Using a human hepatic cell line (HepG2) we determined, the characteristics of AA uptake, the expression levels of the hSVCT systems and characterized the hSVCT promoters. The initial rate of ascorbic acid uptake by HepG2 cells was: 1) Na+ gradient, pH, and energy dependent, 2) saturable with a Km of 10μM 3) not inhibited by membrane transport inhibitors 4) not stimulated by glucocorticoids 5) PKC, PKA, PTK, Ca‐Camodulin dependant, but not NO dependant. Expression of hSVCT1 and hSVCT2 protein and mRNA in both HepG2 cells and human liver was determined; hSVCT2 is expressed at higher levels. Using cloned promoter fragments, transcriptional activity was determined in HepG2 cells and the minimal regions required for basal activity mapped. In addition, using promoter mutagenesis we determined that Hepatic Nuclear Factor sites in the hSVCT1 promoter and Sp1/KLF sites in the hSVCT2 promoter were essential for activities. Results from these studies are the first determination of the AA uptake process, relative expression of the hSVCT systems and characterization of the hSVCT promoters in human liver cells.
Activation of IClswell by TNFα requires CLC‐3 dependent ROS production
Authors: Matsuda James J , Filali Mohammed S , Volk Kenneth A , Lamb Fred S
Published: June 2021

ClC‐3 is a Cl−/H+ antiporter whose biophysical properties are distinct from the swelling activated chloride current (IClswell). ClC‐3 is required for reactive oxygen species (ROS) generation in response to TNFα in vascular smooth muscle (VSM) cells. Because ROS have been shown to independently activate IClswell, we wanted to determine if TNFα activates IClswell in VSM, and if this process was ClC‐3 dependent. Whole‐cell patch clamp recordings were used to study the effect of TNFα on Cl currents in cultured aortic VSM from wild‐type (WT) and ClC‐3 knockout (KO) mice. We found that TNFα (10 ng/ml) activates IClswell in WT VSM but not KO VSM cells under isotonic bath conditions. The effects of TNFα were inhibited by Tamoxifen (10 μM) and PEG‐catalase (1000 U/ml) and were mimicked by hydrogen peroxide (500 μM). Adenovirus‐mediated over‐expression of ClC‐3 in KO VSM induced IClswell in the absence of swelling or TNFα, and these currents were also inhibited by PEG‐catalase. Hypotonicity activated IClswell in both WT and KO VSM and these currents were inhibited (60% at 80 mV) by PEG‐catalase in WT VSM. Finally, expression of dominant negative Rab11 (S25N) in WT VSM inhibited TNFα activation of IClswell, suggesting that activation of IClswell takes place in the endosomal compartment. These results show that TNFα activates IClswell, and that this effect is mediated by ClC‐3 dependent ROS production. Supported by HL62483.
Splice variation alters CBS domain structure and gating of a C. elegans ClC channel
Authors: Dave' Sonya , Sheehan Jonathon , Strange Kevin
Published: June 2021

Eukaryotic CLCs have large cytoplasmic C‐termini containing two Cystathionine‐β‐Synthase (CBS) domains. The role of CBS domains in ClC channel gating and structure is unclear. clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans ClC channel that exhibit striking differences in sensitivity to depolarizing voltages, pH and Cl−. Mutagenesis studies have shown that the C‐terminus of CLH‐3b gives rise to its unique gating properties. Splice variation of the C‐terminus includes the last six amino acids in the second CBS domain (CBS2). Mutating these amino acids to those present in CLH‐3a gives rise to CLH‐3a gating properties. Similarly, CLH‐3b gating properties are induced in CLH‐3a by mutating the last six amino acids of CBS2 to those present in CLH‐3b. To define how these amino acids alter CBS2 structure, we developed homology models based on crystal structures of CBS domains in vertebrate CLCs using Modeller and Rosetta software. Our models show that the six amino acid splice variation is part of the last α‐helix of CBS2. Sampling 10,000 conformations with Rosetta demonstrated that the splice variation in CLH‐3b has a higher probability of forming a helical turn. Thus, the last α‐helix of CBS2 in CLH‐3a is shorter compared to CLH‐3b. We are currently carrying out modeling and mutagenesis studies to determine how CBS2 splice variation alters the channel dimer‐dimer interface and gating.
Association of protein kinase C to the Cav1.2 Ca2+ channel improves muscarinic‐induced contraction of urinary bladder
Authors: Wegener Jörg W , Huster Maria , Frei Eva , Hofmann Franz
Published: June 2021

