miR-200c is upregulated in urine from patients with MGN, MCD and FSGS. (a) Fold change in urinary miR-200c expression normalized to urine creatinine and cel-39 in different renal diseases. N = 27 different patients. * p < 0.05, *** p < 0.001 compared to control. CTRL: control, MGN: membranous glomerulonephritis, MCD: minimal change disease, IgAN: IgA nephropathy, DN: diabetic nephropathy, ANCA: antineutrophil cytoplasmic autoantibody vasculitis, HUS: hemolytic uremic syndrome, FSGS: focal segmental glomerulosclerosis, PREE: preeclampsia. (b) Sequence of human (hsa) and zebrafish (dre) miR-200c. Differences are highlighted in red. The seed region of the miRs are underlined and labeled in green.

miR-200c mimic induces edema, proteinuria, podocyte effacement and loss of endothelial fenestrations. Zebrafish were injected with an miR-Ctrl mimic and miR-200c-3p mimic at the one-to-four cell stage. (a) Phenotype pictures of zebrafish larvae at 96 h postfertilization (hpf). Scale bar = 500 µm. (b) Staining the zebrafish pronephroi with hematoxylin–eosin (H&E) at 96 hpf after miR-Ctrl and miR-200c mimic injection in egg stage. Scale bar = 100 µm and 10 µm in the zoom in (c) Quantification of phenotype by severity of pericardial and yolk sac edema in percentage. Examples of phenotypic changes, grading the normal zebrafish phenotype with no edema P1 and the most “edematous” phenotype with P4 at 96 hpf. (d) Timely fusion of the glomeruli at 48 hpf after miR-200c overexpression in Tg (wt1b:EGFP) zebrafish, which express eGFP under the control of the wt1b promotor. Scale bar = 200 µm. (e) Representative images of an eye of Tg(I-fabp:VDBP:EGFP) fish, which were injected with an miR-Ctrl or miR-200c mimic. Scale bar = 150 µm. Quantification of loss of circulating high-molecular-weight proteins by measuring max. eye fluorescence in the retinal vessel plexus of Tg(l-fabp:VDBP:EGFP) zebrafish larvae at 96 hpf. *** p < 0.001. (f) Top: TEM pictures of the zebrafish GFB at 96 hpf after miR-200c mimic and miR-Ctrl injection in egg stage. White arrows show fenestration in the control zebrafish, which are lost in miR-200c-injected zebrafish. Black arrows show podocyte effacement. Scale bar = 500 nm. Bottom: quantification of loss of endothelial fenestrae and podocyte effacement in percentage of representative fish in each group. * p < 0.05; *** p < 0.001.

miR-200c mimic induces edema, proteinuria, podocyte effacement and loss of endothelial fenestrations. Zebrafish were injected with an miR-Ctrl mimic and miR-200c-3p mimic at the one-to-four cell stage. (a) Phenotype pictures of zebrafish larvae at 96 h postfertilization (hpf). Scale bar = 500 µm. (b) Staining the zebrafish pronephroi with hematoxylin–eosin (H&E) at 96 hpf after miR-Ctrl and miR-200c mimic injection in egg stage. Scale bar = 100 µm and 10 µm in the zoom in (c) Quantification of phenotype by severity of pericardial and yolk sac edema in percentage. Examples of phenotypic changes, grading the normal zebrafish phenotype with no edema P1 and the most “edematous” phenotype with P4 at 96 hpf. (d) Timely fusion of the glomeruli at 48 hpf after miR-200c overexpression in Tg (wt1b:EGFP) zebrafish, which express eGFP under the control of the wt1b promotor. Scale bar = 200 µm. (e) Representative images of an eye of Tg(I-fabp:VDBP:EGFP) fish, which were injected with an miR-Ctrl or miR-200c mimic. Scale bar = 150 µm. Quantification of loss of circulating high-molecular-weight proteins by measuring max. eye fluorescence in the retinal vessel plexus of Tg(l-fabp:VDBP:EGFP) zebrafish larvae at 96 hpf. *** p < 0.001. (f) Top: TEM pictures of the zebrafish GFB at 96 hpf after miR-200c mimic and miR-Ctrl injection in egg stage. White arrows show fenestration in the control zebrafish, which are lost in miR-200c-injected zebrafish. Black arrows show podocyte effacement. Scale bar = 500 nm. Bottom: quantification of loss of endothelial fenestrae and podocyte effacement in percentage of representative fish in each group. * p < 0.05; *** p < 0.001.

TGF-β stimulation induces miR-200c expression in cultured glomerular endothelial cells. (a,b) qPCR for miR-200c detection in cultured human glomerular endothelial cells (a) and human podocytes (b) normalized to U6 RNA. Data are given as fold change compared to unstimulated cells (CTRL) at the respective time point. Cells were stimulated with TGF-β for 6 h, 24 h and 48 h, *** p < 0.001. Reproducible data from three independent experiments.

miR-200c overexpression alters the expression of ZEB1/2 and VEGF-A. (a,b) Cultured human glomerular endothelial cells and podocytes were transfected with an miR-control (miR-Ctrl) or miR-200c mimic (miR-200c), and qPCR was performed. (a) miR-200c-induced downregulation of ZEB1/2 in glomerular endothelial cells. (b) miR-200c-induced downregulation of ZEB1/2 and VEGF-A in podocytes. * p < 0.05, * p < 0.01, n = 4 independent experiments. (c) qPCR showing vegf-aa and vegf-ab mRNA expression after injection of miR-Ctrl or different concentrations of miR-200c mimic in zebrafish. Injections were performed at one two four cell stage. qPCR was performed 120 hpf. * p < 0.05, ** p < 0.01, *** p < 0.001. A pool of 20 zebrafish was used for RNA isolation in each group.

miR-200c overexpression decreases VEGF-A and ZEB1 on protein level. Images show immunofluorescent staining with phalloidin (red), anti-ZEB1 (green in (a,b)) and anti-VEGF-A (green in (c)). Nuclear staining is shown in blue with Hoechst. Scale bar = 25 µm. (d) VEGF protein in supernatant of cultured human podocytes (left panel shows total concentration, right panel shows fold change) after transfection with miR-200c mimic or miR-Ctrl was measured with ELISA. * p < 0.05.