Análisis de resultados de las concentraciones de emisiones de

Experimental strategy

The aims of this thesis are to investigate the biology of galectin-3 in normal and cystic renal development, with particular emphasis on autosomal recessive polycystic kidney disease (ARPKD). Previous studies had shown that galectin-3 is up regulated in aberrant human kidney development, such as, renal dysplasia and polycystic kidney disease. In order to investigate further these preliminary results I focused on the normal expression patterns of galectin-3 throughout nephrogenesis. W ith this information I could now investigate the abnormal expression patterns, and begin to elucidate the significance of any alterations.

Rationale

If I could map the galectin-3 expression patterns, and prove that this was an essential component for normal development, then I could compare what was happening in aberrant development with normal development.

My initial step was to establish reliable sources of normal and abnormal tissue, we were given permission from the Home Office to establish mouse colonies from the background strain, C57/B1. 6J and c p k /c p k mouse, which is the best chracterised mouse model for ARPKD. Both colonies could be maintained simultaneously, and housed on site for accessibility.

Next I aimed to localise the lectin at different developmental stages (mesonephros to adult kidney), and then plan in vitro studies to investigate the effects of the lectin on metanephrogenesis. My specific aims were to investigate:

1. The origin of the different cells which expressed galectin-3 in normal development, and their fate in the normal adult kidney.

2. Establish cpk heterozygote breeding pairs to provide F I progeny that were both homozygous recessive (affected), to provide fresh abnormal kidneys, and heterozygotes (carriers), to continue the colony and provide normal material.

3. The origin of the different cells which expressed galectin-3 in aberrant development, and their fate in the adult kidney.

4. Develop techniques for in vitro cyst culture and cystic epithelial monoculture. Both cultures could be manipulated to look at effect of galectin-3 on cell characterisation, cyst morphology and quantity.

5. Assess the effects of Paclitaxel (a microtubule stabilising agent, which slows the progression of cystogenesis) on galectin-3 expression in abnormal

development in vivo and in cystic culture in vito.

6. Study the potential role of galectin-3 in a folic acid-induced model of acute nephrotoxicity.

Summary

My overall aims, were to establish where galectin-3 is expressed, and how this pattern alters in aberrant development when compared to normal development. Is galectin-3 essential for normal development, and if yes, what aspects of its bioactivity, if any, change during abnormal development and are these modifications reversible? With the in vitro cyst model established, how are these cysts affected following Paclitaxel treatment and is there a relationship between galectin-3 expression and paclitaxel therapy.

Materials

General reagents

The reagents used in this thesis were mainly supplied by BDH Ltd (Poole, Dorset, UK) with the exception of those listed below:

Sigma Chemicals Company (Poole, Dorset, UK) supplied aprotinin, ampicillin, P- mercaptoethanol, bovine serum albumin (BS), bromphenol blue, ethidium bromide, fetuin-agarose beads, gluteraldehyde, haematoxylin, paclitaxel,

phenylmethylsulphonyl fluoride, paraformaldehyde (2 grams paraformaldehyde, 50 ml Ix PBS), phenol. Ponceau S, periodic acid Schiff, soluble fetuin, trizma base, sodium dodecyl lauryl sulphate, and sodium ortho vanadate.

Other companies supplied the following: ABC kit (Dako, High Wycombe, Bucks, UK), 5-bromo-4-chloro-indolyl-phosphate (BCIP) (Promega, Southampton, UK), Citifluor™ (Chemical Labs, University of Kent, Canterbury, Kent, UK), Enhanced Chemiluminescence Reagents (BCL; Amersham Pharmacia, Little Chalfont, Bucks, UK), Histoclear (National Diagnostics, Atlanta, Georgia, US), nitroblue tétrazolium (NBT) (Promega, Southampton, UK), phosphate buffered saline (PBS) (Gibco BRL, Paisley, UK), and nucleotides: deoxy-adenosine triphosphate (dATP), deoxy-

cystosine triphosphate (dCTP), deoxy-guanine triphosphate (dGTP), and deoxy- tyrosine triphosphate (dTTP) were supplied by Promega (Southampton, UK).

