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Over the years, a large number of in vitro models ranging from renal cell lines to isolated perfused tubules have been established. However, epithelial cell lines had to undergo a number of genetic changes in order to survive indefinitely in vitro. On the other hand, the isolated perfused tubule is a complex system to study. As a consequence, primary epithelial cell culture systems are needed to check the physiological significance of studies done with established cell lines and in some cases they might be the only way to study specific tubular cell function characteristics in particular (patho)physiological and pharmacological conditions. Furthermore, the increasing use of transgenic and knock-out mouse models should be supported by an in vitro model to study the cellular phenotype. Major inherent technical advantages of cultured cells for the study of epithelial transport include their ability to be studied in a controlled environment without systemic neurons and hormonal influences, and their easy accessibility for electrophysiological and imaging techniques. However, primary cell culture also has its limitations such as incomplete differentiation of cells or a limited lifespan, among others. The objective of this thesis was to establish a primary cell culture of mouse proximal tubule cells. The primary cells were extensively characterized for their proximal tubule origin. To correlate our findings in primary cell cultures with data in the literature, a detailed description of proximal tubule cell morphology and ( electro-) physiology is given in section l. 2, l. 3 and 1.4. Finally, we used our cell cultures as a model for the study of renal tubulointerstitial fibrosis. The mechanisms involved in tubulointerstitial fibrosis are described in section 1.5. At the end of this chapter, the different aims of this thesis are summarized.

The objectives of this work were: To study some functional properties of the midgut. 1) First of all some essential information was needed on the electrophysiologica l properties of the midgut of 0. cincta. la) To start with, an elemental analysis of 0. cincta hemolymph was performed and an adequate physiological saline was composed, based on the hemolymph composition. lb) Furthermore we wanted to study the electrical potential differences at the basolateral and the apical membrane, i.e. to assess the importance of the conductance of the basolateral membrane for K+ and to make an estimate of the relative basolateral versus apical membrane resistance. If the basolateral resistance is lower than the apical resistance Vbi approximates Ebi (see chapter 1.2.4 ). If the basolateral resistance can be increased, for instance by the application of Ba2+, we could take advantage of this situation to learn more about the electrical events at the apical membrane. (electrogenic pumping mechanisms or conductances of other ions). lc) The measurement of pH in the hemolymph, in the cell and in the lumen, should allow to calculate the electrochemical gradient across the basolateral and the apical membranes. This is important information because pH is an important factor either regulating transporters or offering a gradient across the cell membrane, energizing secondary active transport. The items la, lb and le are covered in chapter IA. 2) to determine the specific transepithelial resistance (Rte) of the midgut epithelium. Despite its small size, the midgut of Orchesella cincta must fulfill all needs of digestion and uptake of nutrients. Inevitably, the composition of the food composition changes as a consequence of digestion and uptake of food, while it moves through the midgut. Transport processes in the epithelium might therefore be adapted to a gradual change in the luminal content at different sites along the midgut. Consequently the Rte might be unequally distributed along the midgut. Because of t he small size of the animal t he Rte had to be determined with cable analysis. One assumption of cable analysis is, that the Rte is uniformly distributed along the tissue. 2a) Because this may not necessarily be the case, we tried to evaluate, whether it is nevertheless possible to find a good estimate of the true Rte if it changes along the midgut and 2b) whether it is possible to distinguish between artifacts due to electrical leakage in the set-up and a true unequal distribution of R,, along the midgut. The items outlined in 2), 2a) and 2b) are covered in chapter IB. 3) Furthermore, a technique for direct measurement of Cd2+ fluxes was introduced at the lab to explore the possibility of a simple self-referencing ion-selective electrode set-up to detect Cd2+ fluxes at the basolateral side of the midgut, which is known to accumulate cadmium.

The Malpighian tubules of the forest ant, F. polyctena, have been studied for many years now by the research group physiology of the Limburgs Universitair Centrum (Diepenbeek, Belgium). In insects these tubules are responsible for the primary urine production. The latter is achieved via secretion, driven by the active transport of KCI from hemolymph to lumen, and passive diffusion of substances. Initially the transport mechanisms involved in this active transport have been investigated. In a subsequent phase the factors involved in the regulation of this active transport were investigated, i.e. the presence of endogenous factors with a direct effect on the primary urine production by the Malpighian tubules was investigated. Both diuretic and antidiuretic factors were found to be present in body extracts of the ants. Some of these factors, i.e. a diuretic peptide (Fop-DP I) and two myotropic peptides (Fop-TK I en Fop-TK II), could be purified. In the present study we tried to further complete this picture. Different factors, both diuretic (Fop-DP and Fop-GP) and antidiuretic (FopADF), were shown to be present in body extracts of the ant and some could be identified. The antidiuretic factor, purified from an abdomen extract, was shown to block the Transport mechanisms involved in the active transport of K+ through the cells of the Malpighian tubules (see chapter 1). The signal transduction pathway used by FopADF could not be clarified. The involvement of adenylate cyclase and / or an increase in intracellular cAMP could clearly be excluded (see chapter 2). A cross-reactivity of Tenebrio molitor Malpighian tubules towards FopADF could be demonstrated by Dr. S. Nicolson (see Note after chapter 1) and a similar antidiuretic factor was shown to be present in an abdomen extract of the honeybee, Apis mellifer (see chapter 4). The diuretic factors, both purified from head/ thorax extracts, were shown to be peptides. For Fop-DP approximately 35% of the amino acid sequence could be determined. Fop-DP acted via an increase in intracellular calcium (see Chapter 3). A Fop-DP related factor was also demonstrated to be present in a head extract of the honeybee (see chapter 4). The second diuretic factor, Fop-cGP, was fully characterized. It is a peptide of 30 amino acids which exerts its diuretic effect in Locust, via an increase in cAMP (see Chapter 6). The purification of Fop-cGP followed a preliminary study trying to establish the presence of CRF-related peptides in the forest ant. The latter were shown to be present in a head / thorax extract of the ant by means of chromatography and an ELISA based on polyclonal antisera to CRF-related peptides from other insects (see Chapter 5). Fop-cGP had the activity of a CRF-like factor but not the amino acid composition. Another factor having a CRF-like structure may be present but still awaits further purification and characterization. Below we tried to give a brief overview of the relevant results for each chapter. A table, added as bookmarker, summarizes the results and is intended as a reader guide. The factors involved in the endocrine regulation of the primary urine production of the forest ant is compared to the situation known for other insects.