Myo1e Impairment Results in Actin Reorganization, Podocyte Dysfunction, and Proteinuria in Zebrafish and Cultured Podocytes

<div><p>Background</p><p>Podocytes serve as an important constituent of the glomerular filtration barrier. Recently, we and others identified Myo1e as a key molecular component of the podocyte cytoskeleton.</p> <p>Results</p><p>Myo1e mRNA and protein was expressed in human and mouse kidney sections as determined by Northern blot and reverse transcriptase PCR, and its expression was more evident in podocytes by immunofluorescence. By specific knock-down of <i>MYO1E</i> in zebrafish, the injected larvae exhibited pericardial edema and pronephric cysts, consistent with the appearance of protein in condensed incubation supernate. Furthermore, specific inhibition of Myo1e expression in a conditionally immortalized podocyte cell line induced morphological changes, actin cytoskeleton rearrangement, and dysfunction in cell proliferation, migration, endocytosis, and adhesion with the glomerular basement membrane.</p> <p>Conclusions</p><p>Our results revealed that Myo1e is a key component contributing to the functional integrity of podocytes. Its impairment may cause actin cytoskeleton re-organization, alteration of cell shape, and membrane transport, and podocyte drop-out from the glomerular basement membrane, which might eventually lead to an impaired glomerular filtration barrier and proteinuria.</p> </div

Transferrin endocytosis assay of cultured podocytes.

<p>A) Podocyte endocytosis with FITC-transferrin in control group. B) Podocyte endocytosis with FITC-transferrin in negative control group. C) Podocyte endocytosis with FITC-transferrin in <i>MYO1E</i> knock-down group. Arrow: podocytes with endocytic FITC-transferrin granules. D) Comparison of podocyte endocytosis in different groups. **Compared with the results from the control and negative control groups, podocyte endocytosis in the <i>MYO1E</i> knock-down group was down-regulated, <i>P</i><0.01.</p

Proliferation of cultured podocytes by MTT assay.

<p>There was a significant decrease in the cell number in <i>MYO1E</i> knock-down podocytes compared to the cell number in control and scramble negative controls at 24, 48, 72, 96, 120, and 144 h. The inhibition of podocyte proliferation in the <i>MYO1E</i> knock-down group was most evident at 48 h.</p

A) Wild type zebrafish at 4 day post fertilization (dpf).Arrow indicating the glomeruli with visible GFP. B) Phenotype of Myo1e morphants at 4 dpf (days post fertilization) with Myo1e specific knock-down morpholino injected.

<p>Note the pericardial edema (arrow) and invisible GFP indicating the destruction of glomerular structure. C) Proteinuria (arrow indicating Vitellogenin at 70kD band) in concentrated incubation medium of zebrafish larvae after <i>MYO1E</i> knock-down. BSA: bovine serum albumin. D) Histological sections at the level of the glomerulus in zebrafish injected Myo1e MO. The expanded Bowman’s space and destroyed glomeruli observed in <i>MYO1E</i> knock-down zebrafish compared with wild type zebrafish.</p

Myo1e and F-actin expression in cultured podocytes.

<p>A) Myo1e expression in proliferative MPC5 at 33°C. B) Myo1e expression in differentiated MPC5 at 37°C. C) F-actin expression in proliferative MPC5 at 33°C. D) F-actin expression in differentiated MPC5 at 37°C. E) Myo1e expression in MPC5 (control). F) Myo1e expression in MPC5 (scrambled negative control). G) Myo1e expression in MPC5 after shRNA knock-down. Arrow: Myo1e expression was down-regulated in G than that in E & F.H) F-actin expression in controlled podocytes. I) F-actin expression in scrambled negative control podocytes. J) Change of F-actin organization in <i>MYO1E</i> knock-down podocytes.</p

Adhesion assay of cultured podocytes.

<p>A) Podocyte adhesion in control group. B) Podocyte adhesion in the scrambled negative control group. C) Podocyte adhesion in the <i>MYO1E</i> knock-down group. D) Comparison of podocyte adhesion assay in different groups. **Compared with the results of the control and negative control groups, podocyte adhesion in the <i>MYO1E</i> knock-down group was down-regulated, <i>P</i><0.05.</p

The results of <i>MYO1E</i> knock-down by real-time PCR and Western Blotting.

