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Authentication of Key Resources
Antibodies: much of our work is focused on the use of antibodies for immunocytochemistry, western blotting andimmunoprecipitation, so this is a major focus for us. We have decades of experience using these reagents and are more than familiar with the unsubstantiated promises of many commercial reagents in particular. We have developed a systematicprocess by which all antibodies, whether home-made, shared by other investigators, or commercial are validated. First, weperform immunocytochemistry on cells or tissues expected to express the protein of interest. If staining is observed (using a variety of procedures including several antigen retrieval techniques), we perform a series of negative controls includingsecondary antibody alone and primary antibody pre-absorbed with a 5-10 fold molar excess of the immunizing antigen at4°C overnight. We always use affinity purified antibodies, typically purified in house using the Pierce purification kit orpurchased as affinity purified products, so that purified IgG subtype fractions from the same species can also be used as anegative control. For cultured cells when required and when relevant, we perform immunostaining on cells that do notexpress the protein, followed by staining on transfected cells that do express the protein. Finally, when available, WT and knockout mice are compared, as for our V-ATPase B1 subunit KO mouse, for example, to validate anti-B1 antibodies and the NCOA7 null mouse to validate anti-NCOA7 antibodies. Next, we perform western blot screening of cells and tissuesexpressing the target protein, to determine whether a band of the appropriate MW is detected. Full-length western blots willbe provided to reviewers for each cell/tissue used, upon submission of manuscripts as now required by several journals. This is a routine procedure in our laboratory. Where possible, different antibodies against the same antigen are used – forexample we typically raise antibodies in different species (e. g., rabbit and chicken) and expect them to give the sameimmunostaining and western blotting patterns. In addition, we also raised antibodies against different peptide sequencesfrom the same protein, or protein complex and use these to verify localization of a given protein. For commercial reagents,we test antibodies against the same protein from different vendors if available. We have a long track record of antibody production and validation, and we have shared our home-made reagents, most notably against many V-ATPase subunitsand aquaporins, with dozens of investigators throughout the world over the past several years.
Cell lines: the cell line to be used here is the M1 mouse cortical kidney collecting duct epithelial cell line (ATCC CRL2038). The original resource was validated by ATCC and we routinely use low passage numbers from frozen stock aliquotsmaintained in liquid nitrogen. All “thawed” aliquots are tested weekly for mycoplasma contamination by DAPI or Hoechst33258 staining and visual inspection. Finally, we routinely test M1 and other cells used in the lab for species specificity to avoid “human” errors that might occur during passaging and rethawing, using PCR amplification of RNAs that show inter-species sequence variability, e.g ., GAPDH. For example, we have other cell lines from collecting ducts (IMCD cells) thatare of rabbit, mouse and rat origin and obviously need to be identified correctly. There is now a test product called “Authentikit” on the market that does essentially the same thing, and is recommended by ATCC.
Serum for culture media: this is one of the major variables than can lead to different results in labs throughout the world,There are numerous sources of the fetal bovine serum that we need to add to our culture medium and we strive to reducevariability by purchasing our stock from the same source, Atlanta Biologicals, and purchasing many bottles from the samelot number to supply the lab for over a year. Prior to purchasing FBS, we receive small test samples from different lots fromvendors, and test these lots in several typical assays that we use in the lab. In the case of the current proposal, we look atcell growth and establishment of a tight epithelial monolayer by monitoring transepithelial resistance in our cell culturechamber made for this purpose. We look at response of cells to hormonal stimulation by measuring cAMP levels with anELISA assay, and we examine V-ATPase expression and distribution by immunofluorescence and western blotting. When we identify a “good” lot, we purchase at least 24 x 500 ml bottles, sufficient for about 18 months at existing usage levels,and store them at -80°C. All serum comes with a data sheet providing some information on biochemical and hormonalprofile. Some companies are aware of the more stringent NIH requirements and are beginning to provide more extensiveinformation, but that provided at present by Atlanta falls short of giving actual concentrations of components. It is certainlynot practical or feasible for our lab to perform all of these complex measurements. So at present, our assessment is based on consistent cell growth, responses to treatments, and protein expression patterns.
Drugs and chemicals: All commercial drugs/inhibitors etc. used in this study are provided with quality control datasheet that describes the drug chemical properties and lot number. Efficacy is determined using standard assays and by comparison with data from the literature showing efficacy. This information is kept in a central binder that is available for consultation.
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BUDGET JUSTIFICATION
A. Senior/Key Personnel
Dr. Dennis Brown, Ph.D., Principal Investigator (25% effort, 3 calendar months) Dr. Brown is Professor of Medicine, is an internationally recognized expert in kidney epithelial cell biology and in V-ATPase structure and function, and he will oversee all aspects of the proposed studies, in addition to continuing his hands-on involvement in much of the work. In particular, he will perform laser confocal microscopy on cells expressing fluorescent protein constructs, mutant constructs and after protein knockdown and gene editing, using the new Zeiss LSM800 Airyscan confocal microscope. He will supervise the postdoctoral fellow in all aspects of cellular imaging. He will be responsible for the overall design and implementation, and interpretation of the experiments as well as coordinating the activities of the collaborating investigators, consultants and technical staff.
Dr. Sylvie Breton, Ph.D., Co-Investigator (20% effort, 2.4 calendar months) Dr. Breton is a Professor of Medicine who has been working with Dr. Brown since 1994 on many aspects of V-ATPase trafficking and regulation in proton secreting cells of the urogenital tract. She is highly proficient in all aspects of cell biology and will also be involved in the planning an interpretation of many of the proposed studies. Dr. Breton’s lab developed the transgenic mice expressing EGFP in intercalated cells in collaboration with Dr. Raoul Nelson in Utah, and she is assisting Dr. Nair in preparing isolated intercalated cells from these mice for FACS sorting and further analysis. She will supervise many of the whole animalstudies including perfusion of kidneys in situ, intercalated cell activation and acidification assays, and the studies on V-ATPase assembly/disassembly in cell and tissues. She is the senior author on our recently published study identifyingadenosine as a stimulator of intercalated cell proton secretion, which will be extensively used in the work described here.
Dr. Maria Merkulova, Ph.D., Co-Investigator (75% effort, 9 calendar months) Dr. Merkulova is an Instructor in Medicine.She is a highly experienced molecular biologist who has been working in the PMB lab for the past 8 years. She is an experton protein interactions, and was the lead author on the V-ATPase interactome paper that identified new proteins that areassociated with this proton pump, and whose function will be examined in this application. She has several publications inwhich she has elucidated V-ATPase protein interactions using techniques used in Aims 1 and 2, including being the lead author on our recent paper describing the acidosis phenotype in NCOA7 null mice. She will be responsible for studiesinvolving cellular protein knockdown studies, expression and preparation of purified proteins, and the protein interaction assays. She has expertize in the analysis of protein expression patterns based on mass spectrometry data, and will extend this to work with the postdoctoral fellow for RNA seq analysis of isolated intercalated cells.