Muscarinic agonists like carbachol induce contraction of urinary bladder. This contraction is blocked by L‐type Ca2+ channel antagonists and is abolished in detrusor muscle from Cav1.2 deficient mice, demonstrating that this type of contraction depends on functional L‐type Ca2+ channels. However, it is unclear how muscarinic stimulation couples to the L‐type Ca2+ channel. In this study, we present several lines of evidence that protein kinase C is involved in this signalling pathway: (i) carbachol‐induced contraction was attenuated by the protein kinase C inhibitor Gö6850; (ii) contraction and (iii) intracellular Ca2+ signals induced by carbachol were increased after activation of protein kinase C with phorbol ester; (iv) these effects of phorbol ester were inhibited by Gö6850; (v) co‐immunoprecipitation using a Cav1.2 antibody and (vi) analysis of membrane preparations using blue native gelelectrophoresis revealed an association of protein kinase C to the Cav1.2 channel if the muscles had been stimulated by carbachol; (vi) phosphorylation of the Cav1.2 channel was increased in carbachol‐stimulated preparations and attenuated by Gö6850. These results suggest that association of protein kinase C to the Cav1.2 Ca2+ channel participates, at least partially, in the signalling pathway from the muscarinic receptor to the L‐type Ca2+ channel during micturition.
Membrane Trafficking of TRPM4 in Smooth Muscle Cells
Authors: Crnich Rachael Carissa , Gonzales Albert Louis , Earley Scott
Published: June 2021

The melastatin Transient Receptor Potential (TRP) channel TRPM4 mediates pressure‐induced smooth muscle depolarization and vasoconstriction of cerebral arteries. Interestingly, recent reports show that several members of the TRP superfamily are gated by directed movement of channel protein into and out of the plasma membrane. We hypothesized that activators of TRPM4‐dependent currents such as elevated intracellular Ca2+ and protein kinase C (PKC) induce surface expression of TRPM4 protein, thus increasing the number of functional channels present in the membrane. We created a fusion protein consisting of green fluorescent protein (GFP) covalently linked to the carboxyl terminus of TRPM4 (TRPM4‐GFP) and transiently expressed this construct in the smooth muscle‐derived A7r5 cell line. In whole‐cell patch clamp experiments, cells expressing TRPM4‐GFP exhibit electrophysiological properties similar to those of native TRPM4 channels. Live‐cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis of cells expressing TRPM4‐GFP indicate that channel protein is packaged in discreet, mobile vesicles, consistent with a potential role for vesicular trafficking in TRPM4 regulation. Preliminary cell surface biotinylation assays reveal surface expression of TRPM4‐GFP. These techniques will be used to investigate the effects of Ca2+ and PKC on channel mobility. AHA0535226N
Inhibition of TRPM3 channel by anti‐depressant and anti‐psychotic drugs
Authors: Majeed Yasser , Naylor Jacqueline , Zeng Fanning , Jones Clare , Beech David J
Published: June 2021

