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The Role of Palladin in Podocytes

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The Role of Palladin in Podocytes BASIC RESEARCH www.jasn.org The Role of Palladin in Podocytes Nadine Artelt,1 Tim A. Ludwig,1 Henrik Rogge,1 Panagiotis Kavvadas,2 Florian Siegerist ,1 Antje Blumenthal,1 Jens van den Brandt,3 Carol A. Otey,4 Marie-Louise Bang,5,6 Kerstin Amann,7 Christos E. Chadjichristos,2 Christos Chatziantoniou ,2 Karlhans Endlich ,1 and Nicole Endlich1 1Department of Anatomy and Cell Biology and 3Central Core and Research Facility of Laboratory Animals (ZSFV), University Medicine Greifswald, Greifswald, Germany; 2National Institute for Health and Medical Research (INSERM), Unité Mixte de Recherche (UMR)-S1155, Tenon Hospital, Sorbonne Universités, Paris, France; 4Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill (UNC), Chapel Hill, North Carolina; 5Institute of Genetic and Biomedical Research, UOS Milan, National Research Council, Milan, Italy; 6Humanitas Clinical and Research Center, Rozzano, Milan, Italy; and 7Department of Nephropathology, University Medicine Erlangen, Erlangen, Germany ABSTRACT Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major 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. Methods We 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 inves- tigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld2/2 mice) by immunoflu- orescence 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 fila- ment staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and a-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin 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 early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld2/2 mice developed glomeruli 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-associated protein, whichwasfirst described by Parast and Otey in fibroblasts and ep- Received September 28, 2017. Accepted March 28, 2018. 1 ithelial cells, plays a pivotal role in the stability and Published online ahead of print. Publication date available at dynamics of the actin cytoskeleton. It has already www.jasn.org. been reported that different palladin isoforms are Correspondence: Dr. Nicole Endlich, Department of Anatomy expressed in a tissue- and development-dependent and Cell Biology, University Medicine Greifswald, Greifswald, way.1–3 Currently, UniProt describes seven palladin Germany. Email: [email protected] transcript variants (72–152kD)inmicethatare Copyright © 2018 by the American Society of Nephrology 1662 ISSN : 1046-6673/2906-1662 JAmSocNephrol29: 1662–1678, 2018 www.jasn.org BASIC RESEARCH generated by alternative splicing and start-sites.3 However, it Significance Statement seems likely that additional isoforms exist. It was shown that the knockout (KO) of palladin, which is Podocytes play a key role in the formation of a proper glomerular highly and ubiquitously expressed in mouse embryos, resulted filtration barrier. Their complex 3D morphology is highly dependent in severe tube closure defects and embryonic lethality before on an intact actin cytoskeleton. This manuscript describes the role of palladin, an essential actin-binding protein, for podocyte function in 4,5 embryonic day 15.5. This might be caused by a disturbance vitro and in vivo. The results demonstrate that cultured podocytes of the proliferation and differentiation as well as by a reduction developed disorganized actin filaments and smaller focal adhesions of cell adhesion in neuronal cells of the palladin-KO mice.5 In after the knockdown of palladin. Moreover, the podocyte-specific cell culture, it was observed that palladin downregulation leads palladin-knockout mouse showed glomeruli with a disturbed mor- to a reduction of robust stress fibers in rat choriocarcinoma phology and a mild effacement of podocyte foot processes. After fi the injection of nephrotoxic serum, a model for GN, palladin- (Rcho-1) cells and cultured mouse embryonic broblasts as knockout mice developed higher levels of proteinuria than controls. well as to a disruption of stress fibers in human glioblastoma Taken together, our results demonstrate an important role of pal- (U251) cells.1,6 Moreover, the neurite outgrowth of neuroblas- ladin for podocyte architecture as well as for proper filtration. toma cells and the growth cone formation were diminished after the knockdown of palladin.7 podocytes in vitro and in vivo. Our study reveals that palladin Recently, studies have revealed that palladin carries not only is functionally important for the morphology and behavior of specific binding sites for F-actin and the actin-binding protein podocytes in vitro as well as in vivo. 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 dynam- METHODS ics.10,11 Taken together, these results showed that palladin is a scaffolding protein regulating actin nucleation and polymer- Cell Culture ization as well as cell adhesion in different cell types. Azatov Conditionally immortalized podocytes (CLS Cell Line Service, et al.12 further showed that palladin modulates force genera- Germany) were handled as described previously.18 All exper- tion and mechanosensitivity in tumor-associated fibroblasts. iments were performed using differentiated podocytes. Another interesting finding is that the function of palladin Knockdown of palladin (PalldKD) was achieved using Silencer depends on the phosphorylation status. Asano et al. demon- Select siRNA Palld1 siRNA (s90889), Palld2 siRNA (s90890), strated that palladin becomes phosphorylated by ERK after and control siRNA (4390846), respectively (Ambion; Thermo stimulation with the epidermal growth factor (EGF). It was Fisher Scientific, Waltham, MA). For transfection, the K2 Trans- speculated that the ERK pathway might be involved in the fection System (Biontex, Germany) was used according to the EGF–mediated cell migration.13 The data further suggest manufacturer’s instructions. After 72 hours cells were used for that palladin has an antimigratory function, which was also experiments, except for regulation studies of cytoskeletal genes postulated by Chin and Toker6 who studied the role of palladin that were performed on double-transfected PalldKD cells. in breast cancer. For a long time, it has been well known that the morphology Immunocytochemistry and function of podocytes, a terminally differentiated cell type Cultured podocytes were fixed with 2% paraformaldehyde and in the glomerulus which is part of the glomerular filtration incubated with the following primary antibodies: palladin barrier, are highly dependent on the actin cytoskeleton. These (Proteintech Group, UK), synaptopodin (Progen, Germany), cells express actin specifically in a cortical net near the plasma vinculin (Sigma-Aldrich), and a-actinin-4 (immunoGlobe, membrane as well as in filament bundles spanning two neigh- Germany). Alexa Fluor 647-conjugated (Thermo Fisher Sci- boring podocyte foot processes.14 A disturbance of the actin entific) or Cy3-conjugated secondary antibodies (Dianova/ cytoskeleton, e.g., by the loss or mutation of actin-binding Jackson Immuno Research, Germany) were used. F-actin proteins such as a-actinin-4 and CD2AP, severely influences was stained with Alexa Fluor 488–phalloidin (Thermo Fisher the morphology and function of foot processes and the integ- Scientific). Images were taken by a Leica TCS SP5 confocal rity of the glomerular filtration barrier.15,16 laser scanning microscope (Leica Microsystems, Germany). In a recent study, we showed that the expression of palladin in the kidney is essentially restricted to podocytes and ar- Actin Dynamics and Focal Adhesion Analysis teries.17 However, nothing is known about the role of palladin Confluent podocytes were incubated with latrunculin A, cy- in kidney function. Therefore, this study focused on the ex- tochalasin D, and jasplakinolide, respectively (final concen- pression and function of palladin in podocytes of mouse and trations 0.5 mM; Sigma-Aldrich). Podocytes were stained human kidney as well as on the behavior of epithelial cells in for F-actin, nuclei, and palladin to confirm knockdown effi- living zebrafish embryos, a well established animal model. Be- ciency. To investigate the actin cytoskeleton, .150 podocytes cause
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