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glomerular capillary convolute

magnetic bead Bowman capsule vas efferens proximal tubulus Collagenase A (30 minutes / 37°C) digested tissue perfused kidneys © Blutke 2007

3. Research design and methods

To address the question, if common characteristic patterns of morphological and functional glomerular alterations, displayed by both GIPRdn- or bGH-transgenic mice (refer to chapters 2.4.2 and 2.4.3), would also find a reflection in detectable common glomerular gene expression profiles, differential transcript profiling analyses were performed on samples of isolated kidney glomeruli, generated from male GIPRdn- or bGH-transgenic animals, as well as from their corresponding non-transgenic littermate controls (figure 3.1). The investigated animal models are bred on different genetic backgrounds (chapter 3.2.1) and in both models different mechanisms are responsible for the development of renal alterations (diabetes mellitus vs. overexpression of the GH-transgene). In the context of the present study, the term

“group” refers to either GIPRdn-, or bGH-transgenic (tg) animals and their corresponding non-transgenic wild-type littermate controls (wt), respectively the sample materials generated from these animals. Sample materials were generated from pairs of animals of the respective groups and also analyzed pairwise. As a matter of principle, a pair of animals consisted of a transgenic animal and an associated non-transgenic wild-type littermate control (tg/wt-pairs). If the numbers of male transgenic and non-transgenic animals in a respective litter allowed for more than a single possible combination, assignment of transgenic animals and associated littermate controls to tg/wt-pairs was performed by lot. For generation of sample materials, both animals of a respective tg/wt-pair were sacrificed at the same day of age. Sample materials of two cohorts of animals were generated: samples of isolated glomeruli of animals of the “Array Cohort” were investigated by performance of microarray experiments. For performance of real-time PCR confirmation experiments, samples of an additional second independent cohort, termed “Independent Control Cohort” were generated. Investigations were performed in two defined early stages of comparable glomerular alteration, displayed by transgenic animals of both models: First, the stage of glomerular hypertrophy (stage I) and second, the stage of glomerular hypertrophy with onset of micro-albuminuria (stage II). Stringent criteria for assignment of animals to either the first, or the second stage of investigation were defined in order to ensure the comparability of the respective stages in both groups, which was considered to be a crucial prerequisite for meaningful interpretation of analysis results. Assignment to the stages of investigation was performed in

dependency on the degree of glomerular alteration that was detected in the transgenic animal in comparison to its associated wild-type littermate control. In pairs of animals assigned to stage I and II of the Array Cohort, the transgenic mouse was required to display an increase of the mean glomerular volume of at least 40%, compared to its associated control animal. For that purpose, the mean glomerular volume was determined by methods of quantitative stereology, using histological samples of cortical kidney tissue. Since this approach implicated the necessity of accession of kidney tissue of both partners of a respective pair of animals distinguished for assignment to stage I, sacrifice of both mice and glomerulus isolation from their kidneys had to be performed prior to determination of the mean glomerular volumes. In addition to the criterion of glomerular hypertrophy for investigation in stage I, it was required, that neither the transgenic mouse, nor the associated wild-type control displayed albuminuria, verified by recurrent performance of sodium dodecyl sulphate polyacrylamide gel electrophoresis-based urine protein analysis (figure 4.7). In pairs of animals assigned to stage II, the transgenic mouse was required to display glomerular hypertrophy, as well as the onset of persistent albuminuria, whereas the corresponding wild-type mouse was not allowed to show any positive result of albuminuria (figure 4.7). Only sample materials derived from animals that did actually meet the criteria for investigation in the respective stages of glomerular alteration were used for further analyses. After performance of glomerulus isolation from the respective pairs of animals, total RNA was extracted from the glomerulus isolates and routinely processed for microarray analysis, using standard methods. Array data were analyzed to identify transcripts of differentially expressed genes (tg vs. wt) in the respective groups and stages of investigation. Then commonly differentially expressed genes (intersections of congeneric differentially expressed genes in both groups in comparable stages) were identified, representing common patterns of glomerular gene expression profiles in comparable early stages of glomerular alteration, presumably independent of the different expressed transgenes or genetic backgrounds of both different animal models. For revelation of their biological function in the context of molecular pathogenesis of glomerular hypertrophy and albuminuria, bioinformatical analyses were performed. Results from microarray experiments were confirmed, using quantitative real-time PCR. Additionally, sample materials were generated for performance of immunohistochemistry and in situ hybridisation in further studies to demonstrate the cellular distribution of selected proteins and/or transcripts in the glomerulus.

Confirmation of results by real-time PCR (3.9.) Bioinformatical analysis (3.10)