Tissue culture equipment

Media

Dulbecco’s Modified Eagle Medium / F12 mix (DMEM / F12) was supplied by Gibco BRL.

Media supplements

Sigma Chemicals Company supplied: insulin transferrin selenium (ITS), dimethyl sulfoxide (DMSO), human recombinant Epidermal Growth factor (EOF), antibiotics solution, and hydrocortisone, triiodothyronine (T3). Gibco BRL supplied the heat inactivated fetal calf serum.

Tissue culture plasticware

Philip Harris (London, UK) and Nunc (Naperville, Illinois, US) supplied the petri dishes (30mm and 90mm), 6 well and 96 well plates. The Millipore Corporation (Bedford, Massachusetts, US) supplied Millicell™ -CM filters.

Animal husbandry

I utilised normal mouse strains, C56B1. / 6J or (CBA x C57B10) F I (Charles River mouse farms, UK). Known heterozygous cpk/-\- mice were obtained from the Jackson Laboratories (Bar Harbor, Maine, US).

ARPKD affected mice are homozygous recessive (cp k/cp k), and hence are the F I progeny from mating two heterozygotes. Statistically half of the litters from parents that carry the mutation will be heterozygotes, one quarter will be affected

homozygotes and one quarter, normal mice that do not carry the mutation. Since there is no genetic test available as yet, the only route to identification is by breeding and looking for affected offspring within a litter, in other words, trial and error mating. This is how the cpk colony was established and maintained. Eventually approximately 30 young breeding pairs made up the colony, of which 20 would be identified as heterozygotes, with the other pairs at the, ‘to be identified’ stage. Mice were kept with their mothers until they were weaned at 21 days old. Both the Home Office, and the Institute of Child Health Animal Welfare Committee, approved all studies involving animals.

Pregnant C57B1 /6J normal mice were killed by cervical dislocation on embryonic day I I (E l 1) through to E19, and embryos were removed under aseptic conditions.

Kidneys were dissected out from c p k /c p k homozygous offspring and their phenotypically normal littermates at 1, 2, 3, and 4 weeks of age, as well as from normal mouse strains at the same ages.

Following dissection tissue for immunohistochemistry and in situ hybridisation were placed into 4% paraformaldehyde (PF), tissue for protein, RNA and DNA analysis were placed into liquid nitrogen, tissues for cell / organ culture were placed into L I 5 medium at 4®C and tissues for electron microscopy were placed into gluteraldehyde.

Enzymes

Restriction endonucleases were obtained from Gibco BRL. T7 and T3 RNA polymerase, Digoxigenin-UTP RNA labelling kit, RNase A and proteinase K

were supplied by Boehringer-Mannheim (Germany). RNase inhibitor, T4 DNA ligase, RNase A, Taq polymerase and klenow enzyme were obtained from Promega.

Boehringer Mannheim supplied the N-glycosidase F deglycosylation kit, which was used for the enzymatic deglycosylation of galectin-3 protein, prior to western blot analysis.

Microscopy

Light and fluorescent microscopy was performed on a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany) using objective lenses of 5x, lOx, 20x, 40x and 63x (oil immersion) magnification. Specimens were photographed using Fuji Super G

100 colour negative, Kodak Ektachrome 64 colour positive film, or taken digitally using either a Progress 5012 or Olympus Camedia C-3030 Digital camera from Olympus, Middlesex, U.K.

Confocal fluorescent microscopy was performed on a Leica Aristoplan microscope and computer confocal laser scanning system (Aristoplan-Leica, Heidleberg, Germany) with oil immersion objective lenses of 16x, 25x, 40x, 63x and lOOx and software interpolation of intermediate magnifications. Images were saved as tagged image format (TfF) files and imported, for labelling, into Adobe Photoshop (Version 5, Adobe Systems Europe, Edinburgh, UK) or Microsoft Powerpoint 2000 (Microsoft Corporation, Seattle, US).

Gel electrophoresis

Gibco BRL supplied agarose. Sigma Chemicals Company supplied: 30% acrylamide, ammonium persulfate, TEMED, and X-ray film: Kodak X-OMAT AR. 3MM

chromatography paper was supplied by Whatmann Ltd (Maidstone, Kent, UK). Amersham Pharmacia supplied, Hybond-P, and ‘Geneclean H®’.