Dr. Anil Nair, Ph.D., Co-Investigator (25% effort, 3 calendar months) Dr. Nair is an Instructor in Medicine, and hasconsiderable experience in the imaging techniques that will be used in the application. He will work with Dr. Brown on confocal imaging and he developed the image-based acidification assays we will use, in collaboration with Drs. Breton and Merkulova. He will be responsible for the cell biological studies that involve FACS sorted intercalated cells and M-1 cells.He also has experience in animal husbandry and currently maintains our existing ATP6V1B1/EGFP hybrid mouse colony.He will be responsible for developing the new chimeric mouse strains that are proposed in this application, and supervising the producing of the DMXL1 null mouse and derivatives thereof. He will supervise the postdoctoral fellow in thesetechniques and will oversee the metabolic cage studies after acid/base challenges and adenosine injection.
B. Other Personnel
TBN, Ph.D., Postdoctoral Fellow (100% effort, 12 calendar months) We will recruit a new postdoctoral fellow who will have two sets of responsibilities. First, to carry out in vivo studies on existing and newly generated mouse models, including metabolic cage studies and isolation of GFP-positive intercalated cells. We will recruit a person with expertize in RNA seq and data analysis to continue to interrogate gene expression patterns in intercalated cells under different conditions and in different animal models. The fellow will also carry out analysis of protein expression patterns in kidneys by confocal imaging, real time PCR and immunoblotting.
Diane Capen, B.S., Senior Research Technologist (25% effort, 3 calendar months) Ms. Capen is highly accomplished in all aspects of light and electron microscopy. She will perform routine cryostat sectioning and immunostaining of cells and tissues. Her special expertise is in conventional EM, immunogold labeling and ultrathin frozen sectioning, and she will also
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perform these procedures on tissues and cells as described in the application. She also assists in routine maintenance of lab equipment and ordering supplies. Fringe benefits at MGH for professional staff are calculated at 36% and at 37% for non-professional staff. C. Equipment None D. Travel Years 1 – 5: Partial travel costs to attend the Annual American Society of Nephrology or the Experimental Biology meeting are requested for Drs. Brown, Breton, Merkulova and Nair) $5,000 per year F. Other Direct Costs 1. Materials and Supplies: $63,300 per year
Tissue Culture Supplies Cell lines (M1), minimum essential medium, fetal calf serum, plasticware, Costar transwell filters, 6-well plates, 10 cm dishes, glass bottomed petrie dishes for confocal microscopy and in vitro IC maintenance, flasks, pipettes, trypsin, antibiotics for selection and maintenance of transfected cells, LN2 for cell storage, CO2 for incubators, biosafety hood filters. $14,000 Molecular Biology and Protein Chemistry Supplies Reagents for cell transfections, site directed mutagenesis, production and purification of GST fusion proteins, pull down assays, qPCR supplies, RT-PCR supplies, western blotting supplies, standard sequencing through MGH Core, RNA seq through MGH Core. $20,500
Microscopy/Imaging Fluorescence Microscopy: Secondary affinity-purified antibodies, colloidal gold coupled to IgGs, commercial primary antibodies against antigens including cellular compartments (e. g., clathrin, EEA1, rab 5, rab11, TGN, lysosomal proteins, actin, actin, tubulin, GFP); supplies for cryostat; blocking reagents; fluorescent-dextrans; glass slides; Eppendorf tubes and freezer boxes; slide boxes; coverslips; Vectashield anti-fade mounting medium; 3 “projector” bulbs for Nikon Xcite microscope illuminators ($300 each), replacement fiber optic cables for XCite lamps ( 2 x 450 each). Electron microscopy: fixatives, osmium tetroxide, uranyl acetate, lead citrate, 100% ethanol, tannic acid, propylene oxide, horseradish peroxidase, embedding resins, glass knives, EM grids, grid storage boxes, storage racks, specimen supports for ultracryomicrotome, replacement filaments and apertures for JEOL 1011microscope, embedding tubes, storage boxes, assorted glassware, plastic syringes, desiccant, Millipore filter units for clearing EM stains, fine tweezers, filter papers. Digital imaging: updates to imaging, analysis and presentation software (Adobe Photoshop, Adobe Acrobat, Volocity, Nikon Elements, Zeiss Zen). Writeable CDs and DVDs for storage of digital images, removable storage thumb drives, 2 TB external hard drives. 25% of monthly MGH file server cost for image and video storage ($1,500/year at $6000/year, total cost). $14,800 Drugs, Chemicals, Fluorescence probes Adenosine, forskolin, cAMP, bafilomycin, concanamycin, lysosomal inhibitors, proteasome inhibitors, cycloheximide, SNARF 5-F, Lysotracker red DND-99 $6,500 General Lab Supplies Glassware, plasticware, paperware. $7,500
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2. Publications Based on our past productivity from this project, we estimate a minimum annual publication cost of $4,000 per year (2 manuscripts) 8. Other Costs Service contracts: the specialized equipment used in this work requires service and maintenance.
1. Electron microscope; 15% of the annual maintenance of $25,000 is requested = $3,750 2. Nikon A1R confocal microscope: 20% of the annual maintenance cost of $16,500 is requested = $3,300 3. Other microscopes: 20% of wide-field microscope maintenance ($1,200 per year) is requested =$240 4. Leica Cryostats: 20% of the annual maintenance cost ($3,000) is requested = $600 5. Centrifuge maintenance is $11,000/year. We ask for 20% of this related to the activities in this proposal = $2,200
Total other costs = $10,450 per year 9. Animal Costs
Mice will be used for the in vivo studies in both of the Aims, for perfusion of tissue for immunocytochemistry, western blotting and RNA analysis, for preparation of tissue slices for in vitro incubation, for the preparation of tissue extracts for pull down, cell fractionation and IP experiments, and for isolation by FACS of intercalated cells. Our new colony of NCOA7-/- will be maintained and bred in the animal faculty for the duration of the work proposed. New colonies of NCOA7 -/-/EGFP, and IC-specific DMXL1-/- mice will be produced. Breeding projections take into account the need to breed heterzygotes to produce WT and null littermates for comparison, and to avoid genetic drift due to homozygote crossing. We will use equal numbers of males and females in our experiments, and animals will be randomly assigned to control and experimental groups. We are unsure about the use of heterzygotes in our experiments and will assess their phenotype and potential utility as we move forward.
