Chapter 5 86 down dynamin translation in a zebrafish embryo model results in proteinuria. Lastly, we show that glomerular dynamin and cathepsin L protein levels are increased in human patients with proteinuric kidney disease. Methods Microarray analysis We analyzed the microarray datasets of Dahl and SHR rats in order to identify differentially regulated cytoskeleton-related genes. The datasets were retrieved from the Gene Expression Omnibus of the NCBI, which is accessible using the GEO Series accession number GSE 13810. This analysis was performed using Affymetrix GeneChip Rat Genome 230 2.0 arrays. Pathway analysis was performed using the Gene Ontology Tree Machine and by global testing in the Kyoto Encyclopedia of Genes and Genomes in order to determine which differentially regulated genes are involved in cytoskeletal regulation.(77, 78) This microarray was performed on glomeruli obtained from animals at 4 and 6 weeks of age. Animal studies: rats Male spontaneously proteinuric Dahl salt-sensitive rats (Dahl) and non-proteinuric spontaneously hypertensive rats (SHR) were obtained from the Charite-University Medicine Berlin.(79) To prevent the early accelerated development of severe hypertension, the rats were fed a low-salt diet containing 0.2 % NaCl by weight content. Tissues were collected from animals at 2, 4, 6, 8, and 10 weeks of age. A total of 67 animals was used, with respectively 7, 7, 7, 8, and 8 Dahl rats per age group and 5, 4, 6, 7, and 8 SHR rats per age group. In each animal, the left kidney was used to isolate the glomeruli using magnetic retraction; these samples were used for mRNA analysis.(80) The right kidney of each animal was partially embedded in paraffin, snap-frozen, and stored at -80 °C. RNA isolation and qPCR RNA was isolated from rat glomeruli using TRIzol (Invitrogen, Waltham, MA). AMV reverse transcriptase (Roche Diagnostics) was used to reverse-transcribe the RNA into first-strand cDNA, which was then analyzed using qPCR with the primer pairs listed in Table 2. HPRT was used as an internal control. qPCR was performed on an iCycler realtime PCR machine with SYBR green supermix (Bio-Rad Laboratories, Hercules, CA), and iCycler IQ 3.1 software (Bio-Rad Laboratories) was used to analyze gene expression and to normalize the data. Dnm1 and Dnm2 mRNA levels are shown as relative to the average Dnm1 or Dnm2 mRNA expression in SHR rats.
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