The Role of Palladin in Podocytes

BASIC RESEARCH www.jasn.org

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 investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld2/2 mice) 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 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 at least seven isoforms of palladin exist that are ex- were analyzed with the software F_Seg.19 pressed in a developmental- and tissue-dependent way, we For quantification of focal adhesion area, cultured podo- further tried to clarify which isoforms are expressed in cytes were stained for vinculin and at least 60 cells per group

J Am Soc Nephrol 29: 1662–1678, 2018 Palladin Regulates Podocyte Function 1663 BASIC RESEARCH www.jasn.org were studied in an automated fashion by the developed custom Generation of PodoPalld2/2 and PodoPalld-R26R software “Focal Contact Segmentation and Analysis Tool,” as Mice described previously.20 To generate a podocyte-specific palladin-KO (PodoPalld2/2) mouse, 2.5P-Cre mice (kindly provided by Dr. Moeller et al.22) Wound Assay and mice containing a loxP site flanked exon (GGGGTTCCCA Podocytes were seeded on a m-Dish (Ibidi, Germany) and AAGAAGTCCA GTAGAACTGC TAGAATTGCC TCTGAT- transfected with control and Palld1 siRNA, respectively. Cell GAGG AGATTCAAGG CACAAAGGAT GCTGTCATCC AA- migration was observed with a Leica DMI 6000B fluorescence GACCTGGA ACGGAAGCTT CGCTTCAAGG AG- microscope (Leica Microsystems) that was heated to 38°C by a GACCTTCT GAACAATGGC CAACCG; Ensemble Gene ID: temperature control system (Life Imaging Services, Switzer- ENSMUSG00000058056 or NCBI Gene ID: 72333, generated land). Images were taken every 10 minutes and processed with by M.-L.B., manuscript in preparation by Mastrotoraro et al.) Volocity software (PerkinElmer). in the palladin gene were mated. For experiments, Podo- Palld2/2 mice with heterozygous Cre-recombinase expres- Isolation of Glomeruli sion were used. Mice without Cre-recombinase expression Glomeruli were isolatedwithmagnetic Dynabeads as described were used as controls (PodoPalld+/+). Experiments were previously.21 done with 6-month-old male mice with C57BL/6 genetic background (at least n=3 of each group). RNA Analysis PodoPalld mice were mated with R26R1M mice (MPI of Im- Samples from transfected cells/glomeruli/kidneys/zebrafish munobiology and Epigenetics, Germany) containing a lacZ gene larvae were processed in Tri-Reagent (Sigma-Aldrich) accord- flanked by loxP sites, to verify podocyte-specificCre-recombinase ing to the manufacturer’s instructions. One microgram of total expression.23 Experiments were performed on 2-month-old 2 RNA was reverse transcribed using the QuantiTect Reverse male/female C57BL/6 PodoPalld-R26R1M (cre+/ , lacZ/lacZ) and 2 2 Transcription Kit (Qiagen, Germany). Transcription of zebra- PodoPalld-R26R1M (cre / , lacZ/lacZ) mice (at least n=3 for each fish RNA was performed using SuperScript Reverse transcrip- group). Mice were housed as described previously.24 Primers for tion (Thermo Fisher Scientific). genotyping are listed in Supplemental Table 1. For RT-PCR Taq Polymerase (Peqlab) was used for cells/ mouse samples and Platinum Taq DNA Polymerase (Thermo Mouse Model of Nephrotoxic Serum–Induced GN and Fisher Scientific) for zebrafish samples, respectively. RT-PCR Analysis of Renal Function was performed on a Mastercycler gradient (Eppendorf AG, Two- to 3-month-old male PodoPalld+/+ and PodoPalld2/2 Germany). mice were injected with nephrotoxic serum (NTS) over 2 con- qRT-PCR was performed on a LightCycler Nano (Roche, secutive days (n=8 per group) or with PBS as control (n=4 per Germany). Primers are listed in Supplemental Table 1. group). Urine samples were collected at days 0, 4, 8, and 12 and blood samples before euthanasia. Proteinuria was measured Western Blot Analysis with a Konelab analyzer (Thermo Fisher Scientific) and nor- Western blot was performed as already described by Kliewe malized to urine creatinine. BUN and plasma creatinine levels et al.20 Briefly, transfected cells/glomeruli/kidneys were solved were measured with an enzymatic method (Konelab analyzer), in RIPA buffer (Sigma-Aldrich) supplemented with Halt Pro- respectively. tease Inhibitor (Thermo Fisher Scientific). Adjusted protein amounts were blotted and the membrane was incubated with Histology primary antibodies against palladin and Gapdh (Santa-Cruz) Mouse kidneys were harvested and fixed. Paraffin sections (4 overnight at 4°C. A goat anti-rabbit IgG-HRP (Santa Cruz) mm) were performed on a Leica SM 2000R (Leica Microsys- was used for 45 minutes and the signal was detected with the tems), stained with hematoxylin and eosin, and analyzed with Clarity Western ECL Blotting Substrate (Bio-Rad). Protein an Olympus BX50 microscope (Olympus Europe, Germany). expression was normalized to Gapdh as a housekeeping Samples were snap-frozen in liquid nitrogen using Tissue- protein. Tek (Sakura, Germany) and cut on a Leica CM-3050-S Cryo- stat. Cryosections of PodoPalld mice (7 mm) were stained for Human Kidney Biopsy Samples F-actin using Alexa Fluor 488–phalloidin. For detection of The use of remnant kidney biopsy material was approved by the b-galactosidase activity, sections of PodoPalld-R26R mice Ethics Committee of the Friedrich-Alexander University of (9 mm) were fixed with 2% glutaraldehyde and incubated in Erlangen-Nürnberg, waiving the need for retrospective con- X-gal solution.25 sent for the use of archived rest material. Colocalization studies were performed on biopsy samples of healthy patients Immunohistochemistry (n=3), patients with FSGS (n=4), and patients with DN After deparaffinization, sections of mouse kidneys and hu- (n=5). Three to seven glomeruli of each biopsy sample were man biopsy samples were rehydrated and unmasked by heat- analyzed. ing in a pressure cooker. The following primary antibodies