SPECIFIC AIM 1 (WT and NCOA7-/-) a) NCOA7 -/- Years 1 – 3: These mice are for expression, trafficking and functional studies in Aim 1A Confocal imaging (immunocytochemistry): 8 WT mice, 8 NCOA7-/- mice Western blotting: 8 WT mice, 8 NCOA7-/- mice In situ PLA assay ± adenosine: 24 WT mice, 24 NCOA7-/- mice (8 x 3 different time points, 0, 10, 20 mins) V-ATPase recycling, FITC-dextran injection: 8 WT mice, 8 NCOA7-/- mice Transcriptional studies: 8 WT mice, 8 NCOA7-/- mice Transalational studies (tissue slices): 4 WT mice, 4 NCOA7-/- mice Immunoprecipitation for assembly assays: 8 WT mice, 8 NCOA7-/- mice Cell fractionation ± adenosine: 24 WT mice, 24 NCOA7-/- mice (8 x 3 different time points, 0, 10, 20 mins) 92 -/- and 92 WT littermates over 3 years (123 pups needed per year x 3 = 368 in all) Breeding, maintenance and housing of NCOA7-/- mouse colony (Years 1-3)
Breeding pairs
Litters/pair
#pups/litter
#pups/ pair/ year
total pups/year
Mice/ cage
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4 2-3 12 30 120 4 30 180 5400 $1.27 $6,858 b) NCOA7 -/- Years 4-5: These mice are for colony maintenance and residual experiments as needed 2 breeding pairs, producing 60 pups
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Breeding, maintenance and housing of NCOA7-/- mouse colony (Years 3-5)
Breeding pairs
Litters/pair
#pups/litter
#pups/ pair/ year
total pups/year
mice per cage
# cages
# Housing days
Cage days
Per diem/ cage
Total/ year
2 2-3 12 30 60 4 15 180 2700 $1.27 $3,429 c) NCOA7-/-/EGFP+ Years 2 - 4: these mice are for experiments using isolated ICs in Aims 1C, D and E V-ATPase protein and RNA expression: 8 WT mice, 8 NCOA7-/- /EGFP+ mice Recovery from acid load: 8 WT mice, 8 NCOA7-/- /EGFP+ mice V-ATPase trafficking assay: 8 WT mice, 8 NCOA7-/- /EGFP+ mice Cell fractionation: 8 WT mice, 8 NCOA7-/- /EGFP+ mice RNA seq: 8 WT mice, 8 NCOA7-/- /EGFP+ mice (4 RNA seq runs from each genotype) Seahorse flux analysis: 8 WT mice, 8 NCOA7-/- /EGFP+ mice (4 Seahorse runs from each genotype) 48 NCOA7-/-/EGFP+ and 48 WT litternates over three years (64 needed per year x 3 = 192 in all) Breeding, maintenance and housing of NCOA7-/-/EGFP mouse colony (Years 2 - 4)
Breeding pairs
Litters per pair
#pups/litter
#pups/ pair/ year
total pups/year
mice per cage
# cages
# Housing days
Cage days
Per diem/ cage
Total/year
3 2-3 12 30 90 4 23 100 2300 $1.27 $2921 d) NCOA7-/-/EGFP+ Year 5: These mice are for colony maintenance and residual experiments as needed 2 breeding pairs, producing 60 pups Breeding, maintenance and housing of NCOA7-/-/EGFP mouse colony (Years 4-5)
Breeding pairs
Litters per pair
#pups/ litter
#pups/ pair/ year
total pups/year
Mice/cage
# cages
# Housing days
Cage days
Per diem/ cage
Total/ year
2 2-3 12 30 60 4 15 180 2700 $1.27 $3,429 SPECIFIC AIM 2 (WT and DMXL1-/-) a) WT Mice Years 2 - 3: These mice are for the trafficking and assembly assays in Aim 2A Confocal, EM imaging and PLA assay 24 WT mice, 24 DMXL1-/- mice (8 x 3 different time points, ± adenosine) IP assembly assays: 24 WT mice, 24 DMXL1-/- mice (8 x 3 different time points, ± adenosine) V-ATPase recycling, FITC-dextran injection: 16 WT mice, 16 DMXL1-/- mice (1 time point ± adenosine) Purchase of 112 WT mice over two years (66 per year at $24 each = $1584 per year) b) DMXL1 -/- Years 2 - 3: These mice are to examine the role of DMXL1 in vivo in Aim 2B Production of DMXL1-/- mouse at HMS Transgenic Core = $7,000 Confocal, EM imaging and PLA assay 24 WT mice, 24 DMXL1-/- mice (8 x 3 different time points, ± adenosine) IP assembly assays: 24 WT mice, 24 DMXL1-/- mice (8 x 3 different time points, ± adenosine) V-ATPase recycling, FITC-dextran injection: 16 WT mice, 16 DMXL1-/- mice (2 time points ± adenosine)
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64 WT, 64 DMXL1 in two years (256 total pups needed, 128 per year, in all to obtain 64 WT and 64 null mice) Breeding, maintenance and housing of DMXL1-/- mouse colony (Years 2-3)
Breeding pairs
Litters per pair
#pups/litter
#pups/pair/ year
total pups/year
mice per cage
# cages
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Per diem/ cage
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5 2-3 12 30 150 4 38 180 6840 $1.27 $8,686 c) DMXL1 -/- Years 4 - 5: These mice are for maintenance of the colony and residual experiments as needed 2 breeding pairs, producing 60 pups Breeding, maintenance and housing of DMXL1-/- mouse colony (Years 4-5)
Breeding pairs
Litters per pair
#pups/litter
#pups/pair/ year
total pups/year
mice per cage
# cages
# Housing days
Cage days
Per diem/ cage
Total/year
2 2-3 12 30 60 4 15 180 2700 $1.27 $3,429 d) DMXL1-/- Years 4 - 5: These mice will be used for studies on isolated ICs lacking DMXL1 in Aim 2B Cell fractionation: 8 WT mice, 8DMXL1-/- mice V-ATPase protein and RNA expression: 8 WT mice, 8 DMXL1-/- mice Recovery from acid load: 8 WT mice, 8 DMXL1-/- mice V-ATPase trafficking assay: 8 WT mice, 8 DMXL1-/- mice RNA seq: 8 WT mice, 8 NCOA7-/- mice (4 RNA seq runs from each genotype) 80 WT, 80 DMXL1-/- in two years (320 total pups needed, 160 per year, in all to obtain WT and null mice) Breeding, maintenance and housing of DMXL1-/- mouse colony (Years 4-5)
Breeding pairs
Litters per pair
#pups/litter
#pups/pair/ year
total pups/year
mice per cage
# cages
# Housing days
Cage days
Per diem/ cage
Total/year
5 2-3 12 30 150 4 38 180 6840 $1.27 $8,686 Total animal cost/year Year 1 = $6,848 housing Year 2 = $18,465 housing; $1,584 purchase; $7,000 HMS transgenic facility Year 3 = $18,465 housing; $1,584 purchase Year 4: 18,465 housing Year 5: 18,973 housing
