Fig 1: NTS targets nephrin when miR-204-5p expression is reduced. Immortalized mouse podocytes were treated with the miRNA control or miR-204-5p mimic or inhibitory sequences or left untreated prior to the addition of 1:200 nephrotoxic serum (NTS). The cell lysates were analyzed by Western blot (A) and probed for the podocyte’s markers nephrin, neph1, and podocin. Actin was used as a loading control. The blots are quantified in (B–D). The miR-204-5p sequence expressions were analyzed by real-time PCR, and the data were normalized to the expression of miR-16-5p (E). The presence of anti-nephrin antibodies in NTS was analyzed by Western blot (F) under native and reduced and denatured conditions. The immunofluorescence images in (G) show mouse podocytes transfected with miR-204-5p mimic, inhibitor, or control sequences prior to NTS treatment. The images were obtained using a Stedycon Abberior (STED) (Göttingen, Germany) and Olympus 1X81(Tokyo, Japan) confocal microscope and 100X objective lens. Nephrin is stained in green, actin is stained in red and the nucleus is stained with DAPI (blue). The scale bar represents 10 μm. The experiments were repeated 4 times (n = 4), and p values < 0.05 were considered significant, **** p < 0.0001.
Fig 2: Nephrin degradation is mediated by an aspartyl enzyme. Immortalized mouse podocytes were transfected with miR-204-5p mimic, inhibitor, or control sequences and treated with protease inhibitor cocktail before treatment with NTS. As shown by the Western blot (A) and quantitation (B), protease inhibition prevented the NTS-induced degradation of nephrin, even when miR-204 was inhibited. Mouse podocytes treated with the miR-204-5p inhibitory sequence and various protease inhibitors before NTS treatment showed that the aspartic acid protease inhibitor Pepstatin A rescued nephrin from degradation (C), and this was evident through Western blot quantitation (D). The experiments were repeated 4 times (n = 4). p values < 0.05 were considered significant ** p < 0.003, **** p < 0.001.
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