1664 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1662–1678, 2018 www.jasn.org BASIC RESEARCH were used for immunofluorescence staining: palladin, palla- Staining and Live Cell Imaging of Zebrafish Larvae din622 (Palld622), synaptopodin, a-actinin-4, and nephrin Larvae were fixed in methanol/acetone (1:1) and incubated (Progen). Alexa Fluor 488– andCy3-conjugatedsecondary with rabbit anti-palladin622 antibody and Alexa Fluor 488– antibodies were used. Images were acquired using a Leica phalloidin. For palladin detection, larvae were incubated with TCS SP5 confocal laser scanning microscope and super- Cy3-conjugated anti-rabbit secondary antibody. resolution microscope (Elyra PS.1; Carl Zeiss Microscopy, For in vivo observation, zebrafish larvae at 30 hours post- Germany). fertilization were used and images were taken every 12 minutes For immunohistochemistry (IHC), the Vectastain kit (Vec- for 1 hour using a Leica TCS SP5 confocal LSM. tor Laboratories) was used following manufacturer’s instructions and palladin was detected with palladin antibody. Statistical Analyses All data are given as mean6SD or 6SEM, analyzed by un- Analysis of Glomerular Morphology on paired t test with repeated measurements. Differences were Semithin Sections regarded as significant at a P value ,0.05. Kidneys were harvested,24 embedded in Epon 812 (SERVA, Germany), and semithin sections were cut and stained with Richardson’s (Azur II/Methylene blue). At least 50 glomeruli RESULTS of PodoPalld+/+ mice and PodoPalld2/2 mice were catego- rized into glomeruli with (1) normal morphology, (2) dilated Palladin Is Expressed in Podocytes In Vivo and In Vitro capillaries, and (3) affected podocytes (podocytes with cyst RT-PCR as well as IHC of mouse kidney sections and cultured and enlarged subpodocyte space). mouse podocytes (CPs) demonstrated that podocytes express For qualitative ultrastructural investigations using electron palladin (Figure 1). By double staining of cultured podocytes microscopy, ultrathin sections were cut and contrasted with with antibodies against the podocyte-specific and actin-associated 5% uranyl acetate and lead citrate. proteins synaptopodin and palladin, we found that both proteins are colocalized in dense bodies along actin filaments (Figure Podocyte Foot Process Effacement 1C). Because it was also reported that palladin is a part of focal Measurement Procedure adhesions, we stained for palladin and for the focal adhesion The slit diaphragm density was measured by structured illu- protein vinculin. As shown in Figure 1C, palladin is also ex- mination microscopy as described recently.26 Briefly, image pressed in focal adhesions. stacks of 20 glomeruli in three individual mice per group To determine the specific isoforms (UniProt ID Q9ET54 were acquired using a Zeiss Elyra PS.1 system equipped and D3Z1J5, Supplemental Figure 1A) that are expressed in with a 633 (NA 1.4) oil immersion objective. Z-Stacks podocytes of mice in vivo and in vitro, we performed western were recorded with a size of 2430 pixel2 (78.35 mm2) blots of isolated glomeruli as well as cultured podocytes (Sup- with a slice-to-slice distance of 0.2 mm over approximately plemental Figure 1B). Because antibodies often selectively 3.5 mm using the 561 nm laser. 3D structured illumination bind specific isoforms, we further performed qRT-PCR. As microscopy reconstruction was performed with the Zeiss shown in Supplemental Figure 1B, a strong signal was found ZEN Software. For automatic assessment of the slit dia- in isolated glomeruli but not in cultured podocytes by using phragm density, multiple areas per image stack with a plan primers for exons 2–3. This signal may correspond to the view on the slit diaphragm were segmented and measured 60-kD isoform identified in the western blots or to a new using a custom-made FIJI-based macro.26 The slit dia- N-terminal isoform. phragm density was stated as length of the slit diaphragm AsshowninSupplementalFigure1B,podocytesin vivo 2 per glomerular capillary area in mm 1. Means of both express 50-, 60-, and 73-kD isoforms in contrast to CP that groups were compared using unpaired t test using Prism 5 expresses 50-, 73-, and 140-kD isoforms. (GraphPad). Knockdown of Palladin Affects the Actin Cytoskeleton Zebrafish and Focal Adhesions Zebrafish of the AB background were bred in a pH- and tem- To investigate the role of palladin in the morphology and sta- perature-controlled facility as previously described.27 bility of the actin cytoskeleton, palladin-knockdown (PalldKD) experiments were performed. After the transfection Injection of Morpholinos of CPs with two different siRNAs, Palld1 and Palld2 siRNA, the Morpholino (MO) injection into fertilized zebrafish eggs was almost complete loss of the protein and mRNA expression described by Kotb et al.28 MOs were synthesized by Gene Tools could be shown by immunofluorescence staining as well as LLC (Philomath) and the following sequences were used: by western blot (Figure 2, A and B) and RT-PCR/qRT-PCR control MOs (CtrlMO) 59-CCTCTTACCTCAGTTACAATT- (280%64%, n=4, P,0.001). TATA-39 and palladin MOs (PalldMO) 59-TGTCATTC- To study the influence of palladin on the stability of the actin CAGCTCCCGTCCTGCAT-39. cytoskeleton, we compared the actin cytoskeleton stained with