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Major Equipment of Program in Membrane Biology
A. Microscopy, digital imaging1 Zeiss LSM800 confocal microscope with Airyscan “super”resolution capability1 Nikon A1R confocal microscope with a 4-‐lasermodule, equipped with an incubation chamber andperfusion system for live cell imaging1 Perkin Elmer Spinning Disk Confocal Microscopeequipped with an incubation chamber and perfusionsystem for live cell imaging1 Nikon Total Internal Reflection (TIRF) imagingsystem with Nikon TE2000U inverted microscopeand 37°C chamber and perfusion system. Alsoequipped with dual excitation system for Fura-‐2 andBCECF ratio imaging1 FRET imaging and analysis system (TILLPhotonics)1 Nikon 90i Microscope with Motorized X-‐Y-‐Z stage,6 Position Fluorescence Turret, and Digital ImagingHead driven by Nikon Elements Advance ResearchSoftware1 Nikon Eclipse 80i fluorescence microscopes withHamamatsu Orca camera1 Nikon TE300 invertedmicroscope equipped withHamamatsu Orca camera and shutter system for realtime fluorescence and ratio imaging measurements.1 JEOL 1011 electronmicroscope with digital camera3 Computer imaging stations with ImprovisionVolocity software, NIS element AR3.02 Leica Ultracut EMUC7 microtomes1 Leica Ultracut S ultracryomicrotome1 Leica EMFCS ultracryomicrotome2 Leica CM3050S cryomicrotomes1 Pelco101 Vibratome Series 1000 sectioning system1 Cressington freeze-‐fracture apparatus3 Olympus inverted microscopes1 Olympus BH2 upright teaching microscope1 X-‐O-‐mat facility (shared)1 Olympus BH2 fluorescence scope with Spot camera1 Leica AFS freeze-‐substitution system1 Cell microinjection system (Femtojet) withmicromanipulation (InjectMA NI.2)
B. Tissue Culture1 Vapor pressure osmometer (Vapro5520)1 Electro square porator (ECM830)5 ClassII A/B3 biological safety cabinet1 Primary cell culture (BL1/BL2) (9 x 9 ft)1 Autoclave facility (shared)7 Nitrogen Dewars 35 litres2 Cell culture room (BL1/BL2) (15 x 12 ft)10 Forma Scientific water jacketed CO2 incubators2 Shakers for hybridoma and suspension cells
C. Spectroscopy and fluoroscopy1 DTX 800 multimode detector (fluorescence
intensity, absorbance (visible) and glow luminescence for 96-‐ to 384-‐well plates) 1 Luminometer (Centro xs LB960) 2 Bio-‐rad smart spectrophotometer 3000 1 Hi-‐Tech Scientific SFA-‐20 Stopped-‐Flow Accessory with Photo Technology dual wavelength Spectrofluorometer
D. FPLC and Electrophoresis1 AKTpurifier system (FPLC) with automatic fraction collector and UPC-‐900 detector 1 LKB isoelectric focusing apparatus 2 Tube gel IEF 2-‐D systems 10 Bio-‐rad mini-‐trans-‐blot cell 1 Bio-‐rad semi-‐Dry transfert system 2 Invitrogen iBlot Dry blotting system 1 peristaltic pump 1 Bio-‐rad gradient former 1 Cell cracker homogenizer
E. Molecular Biology1 MMI Cellcut laser microdissection system 1 Applied Biosystems 7300 Real Time PCR 1 ABI Prism 7900HT sequence detection system 1 UVP Gel bioimaging system 1 Agilent 2100 BioAnalyser 8 PCR thermal recyclers (Perkin Elmer) 1 Electroporation -‐ Cellzap II 1 Isotemp standard lab incubator 2 Vacuum oven, 1.5 cu ft. 1 Incubator shakers (orbit/environmental) 1 Fotodyne, UV MP-‐4 system
F. In vivo study apparatus1 Small animal surgical procedure room with biosafety hood 1 TEC-‐3 anesthesia machine for small animals 12 Rodent metabolic cages 1 TEC-‐3 isoflurane vaporizer systems 1 Non-‐invasive blood pressure tail cuff system 1 Vapor pressure osmometer (Vapro5520) 4 Syringe Minipump unit for microinjections 5 High intensity illuminator 1 Dissecting microscopes with high power magnification 2 Chirurgical dissection microscopes 1 In vivo heat controlled perfusion system with peristaltic pump. 1 In situ tissue dissecting system
G. Radiolabeing and ligand binding measurement1 Commissioned radioactive room with hoods 1 Freezers -‐20°C 1 Refrigerator 1 Scintillation counter (LS6500) 1 Gamma counter SYS W/ACC MINAX 1 Geiger counter (Ludlum model 3) with GM probe 1 Filtration binding systems
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H. General Laboratory Equipment 3 Mettler AE 200, Sartorius and OHAUS Balances 1 Mettler Micro M3 Balances 1 Ultracentrifuges (Beckman L8-‐80M) 1 Tabletop ultracentrifuge (Beckman Optima TLX) 1 Medium speed centrifuge (Sorvall RC5C) 1 Medium speed centrifuge (Beckman Avanti JE) 1 Low speed centrifuge (Beckman J6-‐MC) 3 Tabletop centrifuges (Beckman Allegra X-‐22) 9 Tabletop eppendoff microcentrifuges 2 Cold room facilities (12’x12’) 26 Refrigerator/Freezer, 5.6 cubic feet (under bench) 10 Freezers upright (-‐20° C) 7 Freezers upright (-‐85° C) 2 pH meters 4 Tabletop orbital shaker 5 Tabletop orbital rotator 6 Tabletop rotator 12 Water baths 1 Milli Q pure water systems 1 Polytron Generator, Brinkmann 1 Sonicator ultrasonic processor XL 1 Ice machine I. Computer Facilities 1 Appleshare network with file server housing Prism, Volocity, Element, IPlab softwares 35 Macintosh computer stations 5 PC computers 5 Laser printers 2 Deskjet color printers (Epson or HP) 1 Epson high definition flat bed gel and photo scanner
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Other FACILITIES AND OTHER RESOURCES
Description of the Scientific Environment: The Principal Investigator and his colleagues are all full-time faculty members of the Program in Membrane Biology
(PMB), Center for Systems Biology (CSB), Division of Nephrology at the Massachusetts General Hospital and Harvard Medical School (HMS). The Scientific Environment of the PMB/Division of Nephrology/CSB and HMS is outstanding, and this environment will greatly contribute to a high probability of success for this project, as detailed below.