<p>A, B) Results of <i>MYO1E</i> knock-down by real-time PCR. C, D) The result of Western blotting of control, negative control, and <i>MYO1E</i> knock-down. **Compared with the results of control and negative control, Myo1e expression was noted to be down-regulated by real-time PCR and Western blotting, <i>P</i><0.01.</p

Expression of Myo1e in human and mouse tissues.

<p>A) Myo1e expression in normal human kidney. Scale bar: 100μm. B) Myo1e expression in normal human kidney. Scale bar: 20μm. C) Co-expression of synaptopodin and Myo1e in normal human podocytes observed by immunofluorescence and confocal microscope. Scale bar: 10μm. D) mRNA expression of Myo1e in multiple mouse tissues by RT-PCR. 1=purified glomeruli, 2=kidney without glomeruli, 3=lung, 4=brain, 5=testis, 6=spleen, 7=heart, 8=muscle, 9=total kidney, 10=β-actin. Myo1e was expressed abundantly in kidney (both in glomeruli and other tissue within the kidney) E) mRNA expression of Myo1e in multiple mouse tissues by Northern blot. 1=heart, 2=brain, 3=spleen, 4=lung, 5=liver, 6=muscle, 7=total kidney, 8=testis.</p

Detachment assay of cultured podocytes.

<p>A) Compared with the results before treatment, the number of cells attached on bottom of the cultural bottle was significantly decreased in the three groups after PAN treatment. This phenomenon was more evident in <i>MYO1E</i> knock-down podocytes compared with the control and negative control groups after PAN treatment. B) The differences among the control, negative control, and <i>MYO1E</i> knock-down groups. **Compared with the results in the control and negative control groups, podocyte detachment in the <i>MYO1E</i> knock-down group was down-regulated, <i>P</i><0.01.</p

Vascular expression and genetic ablation of the <i>Gpr116</i> gene in mouse.

<p>A. <i>Gpr116</i> mRNA expression in the published organ-specific EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref045" target="_blank">45</a>]. B. <i>Gpr116</i> mRNA expression assessed by qRT-PCR in EC from 3-weeks-old and 3-months-old ROSA^mT/mG x Tie2-Cre mice. Results are normalized by brain EC expression. Error bars represent SD. (n = 3 mice per genotype). C. <i>Gpr116</i> mRNA expression in the published brain-specific vascular and EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref046" target="_blank">46</a>]. D. Schematic representation of the area targeted by homologous recombination in the <i>Gpr116</i> locus. Dotted lines indicate the regions of homology in between the Gpr116 locus and the cassette. The dark grey arrow indicates the position of WT primers: both are located in the untranslated region of exon 21, but the area recognized by the forward primer is lost in the mutant allele. The light grey arrow represents the knockout primer, specific for the cassette. Critical Gpr116 domains (SEA, IgG, GAIN and transmembraine, TM) are indicated above the corresponding encoding exons. E. Example of genotyping PCR products on genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. WT primers amplify a 325-bp fragment in the 3´UTR exon 21 of <i>Gpr116</i> gene representing the wild type allele. The 401 bp band is specific for the mutant allele. F. Example of genotyping PCR products using genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. LacZ primers amplify a 210 bp fragment in LacZ gene present in the insert replacing exon 4 to 21. G. <i>Gpr116</i> exon 17–18 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). H. <i>Gpr116</i> exon 2–3 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). I. mRNA detection by RNAscope in brain cortical capillary vessels from <i>Gpr116</i> WT (top row), knockout (middle row) and ROSA^mTmG X Tie2-Cre mice (lower row) at 3 weeks. On the left column, note that only the probe signal (red) and the nuclear staining (blue) are visible. On the right column, an endothelial staining (green) is merged to the probe and the nuclear signal: a CD31 antibody staining is on the two upper rows, while Tie-2 Cre mediated GFP is on the lower row. (n = 1 mouse per genotype).</p