Chapter 6 110 Table 2. Glomerular gene expression in Dahl rats compared to SHR Gene name Symbol Region of rat chromosome Fold change Aldo-keto reductase family 1, member B8 Akr1b8 4q22 -4.5 Similar to interferon regulatory factor 10 RGD1562711 3q41 -3.9 Acyl-Coenzyme A oxidase 2, branched chain Acox2 15p14 -3.7 Similar to RIKEN cDNA 4921520P21; DMRTC1 LOC363483 Xq31 -3.4 Transmembrane protein 14A Tmem14a 9q13 -3.0 Glomerular Tmem14a mRNA and protein expression is diminished in spontaneously proteinuric rats before onset of proteinuria Glomerular mRNA and protein expression of Tmem14a were investigated in Dahl rats and compared to spontaneously hypertensive rats at 2, 4, 6, 8, and 10 weeks of age. Of note, Dahl rats develop significant proteinuria from 6 weeks of age. (Figure 1A) When comparing the two groups at each time point, glomerular Tmem14a mRNA expression is consistently significantly lower in the Dahl rats (p < 0.0001 at every time point, Figure 1A). Looking at the difference in expression between the different time points in only the Dahl rats, expression starts high and decreases quickly; at 2 weeks of age expression is significantly higher than at all other time points (p < 0.0001), but no significant difference was seen between the other time points. Doing the same in the spontaneously hypertensive control, glomerular Tmem14a mRNA expression was significantly higher at a younger age at weeks 2, 4, and 6 compared to week 10 (p < 0.01, p < 0.05, and p <0.0001 respectively). At weeks 2, 4, and 6, expression was significantly higher than at 8 weeks of age (p < 0.05, p < 0.05, and p < 0.001 respectively). After 2 weeks of age Tmem14a protein expression was lower in Dahl rats than in SHR at all time points. This was significantly so at 4 and 8 weeks of age (p < 0.001). At later timepoints, no significant differences in glomerular Tmem14a protein expression were seen. So, in Dahl rats, glomerular Tmem14a mRNA and protein expression is diminished before onset of proteinuria. Knocking down tmem14a mRNA translation results in proteinuria in zebrafish embryos The functional role of TMEM14A in the development of proteinuria was further investigated using a zebrafish embryo model. First, the zebrafish homologue of TMEM14A, zgc:163080, was knocked down by blocking its mRNA translation through morpholino injection. Then, a mixture of 3 and 70 kDa dextran tracers was injected. Puromycin aminonucleoside (PAN) injected zebrafish were used as a positive control for inducing proteinuria. PAN is a validated method to induce proteinuria in this zebrafish embryo model (40, 43). If the injected dextran tracers pass the GFB, they are subsequently reabsorbed by proximal tubular epithelial cells.
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