Fig. 6: Overexpression of non-functional I93M-UCH-L1 perpetuates podocyte injury and ubiquitin accumulations in experimental MN.

Experimental MN was induced in UCH-L1 overexpressing (WT OE and I93M OE) and respective littermate (Ctrl) mice. Kidneys were collected on day 14. a, b Urinary albumin to creatinine ratio, pooled values of 5 independent experiments (WT-UCH-L1, n ≤ 18 Ctrl or n ≤ 15 OE, per time) or of 2 independent experiments (I93M-UCH-L1, n ≤ 9 Ctrl or n = 10 OE, per time), mean + /-SEM, *p = 0.0434, **p = 0.0062, two-tailed Mann Whitney U test. c Stainfree SDS-PAGE from 1 experiment of creatinine-adapted urines depicts loss of high molecular weight proteins in I93M OE mice, albumin ~63 kDa. d Glomerular quantification of relative nephrin abundance (normalized to β-actin) to Ctrl. Pooled values of 4 independent experiments (WT-UCH-L1, n = 14 Ctrl or n = 9 WT OE) or of 3 independent experiments (I93M-UCH-L1, n = 7 Ctrl or n = 9 I93M OE), mean + /-SEM, *p = 0.0115, two-tailed Mann Whitney U test. e Left: STED analyses of nephrin meanders. Right: electron microscopy (EM) of glomerular filtration barrier integrity, arrowheads = foot processes, PC = podocyte. f Quantification of foot process effacement by PEMP analyses of filtration slit density (FSD) length per area (µm⁻¹). Pooled values of 1 experiment (WT-UCH-L1, n = 24 Ctrl or n = 36 WT OE; I93M-UCH-L1, n = 23 Ctrl or n = 35 I93M OE), mean + /-SEM, **p = 0.0041, two-tailed Mann Whitney U test. g Quantification of podocyte loss by counting p57+ podocytes per glomerular tuft area. Pooled values of 2 independent experiments (WT-UCH-L1, n = 350 Ctrl or n = 198 WT OE) or of 1 experiment (I93M-UCH-L1, n = 150 Ctrl or n = 128 I93M OE) as mean + /-SEM, ****p < 0.0001, two-tailed Unpaired t-test. h Quantification of glomerular polyubiquitinated (pUB) proteins normalized to β-actin relative to Ctrl. Pooled values of 4 independent experiments (WT-UCH-L1, n = 13 Ctrl or n = 9 WT OE) or of 2 independent experiments (I93M-UCH-L1, n = 7 Ctrl or n = 9 I93M OE) are shown as mean + /-SEM, *p = 0.0164, two-tailed Mann Whitney U test. Confocal images from 2 independent experiments with n = 3 of (i) ubiquitinated proteins (green) in podocytes. Arrowheads = ubiquitin aggregates, nephrin (red) depicts the glomerular filtration barrier or of (j) inclusion bodies, aggresomes = arrowheads), wheat germ agglutinin (WGA, red) highlights the podocyte plasma membrane. Podocytes = asterisks, DNA (Hoechst, blue). Source data are provided as a Source Data file.