Molecular size markers and cloning vectors and bacterial

strains

RPN 800 rainbow marker was purchased from Amersham Pharmacia. 1 Kilobase (K) DNA marker was purchased from Gibco BRL. A galectin-3 cDNA insert of 883 base pair (bp) was ligated to Bluescript KS vector and generously donated by Francois Poirier. (ICGM, Paris, France). Competent X L-1 blue M R F cells were purchased from Stratagene (California, US).

Western blotting equipment

Mini-Protean H cell gel electrophoresis tanks, trans-blot SD semi-dry transfer cell and Polyvinylidene diflouride membrane (PVDF) were purchased from Bio-Rad, supplied by Gibco BRL. BCA Protein Assay Kit was purchased from Pierce (Dlinois, US).

Antibodies and recombinant proteins

Antibodies against galectin-3 consisted of the following:

Galectin-3 rabbit polyclonal affinity purified antibody (plus pre-immune serum). This antibody was raised against the full length of hamster galectin-3 protein and has been shown to be detect a single band (seen as a doublet at high concentrations) on

immunoblot (Foddy, Stamatoglou, & Hughes 1990). This antibody cross-reacts with canine galectin-3 (Bao & Hughes 1995). Monoclonal rat anti mouse galectin-3, and rabbit polyclonal antibody raised against the galectin-3 Carbohydrate Recognition Domain (CRD).

Galectin-3 proteins used include: recombinant hamster galectin-3 protein, A1-93, containing the C-terminal domain but lacking most of the N-terminal domain of full length galectin-3. A mutant protein, named SS, similar to full length galectin-3, except for a mutation, where residue A rgl39 has been changed to serine.

All of the above antibodies and recombinant protein were generously supplied by Dr(s) Colin Hughes and Suleman Bawmania, National Institute for Medical Research, Mill Hill, London, throughout the duration of this study.

Pax-2, a transcription factor with paired box DNA binding domain expressed in the developing kidney, (Gruss & W alther 1992): rabbit polyclonal antibody purchased from Zymed, (CA, US), raised against a GST fusion protein derived from the C- terminal domain (aa 188-385) of the murine Pax-2 protein. This antibody is specific for Pax-2 and does not cross react with other members of the Pax family

(Rothenpieler & Dressier 1993). Two proteins are detected in mouse kidney lysates with this antibody: a major band at 46 kDa (Pax-2) and a minor band at 48 kDa (Pax-

2)

PCNA: (Ab-1; Oncogene Science, Cambridge, Massachusetts, US): mouse

monoclonal antibody (lgG2) to the human DNA-polymerase 5-associated protein that is expressed at high levels during S phase (Bravo et al. 1987). This gene is highly conserved across different species {Suzuka et a l, 1989

Monoclonal anti-p-actin (mouse IgG l isotype) was purchased from Sigma. It is derived from the A C -15 hybridoma produced by the fusion of mouse myleoma cells and splenocytes from an immunised mouse. This antibody recognises an epitope located on the N-terminal end o f the p-isoform of actin. Actins are one of two major cytoskeletal proteins, the other being myosin, which are implicated in cell motility, p- actin is one of the most conserved proteins in eukaryotes.

Secondary biotinylated antibodies which combined anti mouse / anti rabbit IgG were included in the ABC kit and secondary goat anti-rabbit horseradish-peroxidase (HRP), were purchased from DAKO. Fluorescent FTTC (fluoroscein isothiocyanate) and TRTTC (tetramethylrhodamine isothiocyanate) conjugated antibodies raised against rabbit and mouse IgG, and rhodamine conjugated anti-digoxigenin were purchased from Boehringer-Mannheim (Mannheim, Germany).