TRPM3 is a member of the TRP super‐family of cation channels about which relatively little is known (Oberwinkler 2007 Handb Exp Pharmacol 179, 253–67). Putatively it is a novel chemical‐sensing channel and thus we have searched for chemicals that affect TRPM3, either as endogenous modulators or pharmacological agents. For these experiments we used tetracycline‐inducible human TRPM3‐ or TRPM2‐expressing HEK 293 cells studied using fura‐2 to measure calcium‐entry in real‐time 96‐well plate assays. Because TRPM3 is expressed in the nervous system we investigated anti‐psychotic and depressant drugs. The anti‐depressants imipramine and fluoxetine caused concentration‐dependent inhibition, showing IC50s of 91 μM and 42 μM respectively. Neither drug had effect on TRPM2 or the endogenous ATP response of the cells. The anti‐psychotic drug chlorpromazine was also inhibitory on TRPM3 (IC50 28 μM) and TRPM2, but not on the ATP response. The effects of these drugs on TRPM3 may have clinical relevance or indicate lead compounds for the development of potent TRPM3 blockers.
Normotensive menopausal rats: Effects of Enalapril on Sexual Hormones and on Nitric Oxide System Activity in the Kidney, Heart and Aorta
Authors: Zilberman Judith Miriam , Veiras Luciana , Minotti Natalia , Bianciotti Agustina , Villamil Alberto , Costa Maria Angeles , Arranz Cristina
Published: June 2021

Studies in animal models suggest that sexual hormones, oxidative stress, and impaired nitric oxide (NO) system and renin‐angiotesin system activation play a role in hypertension during menopause. However, the involved mechanisms are still unknown.

Objective: To evaluate the effects of enalapril, an inhibitor of the angiotesin‐converting enzyme (ACE), on systolic blood pressure (SBP), sexual hormones and NO syntase (NOS) activity in normotensive menopausal rats.

Wistar rats were ovariectomized, and ten weeks later were divided into two groups: Experimental (E) treated with enalapril for 30 days (15 mg/Kg/day) and Control (C) with tap water. We measured: indirect SBP, androgen, estradiol and FSH plasma levels, and NOS activity (14C‐arginine) in the kidney, aorta and heart at the end of treatment.

Results: treatment with enalapril did not induce any changes in SBP, estradiol, androgen and FSH plasma levels.

NOS activity (pmol/min 100g tissue), * p<0.01 vs Control

Conclusions: The ACE inhibition did not modify SBP or sexual hormones in normotensive menopause rats, but treatment with enalapril improved NOS activity significantly in all the studied tissues. This fact could improve renal and cardiovascular function during menopause.
AICAR inhibits inflammation in MRL/lpr mouse mesangial cells
Authors: Radjavi Ali , Edelestein Rachel , Peairs Abby , Giri Shailendra , Reilly Christopher M
Published: June 2021

MRL/MPJ‐Faslpr (MRL/lpr) mice develop lupus‐like disease similar to human systemic lupus erythematosus, including immune complex deposition in the kidney which triggers the release of proteolytic enzymes, inflammatory mediators, cytokines, reactive oxygen species, and nitric oxide. Recent reports show that 5‐amino‐4‐imidazole carboxamide riboside (AICAR), a pharmacological activator of AMP‐activated protein kinase (AMPK), inhibits the LPS‐induced production of pro‐inflammatory cytokines. In our current studies, we sought to determine the mechanism by which AMPK decreases inflammation. Cultured mesangial cells from MRL/lpr mice were treated with AICAR and stimulated with LPS/IFN‐γ. AICAR dose dependently decreased iNOS, TNF‐α, and IL‐6 production in LPS/IFN‐γ stimulated mesangial cells. In pursuit of a mechanism we showed that AMPK activation induces phosphatase and tensin homolog (PTEN) expression which inhibits LPS‐induced PI3K/Akt/mTOR signaling inflammatory cascade. An adenosine kinase inhibitor blocked the ability of AICAR to activate AMPK and its downstream target PTEN, preventing inhibition of Akt. Interestingly, the p38 and IκB/NF‐κB signaling pathways were unaffected by AICAR treatment. Taken together, these observations suggest that AICAR specifically targets Akt signaling via PTEN activation and that AMPK may serve as a therapeutic target in inflammatory disease.
Small‐Molecule CFTR Inhibitors Slow CYST Growth in Culture and Mouse Models of Polycystic Kidney Disease
Authors: Yang Baoxue , Sonawane N. D. , Zhao Dan , Somlo Stefan , Verkman A. S.
Published: June 2021