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Figure 1. Palladin is colocalized with F-actin, synaptopodin and vinculin in podocytes. (A) Staining of murine kidney sections with anti- palladin antibody indicated a strong expression of palladin in podocytes. Scale bar represents 10 mm. (B) Additionally, palladin expression in kidney, glomeruli, and CP was conﬁrmed by RT-PCR using palladin primers spanning exons 18–20. (C) Double staining of cultured podocytes showed that palladin is colocalized with F-actin, synaptopodin, and vinculin. Scale bar represents 20 mm. DAPI, 49,6-diamidino-2-phenylindole.

Alexa Fluor 488–phalloidin of PalldKD podocytes and con- in PalldKD podocytes was smaller than in Ctrl (0.63 mm2 trol-transfected podocytes (Ctrl) by immunofluorescence. versus 0.88 mm2). Furthermore, we found by qRT-PCR that PalldKD podocytes developed fewer parallel actin filaments the mRNA of the focal adhesion protein vinculin was signifi- in contrast to Ctrl. The reduction of actin filaments was associ- cantly downregulated by 30%612% (n=7, P,0.05) in ated with a significantly reduced expression of the podocyte-spe- PalldKD podocytes. In contrast, the mRNA expression of cific and actin-binding proteins synaptopodin and a-actinin-4 b-actin (92%611%), talin-1 (90%612%), b1-integrin mRNA (Figure 2, C and D, Supplemental Figure 2). (90%67%), and Rac1 (100%613%) was unaffected in Moreover, we analyzed the influenceofpalladinonthe PalldKD podocytes (Figure 2D). In summary, our data show number and size of focal adhesions. For determination of that the loss of palladin leads to a higher number of small focal the size of the focal adhesions, we quantified the areas of the adhesions and to a concomitant reduction of vinculin. focaladhesionsusing custom-designed image analysissoftware (cf. Methods section). Our analysis revealed that PalldKD po- Knockdown of Palladin Influences the Migration of docytes possessed more focal adhesions with an area of #0.5 Podocytes and Actin Polymerization mm2 and fewer focal adhesions with an area of $1 mm2 com- Because the actin cytoskeleton was affected by the PalldKD, we pared with Ctrl (Figure 2E). Interestingly, the percentage of studied the dynamics of the polymerization/disassembly of focal adhesions with an area of 0.75 mm2 remained un- actin filaments by the use of two differently acting inhibitors changed. We found that the average area of focal adhesions of actin polymerization, cytochalasin D and latrunculin A.

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Figure 2. Palladin knockdown leads to a downregulation of cytoskeletal proteins in cultured podocytes. For studying the role of palladin in vitro, CPs were transfected with siRNA. (A) To conﬁrm the knockdown efﬁciency, podocytes were stained for palladin. Scale bar represents 50 mm. (B) The strong reduction of palladin expression was also shown by western blot (20 mg/lane), RT-PCR, and qRT- PCR (palladin primers spanning exons 18–20). Palladin mRNA expression of Palld1 or -2 siRNA-transfected podocytes was normalized to Gapdh and control-transfected (Ctrl) podocytes (means6SD, n=4, ****P,0.001). (C) Cultured mouse PalldKD podocytes showed