Lectins

FTTC, TRITC and biotin conjugated lectins were obtained from Sigma. Lectins are proteins or glycoproteins, often derived from natural sources such as plants, which bind to specific carbohydrate residues. The specificities of lectins used in this thesis were: Arachis hypogaea (peanut) - P-gal(l-3)galNAc and Dolichos biflorus - a - galNAc. In the kidney, lectins can be used to identify different parts of the mature nephron. Arachis hypogaea specifically detects the collecting ducts and Dolichos biflorus specifically detects the ureteric bud and its derivatives(Holthoffer et al. 1981;Verani, Walker, & Silva 1989).

Methods

Immunohistochemistry

Basic principles

Immunohistochemistry is a well established technique that allows detection of protein epitopes in a tissue section using an antibody - antigen reaction as means of

visualisation of this complex. (Coons, Leduc, & Connolly 1955). For conventional immunohistochemistry using tissue sections, the tissues are first fixed to preserve morphology, then dehydrated and embedded in a wax block. Thin sections of the block ( 4 - 6 pm) are cut on a microtome and transferred to a pretreated microscope slide. Next, the sections are rehydrated, pre-treated to increase penetration of the antibodies (optional), blocked to avoid non-specific binding and the antibody is applied. The antibody is then detected using secondary antibodies or other amplifying secondary detection techniques (see ABC below). The distribution of the protein can be determined using fluorescent or light microscopy.

Two types of antibody are commonly used: monoclonal antibodies and polyclonal antibodies. Antibodies are produced as part of the immune response to foreign antigens. The easiest to produce, polyclonal antibodies, are usually generated in rabbits, but also using other species. Production of antibodies involves recurrently immunising animals, with the antigen of interest over a period of several months. Antigen specific B-lymphocytes are stimulated when the antigen binds to cell surface receptors, resulting in the proliferation of the antigen - stimulated B-lymphocytes. This results in the secretion of increasingly large amounts of antibody. Many different B-lymphocytes will be stimulated this way, to a variety o f antigenic determinants, giving rise to different clones of B-Lymphocytes. The different antibody molecules are all secreted, giving what is called a “polyclonal” response, since different epitopes may be targeted.

Monoclonal antibodies are produced when the activated B-Lymphocyte is fused with a myeloma B-lymphocyte, and hybrid cells are cloned. These cells are immortalised

hence produce unlimited amounts of antibody. The clones also come from a single hybrid cell, and therefore give rise to identical antibodies. Rats and mice are used in this technique.

Fixation, wax embedding and sectioning

Samples of kidney tissue were fixed for between 1 and 24 hours, depending on size of sample, in 4% paraformaldehyde dissolved at 65°C) ( can be prepared in advance and stored at -20^C). The fixed samples were then washed twice with saline (0.83% NaCl in water) and dehydrated through 30%, 50%, 70%, 85%, 95% and 100% (twice) ethanol. Solutions were changed at 30 minute intervals and were kept at 4^C until the 70% stage, at which point the protocol was continued at room temperature.

Next the samples were immersed in Histoclear, twice, for 30 minutes each followed by a 1:1 Histoclear : wax mix at 60^C for 1 hour. The wax was then changed 3 times, each for 30 minutes at 60^C. Samples were then transferred to a mould, where they were orientated and the wax allowed to set for 4 hours. Specimen blocks were then stored at room temperature until sectioning.

Sections of between 4 - 6 |nm in thickness were cut and floated in a water bath at 45®C until any creases disappeared. The sections were then picked up on glass slides, which had been pretreated with poly-L-lysine. Slides were dried at 37®C overnight and stored at room temperature.

Rehydration and pretreatment

Sections were dewaxed and rehydrated. The slides were placed in a rack and sequentially passed through Histoclear twice for 5 minutes each and a graded alcohol series of 100% twice, 95%, 85%, 70%, 50% and 30% for 5 minutes each. The slides were then placed in milliRo water for a further 5 minutes

The next step was to treat the sections to improve antibody penetration. Preliminary experiments showed that this was most successfully achieved by microwaving the sections whilst immersed in a solution of citric acid (2.1 g citric acid in 1 litre milliRo water, adjusted to pH 6.0), which has been reported to cause antigen unmasking (Shi, Key, & Kalra 1991). Treatment with proteinase K (0.1 g/ml) was also used to achieve

this, but was not effective in this case. Using citric acid, the specimens were

microwaved at full power in a 700 W oven in a plastic container for several minutes. Once again, the time was optimised for the number and type of specimens, ranging between 2 - 5 minutes for small friable embryonic / cystic tissues to 10 minutes for large less fragile samples. Slides were left to stand and cool for 10 minutes

(occasionally speeded-up by surrounding the container with running water) before washing in running tap water, followed by two short dips in milliRo water then PBS, twice, for 5 minutes.