Polycystic kidney disease (PKD) is a major cause of chronic renal insufficiency. Cyst expansion in PKD involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The Aim of this study is to test if small‐molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. Screening of more than 30 CFTR inhibitor analogs in an MDCK cell cyst model indicated near complete suppression of cyst growth by the tetrazolo‐derivatized thiazolidinone, tetrazolo‐CFTRinh‐172, and the phenyl‐derivatized glycine hydrazide, Ph‐GlyH‐101, without effect on cell proliferation. These compounds also inhibited cyst number and growth by > 80 % in an embryonic kidney cyst model involving 4‐day organ culture of E13.5 mouse kidneys in 8‐Br‐cAMP‐containing medium. Subcutaneous delivery of tetrazolo‐CFTRinh‐172 and Ph‐GlyH‐101 to neonatal, kidney‐specific PKD1 knock‐out mice produced stable and ‘therapeutic’ inhibitor concentrations of >3 μM in urine and kidney tissue. Treatment of mice for up to 7 days remarkably slowed kidney enlargement and cyst expansion, and preserved renal function. These results indicate the involvement of CFTR in renal cyst growth in PKD, and provide proof‐of‐concept for application of CFTR inhibitors to reduce cyst growth in PKD.
Endothelial to mesenchymal transition and circulating cells contribute to fibroblast formation following renal ischemia reperfusion
Authors: Friedrich Jessica , Leonard Ellen , Blazer‐Yost Bonnie , Basile David
Published: June 2021

Renal ischemia reperfusion (I/R) results in loss of peritubular capillaries and increased interstitial fibroblasts predisposing chronic kidney disease (CKD). To determine the source of fibroblasts following I/R, male SD rats were subjected to 30 min of bilateral ischemia and reperfusion for 3, 6, 12, 24 hr and 3d, and 7d. S100A4+ immunohistochemistry identified an increase in fibroblasts in the renal cortex within 6 hour relative to sham‐controls (6.5 ±1.1 vs 24.5 ±1.6 cells/field, p < 0.05) and these values remained elevated for the duration of the study. At 6 hours and 24 hours, ~5.8% of S100A4+ cells localized in vascular structures identified using biotinylated tomato lectin infused i.v. just prior to death. In contrast, only ~0.1% of S100A4 cells localized in tubules. Dual immunofluorescence also identified a population of cells defined as S100A4+/CD31+, further suggesting a potential endothelial → mesenchymal transition state. The contribution of circulating cells was evaluated in Lewis rats following a bone marrow transplant with syngenic EGFP rat donors; this resulted in the identification of a population of EGFP+ / S100A4+ cells after renal I/R. Taken together, these data suggest that fibroblast deposition may arise from endothelial and circulating sources following acute renal I/R injury (Support from NIH DK 63114).
Dose‐response effect of estrogen on the kidney and heart
Authors: Meng Xiaomei , D'Ambrosio Martin Antonio , Liao Tang‐Dong , Yang Xiao‐Ping
Published: June 2021

Recent clinical trials indicate that estrogen replacement therapy (ERT) may increase the risk of cardiovascular diseases. Here we studied whether the adverse effects of ERT are related to the doses used. Ovariectomized (OVX) mice received 17β‐estrodial (E2) at 0.001 (VL, very low), 0.42 (L, low), 4.2 (M, moderate) or 28.3 μg/d (H, high) for 60 days. E2 at moderate and high doses increased plasma estrogen levels by about 4.5‐fold. This was associated with ascites, nephrydrosis, proteinuria and fluid retention in uterus horns. Low dose of E2 restored plasma estrogen and uterus weight to levels similar to normal controls and no fluid retention or renal damage were found in this group of mice. Moderate and high doses of E2 also increased cardiac ANP mRNA expression and PKC and PI3K phospholyration. Aside from diastolic function, blood pressure and cardiac function were not altered by ERT at any given dose. These results suggest that ERT dosage could be an important determinant for a beneficial or detrimental outcome.