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Cytochalasin D as well as latrunculin A induced a disassembly Palladin Knockdown Affects the Morphology of of the actin filaments in PalldKD podocytes compared with the Zebrafish Embryos Ctrl (Figure 3A, Supplemental Figure 3). For quantification, Because the KO of palladin in mice is lethal in utero,4 the we determined the filament length and size distribution with a zebrafish model was used as an in vivo model to study the recently developed image analysis software.19 Actin filaments role of palladin during development. After the injection of were classified into three categories: thin actin filaments, thick specific MOs against palladin (PalldMO) into fertilized zebra- actin filaments, and nonfiber-like F-actin. Under baseline con- fish eggs at the one- to four-cell stage, we found that nearly ditions, PalldKD podocytes had fewer thin actin filaments, 100% of the PalldMO embryos died after 16–18 hours. more nonfiber-like F-actin, a lower number of longer (.10 To increase the survival rate, we injected the PalldMO into mm) filaments, and a reduced total filament length by about the eggsat later time points (eight- to 32-cellstage)togenerate a one third as compared with Ctrl (Figure 3B, Supplemental mosaic phenotype. These PalldMO embryos survived beyond Figure 3). PalldKD podocytes that were treated with cytocha- 16–18 hours (Figure 4A). The knockdown of palladin was lasin D or latrunculin A showed decreased numbers of thin confirmed by RT-PCR and IHC (Figure 4, B and C). filaments (35.2% and 32.1%) in contrast to Ctrl. The number IHC of the embryos revealed that the actin cytoskeleton was of actin filaments with a length between 10 and 30 mmwas disturbed due to the knockdown of palladin. Instead of highly decreased by 46.8%613.7% after cytochalasin D and by organized F-actin bundles in myotomes of control embryos, 43.0%613.7% after latrunculin A treatment over 25 minutes. PalldMO embryos developed only a few actin bundles that were The number of filaments with a width between 1.5 and 3 mm disorganized (Figure 4D). Further, we observed that instead of decreased by 44.3%65.4% after cytochalasin D and by 46.4%6 an intact flat epithelial layer, the epithelial cells of the PalldMO 4.5% after latrunculin A treatment of 25 minutes. However, dur- embryos had a rounded morphology (Figure 4E) and instable ing the incubation period over 25 minutes, the reduction of the cell-cell contacts. Exposing the embryos to very faint mechan- total filament length was similar in Ctrl and PalldKD in cytocha- ical forces resulted in a dissociation of the whole cell layer. lasin D– (39.4%66.0%) and latrunculin A–treated PalldKD cells Beside this, the trajectories of the migration of single epithelial (41.3%66.1%) (Figure 3B, Supplemental Figure 3). cells (insets of Figure 4E) demonstrated that cell migration in Furthermore, we studied the influence of jasplakinolide, an PalldMO embryos is undirected in contrast to that observed in F-actin stabilizing drug leading to a disruption of filaments, on control embryos (Figure 4E, Supplemental Material). the cytoskeleton of PalldKD and Ctrl podocytes. After an in- Summarizing, palladin knockdown in zebrafish embryos cubation of 25 minutes, the disrupted actin filaments vanished leads to disorganized actin filaments, impaired cell migration, completely in PalldKD cells in contrast to the