case_images: PMC5158186_01
case_images: (A), histologic findings in the patient's kidney biopsy. Periodic acid-Schiff (PAS) staining (magnification 200X) shows segmental glomerulosclerosis and interstitial fibrosis with multiple foci of microcystic tubular dilation. (B), Electron microscopy images shows glomerular podocyte foot processes effacement (arrows) (magnification 20000X). (C), serum creatinine and urine protein/creatinine ratio of the patient over a period of a year.
case_images: pone.0167467.g001.jpg
case_images: PMC5158186_01_pone.0167467.g001.jpg
case_images: pone.0167467.g001
case_images: Histological exam demonstrated FSGS with collapsing features (Fig 1A). Electron microgram showed diffuse podocyte effacement (Fig 1B). She was resistant to steroid treatment, creatinine increased (Fig 1C) and after six weeks, the prednisone was tapered.
case_images: (A), pedigree of the patient and nuclear family. Partial ACTN4 genomic sequence from the patient (red circle) shows a heterozygous T793C substitution that results in a Y265H ACTN4 amino acid substitution. The patient's mother (white circle) does not carry this mutation. The genotype of the father (white square) is unknown. (B), mutation prediction software results for Y265H ACTN4 mutation and sequence alignment showing sequence conservation of ACTN4 Y265 across difference species. (C), representative image of coomassie blue stained SDS-PAGE gel showing an F-actin bundling experiment. The distribution of ACTN4 and F-actin in supernatant (S) and pellet (P) after centrifugation is shown. Arrows point out the bundled F-actin in the pellet. 55% of F-actin (42kDa) is bundled when crosslinked by K255E ACTN4 and Y265H ACTN4 proteins while only 32% of F-actin is bundled when crosslinked by WT ACTN4. (D), representative immunofluorescence staining images. Rhodamine phalloidin stains actin (red). GFP tagged ACTN4 is green. Hoechst 33342 stains the nuclei (blue). K255E ACTN4 and Y265H ACTN4 form aggregates within the cells. Scale bar is 5mum shown in WT merged image. 2E, representative contractile force maps for podocyte expressing the different forms of ACTN4. Different colors correlate with different levels of force, with red being the largest degree of force. Arrows are force vectors. Bright field image of each cell is included in top left of each force map. Root mean square value (RMST, an index of contractile force) is plotted. Data are from one of the three independent experiments, and are expressed as mean +- SEM. *P<0.05 indicate significant difference from WT.
case_images: pone.0167467.g002.jpg
case_images: PMC5158186_01_pone.0167467.g002.jpg
case_images: pone.0167467.g002
case_images: We confirmed this result by Sanger sequence (Fig 2A). Provean, Polyphen, and SIFT prediction software predicts this amino acid change as deleterious (Fig 2B). We found more bundled F-actin when F-actin was incubated with either Y265H ACTN4 or K255E ACTN4 compared to the WT ACTN4 (Fig 2B). ACTN4-actin aggregates within the cells (Fig 2). Additionally, we observed a clear increase in the amount of stress fibers present in the mutant ACTN4 podocytes. We observed that podocytes, transfected with either K255E (386.9 +- 43.54 Pa, mean +- SEM) or Y265H (366.8V+- 32.98 Pa) ACTN4 were more contractile than podocytes transfected with WT ACTN4 (151.7 +- 20.22 Pa) (Fig 2D).