Institutional Support: The Massachusetts General Hospital (MGH) has a long-standing tradition of supporting innovative basic and clinical research. The research efforts in the PMB/CSB/Division of Nephrology, and the MGH at large are actively supported by an administrative body dedicated to the preservation of strong scientific research at this institution. This organization includes the Executive Committee on Research (ECOR) and a Scientific Advisory Committee composed of distinguished scientists from across the country. ECOR plays an integral role in strengthening scientific training and expanding the research programs at MGH. The PI, Dennis Brown is Director of the PMB and is a full voting member of ECOR. In 2007, ECOR established a new Office of Research Career Development to re-emphasize the commitment of MGH to its research faculty, both M.D. and Ph.D. and Dr. Brown is the current Director of this office. Furthermore, the fostering of interaction among researchers and scientists within Harvard University and other hospitals and institutions in the Boston metropolitan area has always been a strong part of the scientific endeavor at MGH. These interactions contribute to the highest level of state-of-the-art science at this Institution. The PMB is a subgroup within the Division of Nephrology. It is independently funded and occupies 8,000 square feet of dedicated laboratory research space and modern, state-of-the art equipment. The MGH has recognized the PMB as a major research program within the hospital and has allocated tremendous resources to ensure its continued success and growth, including over $X for the purchase of new laboratory equipment, plus $X to help purchase a new microscope system (Total Internal Reflectance microscopy), $X towards a new Nikon 90i automated stage microscope, $X to upgrade the computational power and purchase 3D image rendering and analysis software for the Spinning Disk Confocal Microscope, as well as other major pieces of equipment. The MGH provided $X to Dr. Brown for the purchase of a 4 year service contract (2015-2018) on its Nikon A1R confocal microscope and just this year with another $X to maintain the new Zeiss LSM800 Airyscan system. The PMB retains close ties with the Division of Nephrology, and Dr. Brown works closely with the Interim Chief of the Division, Dr. Eugene Rhee, to integrate the clinical and scientific responsibilities of members in the Division of Nephrology, including issues of faculty development.
Most of the proposed work in this project will be achieved using available resources within the PMB, and additional resources will be provided by our collaborator in the UK, Dr. Peter Oliver (see letter of collaboration).
Intellectual Rapport: This project will benefit greatly from the intellectual rapport within the PMB, and the Division of Nephrology. The extensive microscopy Core facility within the PMB will be a nucleating force and in addition, the PMB has collaborative work with a number of other groups, allowing for a rich source of interaction not only at the MGH but also throughout the Boston area. In addition to the specific personnel named in the application, other PMB members have contributed greatly to the success of the PMB as a whole over the years. These include Dr. Nicolas Da Silva, who is an imaging expert, Dr. Jodie Babitt, who is an accomplished cell physiologist and practicing nephrologist working on iron metabolism and who is an expert in luciferase promoter assays, and Drs. Jenny Lu and Richard Bouley who are long time colleagues of Dr. Brown working mainly on the cell biology and signaling pathways regulating aquaporin trafficking. The PMB/CSB/Division of Nephrology offer numerous educational forums to encourage intellectual growth and development in basic or clinical research, including weekly PMB research seminars, bi-weekly CSB research seminars, and weekly Division of Nephrology research seminars, in addition to grand rounds. At these seminars, trainees, faculty members and invited speakers present and discuss their published and unpublished data. These sessions provide invaluable discussion from all members of the groups on their individual research projects. In addition, individual lab meetings of PIs are held each week to discuss data and troubleshoot problems. Daily interaction with other investigators is assured by the “no-walls” design of the labs, and the open laboratory plan facilitates interaction and discussion among researchers at all levels. Not surprisingly, many collaborative publications arise from the interactions that “naturally” occur in this interactive environment.
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Resources: Laboratory: The proposed research will be performed in laboratory space in the Simches Research building that has been occupied by the PMB since August 2005. The space is contiguous on one floor and consists of 8,000 sq. ft. of dedicated research space including two confocal microscopy suites, an electron microscopy suite, 3 fluorescence microscope rooms, 3 tissue culture rooms, a whole animal physiology procedure room, a 6-bay microtome room, laser microdissection and FRET imaging suites, designated rooms for biochemistry (including 2 cold rooms), a dedicated tissue embedding room, and wet lab benches and desks for 36 staff. There are 8 offices, a conference room and an administration area. The PI is Director of the PMB and its Microscopy and Cell Culture Core facilities. The technologist associated with this proposal has her own desk and bench, in addition to workspaces within individual equipment rooms.
Services and safety issues: The Environmental Health & Safety office at the MGH ensures the safety of all patients, visitors, and clinical and research staff in all MGH locations, including research laboratories. The EHS provides services related to biosafety, environmental compliance, waste disposal, industrial hygiene, laboratory safety, fire and life safety, and they provide training for employees. The EHS highly qualified personnel provide guidance on how to identify hazardous chemical wastes, and how to dispose safely of toxic wastes. Relevant to this application, the disposal of paraformaldehyde, glutaraldehyde and mercury is under very strict rules, and the MGH must comply with the requirements of the Massachusetts Water Resources Authority (MWRA). These compounds are discarded in separate appropriately labeled bottles. Our laboratory is equipped with three satellite waste disposal areas located within biosafety fume hoods. The waste is picked up weekly and disposed of by the hospital's sub-contracted chemical waste company, Triumvirate Environmental. The EHS at MGH also provide regular mandatory training sessions on various safety issues, including the handling of radioactive material and fire. One of the technologists (not specifically named on this application) in the PMB, Robert Tyszkowski, is an accredited member of the MGH Hazmat team and has numerous local and National licenses and certificates related to his expertize in chemical, radiation and biohazards. He serves this important function for the entire PMB program. He will instruct investigators in the proper use and disposal of protective equipment such as gloves, masks, goggles and lab aprons.
