Transmembrane Protein 14A protects glomerular filtration barrier integrity 109 6 washed in PBS and blocked with 5% Normal Rabbit Serum (NRS) for 1 hour. 5% NRS was aspirated, and the glasses were incubated with primary antibody diluted in 1% BSA in PBS (polyclonal goat anti-TMEM14A) at 4°C overnight. For rat and human kidney tissue, after sectioning at 4 μm, the slides were deparaffinized, dehydrated, and boiled in Tris/EDTA buffer for antigen retrieval for 10 minutes. The slides were washed in PBS and incubated with the primary antibody diluted in 1% BSA in PBS (polyclonal goat anti-TMEM14A 1:200 for rat tissue, 1:150 for human tissue) at 4°C overnight. For all mentioned materials, after incubation of the primary antibody the samples were washed in PBS and incubated for 30 minutes with the secondary antibody ((Polyclonal Rabbit Anti-Goat Immunoglobulins/horse radish peroxidase, Cat no: P0160, AgilentDako, CA, United States). The material was then washed in PBS again and immunoreactivity was detected with diaminobenzidine. After counterstaining with haematoxylin, the material was dehydrated and mounted. For quantification of stained slides in both rat and human material, we used a semiquantitative approach. TMEM14A staining was evaluated on a semi-quantitative scale with a score of 0 to 4 in a blinded manner, subdivided in respectively no podocyte staining (0), 0-10% of the podocytes (1), 10-30% of the podocytes (2), 30-60% of the podocytes (3) more than 60% of the podocytes (4). Statistical analyses Statistical analyses were performed using Graphpad Prism 9.4.1 (GraphPad Software, San Diego, California). Student’s unpaired t-testing was used for comparisons between 2 or 3 groups. When more than 3 groups were compared, one-way ANOVA with Tukey’s post hoc analysis was used. A p-value below 0.05 was considered significant. Results Glomerular gene expression pattern of proteinuric rats Tmem14a was identified as potentially involved in the development of proteinuria through analysis of a previously published microarray dataset comparing Dahl andSHR rats. The top 5 downregulated genes are shown in Table 2. These genes are found in both SHR and Dahl rats. Tmem14a was selected for further analysis based on this differential expression and the availability of a zebrafish homologue to perform experimental in vivo analysis of development of proteinuria, as performed later in this study.
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