Transmembrane Protein 14A protects glomerular filtration barrier integrity 111 6 These cells reabsorb the dextran tracers in endosomes. Through immunofluorescence microscopy, these endosomes appear as small fluorescent droplets in the proximal tubule cells. The number of these proximal tubule reabsorption droplets were counted in a blinded fashion. (Figures 2A through F) No difference was found in the mean number of 3 kDa reabsorption droplets, indicating that tubular reabsorption mechanisms remained intact in all three groups (Figure 2G). A significantly higher number of 70 kDa reabsorption droplets was measured in both zebrafish embryos with a tmem14a knockdown and in the positive control group, indicating loss of GFB integrity (Figure 2H, p < 0.05). The effectivity of tmem14a knockdown in zebrafish was primarily assessed by quantifying its effect on glomerular permeability. Figure 1. Glomerular TMEM14A expression is diminished before onset of proteinuria (A) Urinary albumin excretion is significantly higher in Dahl rats compared to SHR from 6 weeks of age. Y-axis shows urinary albumin excretion in mg/24h, and the X-axis shows animal age in weeks. (B) Glomerular TMEM14A mRNA expression in Dahl rats is significantly lower than in SHR controls at all time points. The Y-axis shows mRNA expression as relative expression compared to Hprt1, and the X-axis shows animal age in weeks. No correlation was observed between TMEM14A expression and urinary albumin excretion in both SHR (r = - 0.80, CI -0.98 – 0.27) and Dahl (r = -0.60, CI -0.97 to 0.60) using Pearson’s correlation coefficient. (C) Glomerular staining for Tmem14a protein is lower in Dahl rats than in SHR after 2 weeks. This difference is significant at weeks 4 and 8 of age. The columns represent mean semi-quantitative score Dahl rats develop proteinuria at 6 weeks of age. Thus, both mRNA and protein expression levels drop before onset of proteinuria. ANOVA with Tukey’s post hoc analysis was used. (D - M) Representative images of glomeruli of spontaneously proteinuric Dahl (D – H) and SHR (I – M) rats at respectively 2 (D and I), 4 (E and J), 6 (F and K), 8 (G and L), and 10 (H and M) weeks of age. (A – C) Horizontal lines indicate mean with SD. Scalebar = 50 µm *p < 0.001
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