Animal: The Simches Research Building has an AALAC-approved viral-free animal facility fully equipped for small and large mammals. It has several full-time veterinarians plus 6 trained staff and animal technicians.
Computer: PMB staff all have iMac computers, fully networked for access to e-mail, library dbs, gene data banks. Filemaker Pro provides easy access to a fileserver from CPUs to experimental protocols, incubation records, digital images, available antibodies, cDNA reagents, plasmids etc. All computers are connected via a file server with Perkin Elmer Volocity software for image analysis through a multi-user site license.
Offices: The PI and Dr. Breton each has an approx. 144 sq ft. office; and Drs. Nair and Merkulova share a 120 sq. ft office.
Other: Major Equipment: A list of major equipment residing in the PMB space is attached separately.
Biostatistics support: The MGH has an established Biostatistics Group, Directed by Prof. Diane Finkelstein, to help researchers with biostatistical analysis as needed during experimental design and post-experimental analysis. This office receives support from many individual programs and grants within the MGH, as well as a contribution of $x from MGH ECOR. This office provides biostatistics support on request for all investigators at MGH, and we in the PMB routinely use their services for initial power calculations and subsequent data analysis.
Major Equipment in the PMB relevant to this application:
1 J EOL 1011 electron microscope with fully digital image capture & processing software 1 Zeiss LSM800 confocal microscope with Airyscan, plus grayscale and color CCD cameras 1 Nikon A1Si R confocal microscope equipped with a 4-laser module and environmental chamber for live imaging
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1 Nikon TE2000U inverted microscope with TIRF capability and Calcein, BCECF and Fura-2 attachments 1 Nikon 80i upright epifluorescence microscope with Hamamatsu camera 1 Nikon 90i with Hamamatsu CCD camera, motorized X-Y-Z stage and 3D analysis software 1 Olympus doubleheaded light microscope for section examination 2 Leica CM3050S cryomicrotomes for routine 4-20 µm cryosections 1 Leica EMFCS ultracryomicrotome for ultrathin frozen sectioning 2 Leica Ultracut EMUC7 microtomes for routine EM sectioning 1 AKTpurifier system (FPLC) with automatic fraction collector and UPC-900 detector 1 Tabletop ultracentrifuge (Beckman Optima TLX) 1 Luminometer (Centro xs LB960) 1 Applied Biosystems 7300 Real time PCR analysis system 1 Commissioned radioactive room with hoods
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PROTECTION OF HUMAN SUBJECTS
Risk to Human Subjects Risks of blood draws: The patient may have a bruise or pain at the site where the blood sample is
taken. There is also a small risk of infection, lightheadedness, and/or fainting. Risks of intranasal theophylline treatment: Theophylline administered via nose spray is generally well
tolerated. However, taking theophylline may cause one or more of the side effects listed below. Common side effects:
Headaches Restlessness
Less common side effects: Nausea Gastrointestinal tract discomfort Irritation of nose
Uncommon side effects: Tremors Seizures Sleep disturbances Palpitations
There may be other risks of theophylline administered as nose spray that are currently unknown. As with any drug, an allergic reaction can occur. Allergic reactions can be mild or more serious, and
can even result in death. Common symptoms of an allergic reaction are rash, itching, skin problems, swelling of the face and throat, or trouble breathing.
Because of unknown risks of theophylline treatment to an embryo or fetus, or to a breastfeeding infant, women cannot take part in this study if they are:
Pregnant Trying to become pregnant Breastfeeding
Informed Consent The patients who agree to participate in the study will be contacted by a Clinical Research Coordinator
for consent. Designated trained research personnel will ensure that all aspects of the study are explained to the participant and will answer any questions related to the study. Participants will be informed that they are free to withdraw their consent and discontinue their participation in the study at any time without prejudice.
Subject Confidentiality/Privacy Following informed consent, subjects will be assigned a unique study identifier upon entry into the
clinical research data capture software. All data and samples will be identified using a numerical codeassigned to individual subjects without access to the full identity of subject.
Participant medical information obtained during the study is confidential and disclosure to thirdparties is prohibited. Only study investigators and authorized staff from the study site’s IRB will have access to research records.
No identifying participant information, including names, will be disclosed in reports, publications, or presentations. At the participant’s written request, medical information may be given to his/her personal physician.
Plans to Inform Subjects of Study Results During the course of the study, participants may be informed of any significant new findings (either
positive or negative) which a reasonable person might consider important when deciding whether to participate or not.
Protection of Human Subjects Page 49
Contact PD/PI: Paunescu, Teodor G.
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Foreseen Future Uses of Personal Data The rights and privacy of participants who participate in research must be protected at all times. The
data and associated documentation will be available to users only under a data-sharing agreement that provides for: (1) a commitment to using the data only for research purposes, and not to identify any individual participant; (2) a commitment to securing the data using appropriate computer technology; and (3) a commitment to destroying data 3 years after research completion.
Data Security All data and samples will be identified using a numerical code assigned to individual subjects without
access to the full identity of subject. Data capture will be conducted using a secure and HIPAA-compliant tool.All study data will be housed electronically in a password-protected and encrypted computer, accessible only by study investigators. This computer has additional security safeguards, such as automated operating systempatch (bug fix) management, anti-virus controls, firewall configuration, and scheduled and automatic back-ups to protect against data loss or theft. Data records will be retained for 3 years after research completion and will be subsequently destroyed by multiple overwrite steps.
Potential Benefits of the Proposed Research To Human Subjects and Others The study population will be inclusive of all adult patients without any restrictions in regard to gender,
race, age, and socioeconomic status. There are no direct benefits to subjects used in this study. It is possiblethat their smell and taste sensations may improve while they are taking the study medication. Other hemodialysis patients, peritoneal dialysis patients, and patients with non-dialysis requiring chronic kidneydisease with smell disorders may benefit in the future from what we learn in this study.
Importance of the Knowledge to be Gained Since no effective treatments are currently available to address malnutrition and cachexia in patients
with advanced renal disease, and no intervention has yet been tested in a clinical trial to improve olfactory function in this patient cohort, knowledge to be gained from our proposed research is of critical importance.
Data and Safety Monitoring Plan Given the adverse effects reported in a previous study with oral theophylline (and described above
under “Risk to Human Subjects”), we decided to have a data safety monitoring board (DSMB), even if noadverse events were attributed to nasal theophylline. Moreover, serum theophylline levels were undetectablethroughout the study period in all 3 ESRD patients that we enrolled in a pilot clinical trial (CT.gov:NCT02479451). These study subjects tolerated nasal theophylline well and no adverse events were reported at 2, 4, and 6 week follow-ups.
