Transmembrane Protein 14A protects glomerular filtration barrier integrity 107 6 The number of proximal tubule reabsorption droplets were quantified manually in a blinded fashion. As a positive control, a section of control zebrafish embryos was injected with puromycin aminonucleoside (PAN, Sigma-Aldrich, St. Louis, MO) at 4 dpf (40). Under physiological conditions, the 3 kDa tracer freely passes the GFB and the 70 kDa does not. After passing the GFB, these tracers are absorbed by proximal tubule cells in endosomes, appearing as small droplets. Hence, observing 70 kDa reabsorption droplets in proximal tubule cells indicates loss of GFB integrity. Reabsorption droplets of the 3-kDa tracer were used to assess the correct location of the proximal tubule cells and whether tubular reabsorption mechanisms are sufficiently intact. Although injecting dextrans of sizes larger than 70 kDa could theoretically provide additional information on the severity of loss of GFB integrity, previous pilot experiments (data not shown) have shown 70 kDa to produce the most consistent and reliable results when fixing the samples 1 hour after injection. We were not able to reliably differentiate between the 70 kDa and larger tracers when maintaining this timeframe. Therefore, in this study, only the validated 3- and 70 kDa tracers were used. Cell culture Immortalized podocytes (Moin Saleem, Bristol, UK, human), embryonic kidney (HEK) 293 cells (ATCC), and human umbilical vein endothelial cells (Huvec) (Lonza, Allendale, NJ, USA) ATCC were used to measure mRNA expression for TMEM14A. Podocytes were also used for localization of TMEM14A protein expression by immunohistochemistry as described below. Immortalized podocytes were cultured in RPMI 1640 Medium with added Penicillin, Streptomycin, Insulin, Transferrin, Selenite (Sigma Chemicals, Dorset, UK) and 10% Fetal Bovine Serum at 33 °C (in 5% CO2) for proliferation and at 37 °C (in 5% CO2) for differentiation. RNA isolation, reverse transcription and qPCR RNA isolation, reverse transcription and qPCR were performed as described previously (93). To quantify gene expression of TMEM14A in different cell lines, RNA from podocytes, HEK, and Huvec cells, and RNA from purified glomeruli and whole kidney tissue was isolated using TRIzol (Invitrogen, Waltham, MA). AMV reverse transcriptase (Roche Diagnostics) was used for reverse transcription into cDNA. Primer sequences are listed in Table 1. GAPDH was used as a housekeeping gene for cell culture experiments. For purified rat glomeruli experiments, Hprt1 was used as internal control. mRNA expression values are given as relative to Hprt1 expression. Quantitative real-time PCR
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