Chapter 6 106 dataset with GEO Series accession number GSE 13810 was accessed through the Gene Expression Omnibus of the NCBI. As described previously, this analysis was performed with Affymetrix GeneChip Rat Genome 230 2.0 arrays (93). Animal studies: rats Animal studies in rats were performed with the same animal material as previously described and in accordance with institutional guidelines (17, 93). Rat experiments were approved by the respective Institutional Animal Care and Use Committee. Dahl and SHR male rats were obtained from Freie Universität Berlin (79). They were fed a low salt diet containing 0.2 percent NaCl by weight to prevent early development of hypertension. To investigate the time relation between Tmem14a expression and the development of proteinuria, groups of rats were studied at 2, 4, 6, 8, and 10 weeks of age. mRNA expression was investigated in a total of 67 animals. For the respective ages stated above the respective number of investigated animals per age group were 7, 7, 7, 8, and 8 for the Dahl rats and a number of 5, 4, 6, 7, and 8 for the SHR group. For protein expression, 2 rats were investigated per age and group. Animal studies: zebrafish Wild-type (WT) AB/TL strain zebrafish (Danio rerio H) were injected with 1 nL of a morpholino (Gene Tools, Philomath, OR) blocking the mRNA translation of the zebrafish tmem14a homologue, zgc:163080, or a scrambled control morpholino during the 1-to-4 cell stage of embryonic development (82). Experiments were performed concurrently with previously described studies (55, 93) All zebrafish experiments were performed prior to the free-feeding stage of embryos and therefore are not considered animal experiments in accordance with EU Animal Protection Directive 2010/63/EU. Glomerular permeability and tubular reabsorption assay A tubular reabsorption assay was performed as described previously (55, 93, 102). This method was adapted from Hentschel et al. and has been previously validated to reliably assess glomerular permeability and tubular reabsorption function (40, 43). In short, a 1 nl mixture of 3 kDa TRITC labelled dextran (100 mg/ml; Invitrogen) and 70 kDa FITC labelled dextran (25 mg/ml; Invitrogen) was injected intravenously at 5 days post fertilization (dpf), fixed one hour after injection in 10% formalin for 24 hours, stored in ethanol 70%, embedded in paraffin, sectioned and examined by fluorescence microscopy.
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