Subjects will have 24-hour telephone access to a physician member of the research team to report any concern for adverse events during the study. Caring clinicians will be provided with a standard three-category (probably related, probably not related, unknown) adverse event form and all adverse events will be reviewed by the DSMB. A Clinical Study Monitor who is not an investigator in the study will monitor in real-timereturning laboratory results and adverse events and will communicate with the DSMB.
The DSMB, including 2 clinicians and 1 statistician, will assess potential adverse events as deemed necessary by the study monitor. An interim analysis for this study is not planned, however the DSMB can stop the study at any time, and the Study Monitor will be in constant communication with the DSMB Chair. A DSMB Charter will be developed, conflicts will be reviewed in detail, and two experienced individuals will serve on the committee. We have extensive previous trial experience in developing DSMB charters, as discussed in the application (2.1.I.)
An initial meeting of the DSMB will be held prior to any participant enrollment in order for the members to review the charter, form an understanding of the protocol and definitions being used, establish a distribution and meeting schedule, review the study modification and/or termination guidelines, and finalize format and protocol-specified statistical methods to be used in reports to be considered by the DSMB. Subsequent DSMB meetings will be held to review and discuss study data as described in the application
Protection of Human Subjects Page 50
Contact PD/PI: Paunescu, Teodor G.
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(Table 7). Any adverse events will be immediately reported to the Institutional Review Board and the funding agency. Publication
We will only publish aggregated results to protect the identity of individual patients. We mandate each aggregated result contain at least 15 subjects to prevent the possibility of re-identification. No identifying participant information, including names, will be disclosed in reports, publications, or presentations.
Protection of Human Subjects Page 51
Contact PD/PI: Paunescu, Teodor G.
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RIGOR AND REPRODUCIBILITY: We have received continuous funding from NIH for more than 25 years, and have aspired to generate robust and reproducible data, using an array of complementaryprocedures to examine biological processes, in vitro and in vivo. In addition, as an editor of anAmerican Physiological Society publication, the PI has contributed to new author guidelines for APSjournals that are intended to ensure the maximum level of reproducibility and rigor in submittedmanuscripts. Among the important factors considered are:Animal studies: we will follow the ARRIVE guidelines for reporting animal studies (Species, strain,sex, age, source, genetic modifications, housing, diet); controls used in the study (littermates, whethermice are purchased or not, conditions of experiment); precise details of procedures (drug formulation,dose, anesthesia, analgesia, euthanasia); minimization of bias (blinding of researchers, randomization).Sex as a biological variable: We are very conscious of this issue: in work presented at the ASNmeeting in 2016, we found significant sex differences between the urine concentrating capacity of micein response to V-ATPase B1 subunit knockout and dehydration (48). Furthermore, we published apaper describing sex differences in aquaporin 1 expression in rat proximal tubules (29). Therefore, wehave actively pursued studies describing differences in the physiology of male and female mice. Thiswill continue in the current application. Age-matched adult males and females will be included in ourexperimental groups for all initial experiments. Only if no significant differences are found will weresort to using males alone, a strategy recommended by NIH during numerous informational events.Statistical analysis: Based on power analysis performed in collaboration with the MGH BioStatisticsGroup led by Prof. Diane Finkelstein, the appropriate number of animals (or cell culture plates) neededto generate statistically significant data showing 20-40% changes in a measured parameter for most ofour assays is between 6 to 8 per treatment group. This calculation was completed based upon some ofour preliminary data (e. g., Fig. 15) showing a 15-45% change in V-ATPase assembly at a power of96% at 5% type 1 error rate (two-tailed P < 0.05, n = 5). A stringent statistical analysis will always beperformed: ANOVA followed by post-hoc Bonferroni or Tukey’s tests for multi-group analysis, andStudent t test for paired or unpaired samples when only two groups are included. Collection, analysisand interpretation of data will be conducted in a “blinded manner” in de-identified samples. This isparticularly important for interpretation of semi-quantitative data.
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VERTEBRATE ANIMALS
All animal studies will be performed at the Massachusetts General Hospital (MGH). Animals will be housed in an AAALAC-approved virus-free animal house in the MGH Simches Research Building according to institutional guidelines. There are 6 full-time veterinarians who monitor the care of the animals on a daily basis. An application for the use of animals for all of the procedures to be used in this proposal was submitted and approved by the MGH Subcommittee on Research Animal Care (SRAC) office. No additional animal studies will be performed prior to obtaining approval.
1. Provide a concise description of the proposed procedures to be used that involve vertebrate animals in the workoutlined in the “Research Strategy” attachment. Identify the species, strains, ages, sex, and total numbers ofanimals by species, to be used in the proposed work.
Aim 1A) Heterozygous female NCOA7 knockout (KO) mice (NCOA7+/-) on a mixed C57Bl/6 x 129Sv background will be crossed with male NCOA7+/-) KO mice on a mixed C57BL/6 x 129Sv background to generate doubleknockouts (NCOA7-/-), heterozygotes (NCOA7+/-), and WT mice at an expected 1:2:1 ratio. We will use homozygous KO mice and littermate WT mice of both sexes for our studies. Cohorts of n=6-8 mice per gender per genotype will be usedbetween 14-16 weeks of age, and kidney tissues will be analyzed by confocal imaging, western blotting, in situ PLAassay, V-ATPase recycling assay, protein and RNA expression studies, immunoprecipitation and cell fractionation studies. Mouse numbers were chosen based on our prior experience with similar studies on V-ATPase regulation to give90% power to detect differences between groups with significance level of 0.05. Total n = 368 mice over 3 years,including mice needed for breeding and mice of undesirable genotype. In years 4-5 we will use two breeding pairs forcolony maintenance and generate 60 pups per year. If necessary (e.g., to perform addition studies required by reviewers orto follow up on new data generated in the first 3 years), we can expand the colony as necessary.
We will generate NCOA7-/-/B1-EGFP chimeric mice in order to isolate ICs that are devoid of the NCOA7 protein.These cells, from male and female mice, will be used for various functional assays in vitro after cell isolation by FACS aswe have previously described for WT B1-EGFP mice. We propose to use 192 mice over a three year period (Years 2-4).Mouse numbers were chosen based on our prior experience of the number of ICS that we can isolate from each mouse byFACS, so that sufficient cells can be used in vitro to give 90% power to detect differences between groups withsignificance level of 0.05. The number of cells required per assay differs according to the techniques used. In year 5 wewill use two breeding pairs for colony maintenance and generate 60 pups per year. If necessary (e.g., to perform additionstudies required by reviewers or to follow up on new data generated in the 3 previous years), we can expand the colony asnecessary.