Blocking steps

The next two steps were designed to eliminate non-specific signal by blocking

endogenous peroxidase and non-specific binding. Slides were dipped in 3% hydrogen peroxide (1 in 10 dilution of 30% solution) in PBS for 15 minutes to quench

endogenous peroxidase and then washed twice in milliRo water and once in PBS for 5 minutes. Next they were immersed in 10% fetal calf serum (PCS) in PBS (blocking solution) for 30 - 60 minutes. At the same time, a humid slide chamber was prepared from a slide box with PBS-soaked tissues.

Primary antibody

Note that all subsequent dilution of reagents was performed using blocking solution (10% PCS in PBS), unless otherwise stated.

In the next step, the primary antibody was applied. Excess blocking solution was carefully wiped off around the tissue and 50 to 100 pi of antibody, was applied to the sections. Optimal antibody concentrations were determined by carrying out a series of dilutions usually between 1 in 50 and 1 in 200, to serial sections. Each section was then covered with a plastic coverslip to ensure even distribution of the antibody and the slides were placed in the humid chamber at 4®C overnight. After incubation, the coverslips were removed and slides were washed three times in PBS for 5 minutes.

Detection and mounting

Two secondary antibody detection systems were used: the commercially available avidin-biotin conjugate (ABC) kit followed by diaminobenzidine tetrahydrochloride

fact that since the intensity of the immunoperoxidase reaction is dependent on

peroxidase activity, the more peroxidase molecules bound to the tissue the greater the sensitivity. Biotin is a small vitamin molecule, whereas avidin is a large glycoprotein found in egg white, and has a high affinity for biotin.

Multiple copies of biotinylated secondary antibody (Reagent C) binds to unlabelled primary antibody, bound to the antigen that it was raised against, for example, galectin-3. The section was then treated with the avidin-biotin peroxidase complex (Reagents A and ) and hence the signal is amplified. Peroxidase activity was then detected by colorimetric changes in DAB. For fluorescent staining, a secondary antibody was applied which had been pre-conjugated to a fluorophore such as fluoroscein isothiocyanate (FTTC) or tetramethylrhodamine isothiocyanate (TRITC).

The secondary antibodies were selected specifically to detect the primary antibodies. Thus, for example, the anti-PCNA antibody was a mouse monoclonal and this was detected using an anti mouse immunoglobulin antibody.

Secondary fluorescent antibodies were applied to the tissue sections at 1 in 200 dilution, covered with a coverslip and incubated in the dark in the humid chamber for one hour at room temperature. The slides were then washed 3 times in PBS and mounted with glass coverslips in C itiflu o r^ . The edges of these coverslips were sealed with nail varnish to prevent drying and the slides were stored in the dark at 4®C until visualised. This technique was often used in combination with propidium iodide. This is a fluorescent dye, which intercalates with nucleic acids. It emits fluorescence, therefore can be visualised by fluorescence microscopy. This technique was adapted from Coles et al. (1993) with minor modifications (Coles, Bume, & Raff 1993). Two - four pg / ml propidium iodide plus 100 p g / m l RNase A (DNase free) in PBS was added for the last 5 minutes of the fluorescent secondary antibody incubation period. Then the protocol was completed as normal.

Using the ABC kit. Reagent C was applied at 1 in a 400 dilution to the sections and covered with plastic cover slips. At the same time a mixture containing reagents A and B, at 1 in 100 dilution, was prepared. After 30 minutes at room temperature in the humid chamber, the slides were washed three times in PBS and reagent A / B

complex was applied using the same technique for 30 minutes. The slides were then washed three times in PBS and immersed in 0.5 g/1 DAB with sterile water. A brown precipitate is produced by the peroxidase in the A / B mixture and this reaction can be