Capítulo III. Las familias, sus perfiles y redes
3.1.1.1 Juan Ignacio Sánchez Espinoza
Introduction
For many years the metanephric kidney has been cultured and manipulated in vitro in order to dissect out the mechanisms o f kidney differentiation and m orphogenesis (Saxén, 1987). In this chapter an in vitro kidney culture m odel is used to extend the observations made in the previous chapter, and to test the function o f macrophages during kidney developm ent. M orphogenesis o f kidneys cultured in this way is described, illustrating the extensive ureteric bud branching and the formation o f tubules during the culture period. Resin histology reveals that mesenchymal-epithelial transitions occur, and wholem ount F 4/80 staining reveals the presence o f macrophages in these cultures, just as
in v iv o . This system provides an excellent model to manipulate the number o f macrophages during the developmental process, and to see whether, by depleting them, the normal pattern o f branching and tubule formation is affected.
K idney organ cultures are an accepted in vitro model for studying kidney developm ent, and were first used as a technique in this regard by Grobstein in the 1950s (G robstein, 1956). This procedure was later somewhat m odified by Saxén and colleagues during the 1960s and 1970s (Saxén e t a l . , 1968; Saxén and Lehtonen, 1978; reviewed in S axén, 1987). The kidney rudiment, which consists o f the ureteric bud and surrounding m esenchym e, is dissected out soon after these two tissues have met in v iv o , and is able
to adjust to its new environment. The kidney rudiment develops, presumably in response to the same reciprocal interactions which occur, between these tw o tissues in v iv o .
Similar organ culture techniques were utilised by K oseki and colleagues (1992) when they show ed that programmed cell death was a naturally occuf-ring phenom enon in the developing metanephric kidney. Even when the metanephric m esenchym e was induced by the ureteric bud in this assay, several cells surrounding each new ly formed epithelial nephron exhibited classic signs o f apoptosis; by exogenously adding Epidermal Growth Factor (EOF) they were able to rescue som e o f this cell death (K oseki et a i , 1992). The previous chapter established that m ost o f the naturally occurring cell death in the developing kidney appears to be cleared by m acrophages, but these cells may also have other roles to play during kidney m orphogenesis besides sim ply being phagocytes. To test out this possibility it was necessary to consider several strategies to deplete macrophages in organ culture, one being to kill them with toxic liposom es. L iposom es, which are microspheres o f one or more lipid bilayers forming aqueous compartments, were pioneered by Gregoriadis (1971) as a means for specific drug delivery. If liposom es containing radioactive tags were intravenously administered to rats, they were removed from the circulation within m inutes, and much o f the radioactivity w as sequestered in the liver, because the liposom es were engulfed by Kupffer cells, the resident macrophages o f the liver (Gregoriadis and Ryman, 1972).
Van Rooijen and colleagues (1984, 1985, 1990) show ed in both rats and m ice, that liposom es were engulfed by macrophages after intravenous injection, and if the liposom es were loaded with a toxin, for exam ple dichloromethylene diphosphonate, then engulfing macrophages were killed, with very little related non-specific cell death. The liposom es are broken down by lysosom al com plexes in the macrophages causing release o f the toxin within the confines o f the engulfing cell. Van Rooijen (1989) found that
large multilamellar liposom es were more efficient at killing macrophages since they were able to “entrap” the drug within separate concentric aqueous compartments. M ost recently the liposome-mediated macrophage elimination technique has been used to test the role o f macrophages in the developing rat eye (Diez-Roux and Lang, 1997), where previous genetic studies have suggested macrophages may be involved in cell killing as well as clearance o f cell death (Lang and B ishop, 1993). This study successfully depleted the eye macrophages using toxic liposom es and, as in the genetic study, the pupillary membrane appeared not to die. This w as show n to be a macrophage specific effect because if macrophage numbers were replenished three days after the toxic liposom e killing, then the pupillary membrane began to regress (D iez-R oux and Lang,
Materials and Methods
An outbred strain o f Albino m ouse, strain C D l (Olac) w as used. Gestational age was calculated on the assumption that conception had occurred at midnight preceding the morning a vaginal plug was seen. Time-mated fem ales, E l 1.5, were killed by cervical dislocation, the uterus was removed, and the embryos were carefully dissected out in phosphate buffered saline (PBS; Oxoid) and staged according to Theiller (1989) and Martin (1 9 9 0 ), using the shape o f the forelimb and hindlimb buds as developmental indicators.
Organ Cultures
Under a dissecting microscope in a tissue culture flow hood, kidney rudiments, which are cylindrical in shape and o f approximately 4 0 0 |im in length, were carefully dissected from embryos at 11.5 days o f gestation (Fig. 4.1). In a petri dish containing L eibovitz’s L-15 medium (Gibco) the embryo was first transected with small iridectomy scissors m idway between the forelimbs and hindlimbs (incision 1); the second cut, again made using small iridectomy scissors, was just above the hindlimbs (incision 2); m ost o f the tissue anterior to the position o f the kidneys was teased away at this stage; the third cut, to remove the developing som ites and neural tube, w as performed using a pair o f 0 .6 hypodermic needles on the ends o f 1ml syringes (incision 3). The remaining piece o f tissue was then placed on its ventral surface exposing the metanephric kidneys lying in close apposition with the mesonephroi (Fig. 4.1 D ). At this stage the ureteric bud has branched from the W olffian duct and just entered the condensed metanephric m esenchym e. It is essential to have both components for the culturing procedure. The metanephric kidney rudiment was gently teased away from the surrounding tissue using
watchmakers forceps and a fine bore needle, and placed on a 0.8cm pre-dampened millicell tissue insert (Millipore) in a four-well culture dish (G ibco). The organ culture was allow ed to flatten down for between 30 minutes to one hour, before 2 0 0 |il o f culture m edium was added to the well.
The culture medium used was D ulbecco’s M odified Eagle Medium/Nutrient M ix F I 2 (1:1) with L-Glutamine, 15mM HEPES (DMEM/F12; Gibco; see Appendix) containing 10% foetal ealf serum (PCS; Sigma) and 1% penicillin and streptomycin (Sigm a). The organ culture absorbs its nutrients from the medium through the filter. The culture dish was put into an incubator at 37°C with 5% CO^ and the medium was changed daily. The organ cultures were view ed every day using a Leica Diaplan m icroscope, and each one was captured digitally via a video camera and the Adobe photoshop programme on a Pow er M acintosh Computer.
The method was based on the work o f Grobstein (1955; 1956) and Adrian W oolf, Institute o f Child Health, London.