Untersuchung des Einflusses von Palladin auf das Aktinzytoskelett von Podozyten

BackgroundPodocyte loss and effacement of interdigitating podocyte foot processes are themajor cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. MethodsWe knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld2/2mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin anda-actinin-4. Furthermore, palladin-knockdownpodocytesweremore susceptible todisruptionof theactin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an earlydevelopmental stage.ComparedwithPodoPalld+/+ controls, PodoPalld2/2micedevelopedglomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld2/2 mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Conclusions Palladin has an important role in podocyte function in vitro and in vivo. J Am Soc Nephrol 29: 1662–1678, 2018. doi: https://doi.org/10.1681/ASN.2017091039 Palladin, an actin-associatedprotein,whichwasfirst described by Parast and Otey in fibroblasts and epithelial cells,1 plays a pivotal role in the stability and dynamics of the actin cytoskeleton. It has already been reported that different palladin isoforms are expressed in a tissueand development-dependent way.1–3 Currently, UniProt describes seven palladin transcript variants (72–152 kD) in mice that are Received September 28, 2017. Accepted March 28, 2018. Published online ahead of print. Publication date available at www.jasn. org. Correspondence: Dr. Nicole Endlich, Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany. Email: nicole.endlich@uni-greifswald.de Copyright © 2018 by the American Society of Nephrology 1662 ISSN : 1046-6673/2906-1662 J Am Soc Nephrol 29: 1662–1678, 2018 generated by alternative splicing and start-sites.3 However, it seems likely that additional isoforms exist. It was shown that the knockout (KO) of palladin, which is highly and ubiquitously expressed inmouse embryos, resulted in severe tube closure defects and embryonic lethality before embryonic day 15.5. 4,5 This might be caused by a disturbance of the proliferation and differentiation as well as by a reduction of cell adhesion in neuronal cells of the palladin-KO mice.5 In cell culture, it was observed that palladin downregulation leads to a reduction of robust stress fibers in rat choriocarcinoma (Rcho-1) cells and cultured mouse embryonic fibroblasts as well as to a disruption of stress fibers in human glioblastoma (U251) cells.1,6Moreover, the neurite outgrowth of neuroblastoma cells and the growth cone formation were diminished after the knockdown of palladin.7 Recently, studies have revealed that palladin carries not only specific binding sites for F-actin and the actin-binding protein a-actinin-1,8,9 but also for other actin-associated proteins such as the focal adhesion protein Lasp-13 or proteins such as VASP or profilin, which are responsible for actin dynamics.10,11 Taken together, these results showed that palladin is a scaffolding protein regulating actin nucleation and polymerization as well as cell adhesion in different cell types. Azatov et al.12 further showed that palladin modulates force generation and mechanosensitivity in tumor-associated fibroblasts. Another interesting finding is that the function of palladin depends on the phosphorylation status. Asano et al. demonstrated that palladin becomes phosphorylated by ERK after stimulation with the epidermal growth factor (EGF). It was speculated that the ERK pathway might be involved in the EGF–mediated cell migration.13 The data further suggest that palladin has an antimigratory function, which was also postulated by Chin and Toker6who studied the role of palladin in breast cancer. For a long time, it has beenwell known that themorphology and function of podocytes, a terminally differentiated cell type in the glomerulus which is part of the glomerular filtration barrier, are highly dependent on the actin cytoskeleton. These cells express actin specifically in a cortical net near the plasma membrane as well as in filament bundles spanning two neighboring podocyte foot processes.14 A disturbance of the actin cytoskeleton, e.g., by the loss or mutation of actin-binding proteins such as a-actinin-4 and CD2AP, severely influences the morphology and function of foot processes and the integrity of the glomerular filtration barrier.15,16 In a recent study, we showed that the expression of palladin in the kidney is essentially restricted to podocytes and arteries.17However, nothing is known about the role of palladin in kidney function. Therefore, this study focused on the expression and function of palladin in podocytes of mouse and human kidney as well as on the behavior of epithelial cells in living zebrafish embryos, a well established animal model. Because at least seven isoforms of palladin exist that are expressed in a developmentaland tissue-dependent way, we further tried to clarify which isoforms are expressed in podocytes in vitro and in vivo. Our study reveals that palladin is functionally important for the morphology and behavior of podocytes in vitro as well as in vivo.