Ctrl (Supple- and early embryonic death. mental Figure 4). Furthermore, it was already reported that palladin influ- Podocyte-Specific KO of Palladin in Mice Disturbs the ences cell migration.6 Therefore, we studied whether the mo- Morphology of Glomeruli and Podocytes tility of podocytes depends on palladin expression. PalldKD Because the KO of palladin in mice is lethal, we generated mice and Ctrl podocytes were cultured in a migration chamber as with a podocyte-specificKO of palladin(PodoPalld2/2) using confluent layers separated into two distinct areas by a the Cre/loxP system. We verified the expression of the NPHS2 pro- removable plastic partition in the middle of the chamber. After moter-dependent Cre-recombinase by an X-gal staining of Podo- 2 removing the partition, the dynamics of podocytes were fol- Palld-R26R1M (cre+/ ,lacZ/lacZ) kidney sections (Figure 5A). Addi- lowed over 22 hours by brightfield microscopy. We found that tionally, we confirmed the palladin KO by IHC, RT-PCR/qRT-PCR, PalldKD podocytes migrated significantly faster into the gap and western blot analysis (Figure 5, B–D). The detection of a (Figure 3C, Supplemental Material). Furthermore, PalldKD weak signal for palladin by RT-PCR is caused by a slight con- podocytes developed highly dynamic structures such as lamel- tamination of the isolated glomeruli with small vessels that still lipodia, ruffles, and ring-like structures, respectively (Figure express palladin (Figure 5, B and C, Supplemental Figure 5). 3D, Supplemental Material). Tostudy the morphology of PodoPalld2/2 glomeruli, kid- Taken together, PalldKD podocytes had fewer actin fibers, ney sections were performed and stained with Alexa Fluor were more susceptible to inhibition of actin polymerization, 488–phalloidin, hematoxylin and eosin, and Richardson’s and exhibited a more migratory phenotype. stain, respectively. The stained sections showed a marked

weaker signals for vinculin and a marked reduction of parallel actin stress fibers in immunofluorescence staining compared with control- transfected podocytes. Scale bar represents 50 mm. (D) Additionally, a significant reduction of vinculin, synaptopodin, and a-actinin-4 mRNA was confirmed by qRT-PCR. No remarkable changes were observed for b-actin, talin-1, b1-integrin, and Rac1, respectively (mean6 SEM, n=7, *P,0.05, **P,0.01, ****P,0.001). (E) Furthermore, focal adhesions were analyzed using podocytes stained for vinculin. PalldKD podocytes showed an increased number of small adhesions (#0.5 mm2) and a decreased number of adhesions with an area of $1 mm2 compared with Ctrl. The percentage of focal adhesions with an area of 0.75 mm2 remained unchanged. DAPI, 4’,6-diamidino- 2-phenylindole; qRT-PCR, quantitative reverse transcription PCR; siRNA, small interfering RNA; WB, Western Blot.

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Figure 3. Palladin knockdown in cultured podocytes influences actin polymerization and stabilization as well as cell motility. (A) CPs were incubated with cytochalasin D (0.5 mM) for 25 minutes. Panels show fixed cells at different points in time, stained for F-actin (green), palladin (red), and nuclei (blue). (B) After 25 minutes, PalldKD podocytes had fewer thin actin filaments and more nonfiber-like F-actin, respectively, compared with Ctrl podocytes. Furthermore, long and wide actin filaments as well as the total actin filament