Aim 2A) Male and female B6129SF2/J wild type mice, 12-14 weeks old, will be purchased from Jackson Labs. for use in our proposed V-ATPase trafficking and assembly assays. We propose to use 112 WT mice over two years (years 2 and 3) for these experiments. Mouse numbers were chosen based on our prior experience with similar studies to give 90% power to detect differences between groups with a significance level of 0.05.
Aim 2B) We propose to use the Harvard Transgenic Core facility to generate a IC-specific DMXL1 knockout mice in order to examine the role of this protein in renal acid/base homeostasis, secondary to its effect on IC function. Anappropriate floxed DMXL1 targeting vector is available from the EUCOMM. They will be used for confocal and electron microscopy imaging, V-ATPase assembly assays, and V-ATPase recycling assays in vivo. We will need to generate atotal of 256 mice over a two year period (years 2-3) for our proposed studies by crossing B1-Cre mice (already in hand)with the IC-specific DMXL1flox/flox mice generated by the HMS Core. In years 4-5 we will use a minimum of two breeding pairs for colony maintenance and generate 60 pups per year. If necessary (e.g., to perform addition studies required by reviewers or to follow up on new data generated in the first 3 years), we can expand the colony as necessary.
Further DMXL1 null mice will be generated to isolate ICs that are devoid of the DMXL1 protein using c-kit affinity isolation (see application for details). These cells, from male and female mice, will be used for various functional assays in vitro after cell isolation by FACS as we have previously described for WT B1-EGFP mice. We propose to generate 320 mice over a two year period (Years 4 - 5). Mouse numbers were chosen based on our prior experience of the number of ICs that we can isolate from each, so that sufficient cells can be used in vitro to give 90% power to detect differences between groups with significance level of 0.05.
Total numbers of mice for the above studies is anticipated to be 1608 over the 5 year period (including mice needed for breeding and mice of undesirable genotype). This number is based on the minimum number of animals to generate statistically valid conclusions (generally n=6/8 per sex per group, based on our prior experience using mouse models to study acid/base homeostasis in vivo and in vitro, to give 90% power to detect differences between groups with significance level of 0.05). Experiments will be performed and reported in both male and female mice. We know that
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mice deficient in the V-ATPase B1 subunit show sex differences in water balance, but so far we have not clearly detected sex difference in acid/base balance. Additional factors contributing to the numbers include the necessity for several rounds of breeding and extra mice of undesirable genotypes that will be generated in the course of our studies in order to generate the desired genotypes of KO and EGFP-expressing strains as outlined above.
2. Provide justification that the species are appropriate for the proposed research. Explain why the research goals cannot be accomplished using an alternative model (e.g. computational, human, invertebrate, in vitro). Mice are used as the experimental animal of choice because of the need to make genetically modified and knockout animals in our studies on the mechanisms of whole animal acid/base balance. The in vivo studies on intact mice are important to determine the effect of the proteins we are studying on kidney and whole animal physiology, but we have kept the numbers of mice used to the minimum needed to attain sufficient power to reach our goals. The use of isolated intercalated cells from WT and KO mice will allow us to perform an increased number of replicates compared to the entirely in vivo studies, because the approximately 100,000 cells isolated from 1 or 2 mice can be used to test several conditions in vitro. There are no suitable “permanent” in vitro cell models of intercalated cells (which is why we need to use isolated primary ICs), but some cells such as the M-1 kidney cells we proposed to use have some features that we will take advantage of to reduce the number of animals used. These are explained in the text, but include the expression and knockdown of V-ATPase subunits of interest, as well as the NCOA7 and DMXL1 interacting proteins that we focus on in this application. 3. Describe the interventions including analgesia, anesthesia, sedation, palliative care and humane endpoints that will be used to minimize discomfort, distress, pain, and injury. Where appropriate, animals will be anesthetized with an intraperitoneal injection of pentobarbital (45mg/kg body weight) or with inhaled isoflurane 1-3% prior to any procedures to minimize discomfort, distress, and pain. At the appropriate time points, animals will be euthanized by intracardiac perfusion of chemical fixative (eg. paraformaldehyde), exsanguination under anesthesia with Isoflurane or pentobarbital, or pentobarbital overdose (200mg/kg IP) consistent with the recommendations of the AVMA Panel on euthanasia. We do not anticipate any unalleviated discomfort, distress, pain, or injury during this study. No survival surgery is proposed.
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Dear Dennis,
I continue to be very excited by your finding that the TLDc family proteins NCOA7 and OXR1 interact with the V-ATPase. My group have been working with TLDc proteins for over ten years and I was very interested to read your paper in Scientific Reports that first showed these new data and therefore very pleased that you contacted me to initiate a collaboration.
Our work together has already been productive with the recent publication of our finding that Ncoa7 null mice have a renal phenotype of dRTA. This is the first time that these proteins have been implicated in renal function, largely because no-one has previously looked at this possibility. As we discussed, I am very eager to collaborate with your group on the V-ATPase association with TLDc proteins because I believe this interaction may have important implications for synaptic vesicle recycling and cell function in the CNS - my area of interest - which of course involves vesicle acidification. We have little idea as to the mechanism of action of the TLDc proteins in oxidative stress or in any other function, and your data provide an entirely new research avenue for us that had not been envisaged previously.
That being the case, I am delighted to say that we have used the anti-V-ATPase antibodies that you sent to us and confirmed your data showing that TLDc family members interact with the V-ATPase in the nervous system. Thus, we are on track to pursue this novel finding and now can now work together to identify the precise domains involved in this association, and to examine the functional consequences of this interaction.
In this respect, I have already sent to you the full-length NCOA7 construct that you are modifying to transfect cultured cells. It can also be used to prepare the specific NCOA7 domains that you propose to express, as well as being used for in vitro translation of protein domains for association assays. My group has also succeeded in making an Oxr1 null mouse, which I can also share with you for your control experiments. Although OXR1 is not your main focus, this gene may be of some interest as a homologous TLDc family member expressed in the kidney. We also have appropriate constructs for OXR1 expression in cell culture.
Professor Peter L. Oliver Department of Physiology, Anatomy and Genetics
University of Oxford Parks Road
OXFORD OX1 3PT
United Kingdom Tel: +44 (0)1865 285861 / 64
Email: [email protected]
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In summary, I am happy to help I any way I can to maximise the chances of success. I wish you good luck with you application, and look forward to our continued productive collaboration.
Best regards,
Peter Oliver
