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Genome-wide tissue gene expression profiling in Juvenile Göttingen Minipigs with customized Agilent microarrays

ABSTRACT: Minipigs have favorable biological characteristics for juvenile toxicity studies to assess efficacy and safety of pediatric drug products. To evaluate at the molecular level the expression of drug targets, drug metabolism pathways or general maturation of organ transcriptomes in minipigs, we used Customized Agilent Micoarrays (Design-ID 050244) for genome-wide gene expression profiling on 9 different tissues from male and female juvenile Göttingen minipigs aged 1 week to 24 months. The gene expression results analyzed in this study are further described in Heckel T. et al. (2015) Functional analysis and transcriptional output of the Göttingen minipig genome. under submission

ORGANISM(S): Sus scrofa

PROVIDER: GSE71441 | GEO | 2015/12/16

SECONDARY ACCESSION(S): PRJNA291208

REPOSITORIES: GEO

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Project description:The Göttingen Minipig is gaining ground as nonrodent species in safety testing of drugs for pediatric indications. Due to developmental changes in pharmacokinetics (PK) and pharmacodynamics (PD), physiologically-based pharmacokinetic (PBPK) models are built to better predict drug exposure in children and to aid species selection for nonclinical safety studies. These PBPK models require high quality physiological and PK/PD data such as protein abundance of drug metabolizing enzymes. These data are available for man and rat, but scarce for the Göttingen Minipig. The aim of this study was to assess hepatic cytochrome P450 (CYP) protein abundance in the developing Göttingen Minipig by using mass spectrometry. In addition, sex-related differences in CYP protein abundance and correlation of CYP enzyme activity with CYP protein abundance were assessed. The following age groups were included: gestational day (GD) 84 - 86 (n = 8), GD 108 (n = 6), postnatal day (PND) 1 (n = 8), PND 3 (n = 8), PND 7 (n = 8), PND 28 (n = 8) and adult (n = 8). Liver microsomes were extracted and protein abundance was compared to that in adult animals. Next, the CYP protein abundance was correlated to CYP enzyme activity in the same biological samples. In general, CYP protein abundance gradually increased during development. However, we observed a stable protein expression over time for CYP4A24 and CYP20A1 and for CYP51A1, a high protein expression during the fetal stages was followed by a decrease during the first month of life and an increase towards adulthood. Sex-related differences were observed for CYP4V2_2a and CYP20A1 at PND 1 with highest expression in females for both isoforms. In the adult samples, sex-related differences were detected for CYP1A1, CYP1A2, CYP2A19, CYP2E1_2, CYP3A22, CYP4V2_2a and CYP4V2_2b with higher values in female compared to male Göttingen Minipigs. The correlation analysis between CYP protein abundance and CYP enzyme activity showed that CYP3A22 protein abundance correlated clearly with the metabolism of midazolam at PND 7. These data are remarkably comparable to human data and provide a valuable step forward in the construction of a neonatal and juvenile Göttingen Minipig PBPK model.

Project description:Description Primary membranous nephropathy (MN) is an autoimmune kidney disease histomorphologically defined by subepithelial deposition of immune complexes in the glomeruli, and exhibits a high risk for end-stage kidney disease. Circulating antibodies against phospholipase A2 receptor 1 (PLA2R1) are detected in 70-80% of MN patients and correlate with treatment response and prognosis. Experimental proof that human PLA2R1-antibodies induce MN is still missing. Methods: In passive transfer experiments, minipigs received plasma or purified IgG from patients with PLA2R1-associated MN or from healthy controls. PLA2R1-antibodies and proteinuria were monitored using Western blot, ELISA and Coomassie staining. Kidney tissues were analyzed using immunohistochemistry, immunofluorescence, electron microscopy and proteomic analyses. Results: We show that minipigs, like humans, express PLA2R1 on podocytes. Human PLA2R1-antibodies bound to minipig PLA2R1 in-vitro and in-vivo. Passive transfer of human PLA2R1-antibodies, derived from patients with PLA2R1- associated MN, led to the development of histomorphologic characteristics of human early-stage MN in minipigs, activation of components of the complement cascade and induction of low levels of proteinuria. In the later phases of disease, development of an autologous phase of disease was observed. Conclusions: Applying a translational approach from humans to minipigs we show that human PLA2R1-antibodies are pathogenic, although in the heterologous phase of disease only low level proteinuria developed. Proteomic samples: Porcine glomeruli were isolated following previously published protocols known for human glomeruli, with slight modifications (Stahl et al., Kidney Int. 1984;26(1):30–34.). A total of six samples of sieved glomeruli from porcine kidney were used. 1. untreated_negative_control Experiment 1 (passive transfer of human PLA2R1 antibody from PLA2R1-antibody positive patient into minipigs) 2. exp_1_passive_antibody_rep1 Same experimental procedure in two animals (replicate 1) (treated with patient antibody) 3. exp_1_passive_antibody_rep2 Same experimental procedure in two animals (replicate 2) (treated with patient antibody) 4. exp_1_passive_antibody_control control animal for Pig A and B (treated with healthy antibody) Experiment 2 (passive transfer of human plasma from PLA2R1-antibody positive patient into minipigs) 5. exp_2_passive_plasma treated animal (treated with patient plasma) 6. exp_2_passive_plasma_control control animal (treated with healthy plasma)