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Figure 4. Knockdown of palladin in zebrafish larvae compromises the morphology and the dynamics of epithelial cells. To study the role of palladin in vivo, fertilized zebrafish eggs were injected with MOs against palladin (PalldMO) or control MOs (CtrlMO). (A) At 2 days postfertilization, zebrafish larvae with palladin knockdown are less developed than the control and untreated larvae, respectively. (B and C) The reduced palladin expression was confirmed by RT-PCR and immunofluorescence staining. The staining shows myotomes of the middle to distal region of the zebrafish larvae. (D) Staining with phalloidin demonstrated reduced F-actin staining of myotomes after knockdown of palladin. CtrlMO larvae developed bundled actin filaments in contrast to a few filaments in PalldMO larvae. (E) Epithelial cells of CtrlMO larvae showed an intact flat epithelial layer, in contrast to PalldMO larvae showing cells with a rounded morphology. In migration studies, one selected cell (red mark) was observed over 1 hour and the cell movement is presented by trajectories (insets). In CtrlMO larvae, the cell migrates in a direction shown by the arrow, whereas in PalldMO larvae the cell does not exhibit directed migration. Scale bars represent (A) 100 mm(C–E) and 25 mm, respectively. dilation of the capillary tuft (asterisk in Figure 6A, graph in Mild Foot Process Effacement and Increased Figure 6B) that affected 20% of the glomeruli (Figure 6B). Susceptibility to Injury in PodoPalld2/2 Mice Moreover, semithin sections of the kidneys revealed an en- The PodoPalld2/2 mice showed morphologic abnormalities. larged subpodocyte space (inset in Figure 6A). For almost For that reason, we checked for differential expression of vin- half of the glomeruli, noticeable deviations as an enlarged sub- culin, a-actinin-4, nephrin, and synaptopodin by immuno- podocyte space and cyst formation were found in podocytes histology and qRT-PCR, respectively. (Figure 6B). By electron microscopy, we observed a mild efface- As shown in Figure 7A, the protein expression of a-actinin- ment in the PodoPalld2/2 glomeruli, shown in Figure 6A. 4 and synaptopodin was not markedly affected in Podo- This means that the podocyte-specificKOofpalladinresulted Palld2/2 mice in contrast to the expression of nephrin. in overt morphologic changes of glomeruli and podocytes in mice. Further, the mRNA of vinculin (230%66%), synaptopodin

length were decreased. Treatment with cytochalasin D reduced the total filament length in PalldKD and Ctrl podocytes similarly. (C) Migration assays were performed to study the migration of PalldKD podocytes over 22 hours. Images taken by brightfield microscopy show different time points (0, 10, 22 hours). The distance between the migrating areas is marked by black lines and revealed that PalldKD podocytes migrated faster into the gap than the Ctrl. After live imaging, podocytes were fixed and stained for palladin to confirm PalldKD. (D) PalldKD podocytes showeddynamic structures such as lamellipodia(asterisks), ruffles (arrowhead), andring-like actin structures(arrows). Scale bars in (A, C, and D) represent 50 mm. DAPI, 4’,6-diamidino-2-phenylindole.

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Figure 5. Confirmation of the podocyte-specific Cre-recombinase expression and palladin KO in PodoPalld2/2 mice. (A) The specific 2 expression of Cre-recombinase in podocytes was verified by the X-gal staining of PodoPalld-R26R1M (cre+/ , lacZ/lacZ) kidney cryosections. The blue staining revealed b-galactosidase–expressing podocytes and confirmed the podocyte-specific expression of Cre-recombinase.

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(229%69%), and nephrin (226%64%) was significantly cultured podocytes significantly reduced the number of actin downregulated in isolated glomeruli of PodoPalld2/2 mice fibers; in addition, the remaining filamentswerelessbundledin in contrast to the controls (n=7, P,0.05, P,0.01, Figure contrast to the control. This finding is in agreement with 7B). To determine more precisely the changes of the foot pro- former studies done with fibroblasts and trophoblasts.1 Dixon cess morphology using staining of the slit diaphragm by super- et al.8 nicely showed that the palladin-induced actin bundling resolution microscopy, we employed the podocyte effacement is associated with the Ig3 and Ig4 domains. measurement procedure on nephrin-stained kidney sections, This study shows that a reduction of palladin is not only which was recently established by Siegerist et al.26 In Podo- associated with a reduction of actin fibers but also with a re- Palld2/2 mice, we found a significant reduction of the slit duced expression of the podocyte-specific and actin-binding diaphragm density compared with the control mice (3.586 proteins synaptopodin and a-actinin-4. This is of specific 2 2 0.06 mm 1 versus 3.2460.06 mm 1, n=58 and n=60 glomeruli interest because it is known that mutations or the loss of a-ac- of three mice per group, P,0.001) (Figure 7C). tinin-4 in vivo lead to severe kidney diseases due to the efface- After the injection of NTS, we found a significant increase of ment of the podocyte foot processes.32 proteinuria in PodoPalld2/2 mice (day 8: 8.6160.82 g/L and To study the role of palladin in actin depolymerization, day 12: 13.2260.86 g/L protein per mM creatinine) compared cultured podocytes were treated with cytochalasin D, latrun- with PodoPalld+/+ mice (day 8: 5.7860.73 and day 12: 9.576 culin A, and jasplakinolide. These substances inhibit actin po- 0.67 g/L protein per mM creatinine; n=8 per group, P,0.05) lymerization by different mechanisms.33–35 Cytochalasin D as shown in Figure 7D. PodoPalld+/+ and PodoPalld2/2 and latrunculin A inhibit actin polymerization by binding ei- mice treated with PBS as controls did not develop proteinuria. ther to the barbed end of actin filaments or by sequestering Taken together, palladin is essential for a proper podocyte actin monomers.33,36 Treatment of palladin-knockdown po- foot process morphology and therefore for an intact filtration docytes with cytochalasin D and latrunculin A significantly barrier. increased the number of short and thin filaments compared with the controls. This supports the findings of Gurung et al.29 Palladin Is Downregulated in Glomeruli of Patients that palladin stabilizes actin filaments and thereby prevents Suffering from Diabetic Nephropathy and FSGS the dissociation of actin monomers from filaments. The in- To find out whether the expression of palladin might be reg- crease of nonfiber-like F-actin under palladin-knockdown ulated in patients suffering from diabetic nephropathy (DN) conditions might also be caused by the loss of the stabilization and FSGS, renal biopsy samples were stained with an antibody of newly formed filaments by palladin. In contrast to the re- against palladin. Palladin was nearly absent in podocytes of sults described by Niedenberger et al.,37 we did not observe a patients suffering from DN and FSGS in contrast to control relocation of palladin to the cell nucleus after the treatment of kidneys (Figure 8). podocytes with cytochalasin D. In the presence of jasplakinolide, we observed a nearly complete loss of all actin fibers in palladin-knockdown cells. In DISCUSSION contrast to cytochalasin D and latrunculin A, the depolymerization mechanism of jasplakinolide is completely different. Palladin, a key protein for actin bundling, nucleation, and Jasplakinolide stabilizes the actin filaments in a similar way to polymerization, influences the morphology and the dynamic phalloidin, resulting in a tension-mediated rupture of the fil- behavior of different cell types.1,4,8,29 Recently, we have shown aments. We hypothesize that palladin blocks the binding sites that palladin is highly expressed in the kidney, especially in forjasplakinolide and thereforepreventsthe disruption of actin podocytes.8,17,30 Furthermore, it was shown that the isoform 4 filaments in palladin-expressing cells. Furthermore, because it of palladin is significantly upregulated in ANCA-GN.31 is described that palladin has a formin-like function,29 our Because the morphology and function of podocytes are results suggest that palladin is also essential to nucleate new highly dependent on the actin cytoskeleton, we focused our actin filaments. investigation on the role of the actin-binding and regulating Because palladin is also expressed in focal adhesions1,17 and protein palladin. We found that the knockdown of palladin in detachment of podocytes plays a central role in chronic

2 2 Podocytes of PodoPalld-R26R1M (cre / ,lacZ/lacZ) mice without Cre-recombinase showed no staining. Sections were counterstained using nuclear fast red. Scale bar represents 50 mm. (B) In PodoPalld2/2 mice, palladin KO was confirmed in isolated glomeruli by RT-PCR (palladin primers spanning exons [E] 11–12 and 18–20), qRT-PCR (palladin primers spanning E 18–20), and western blot (8 mg/lane). Palladin expression was normalized to Gapdh in the qRT-PCR analysis (mean6SD, n=3, ****P,0.001). (C and D) Podocyte-specificKOis shown by immunofluorescence staining of kidney paraffin sections. Pictures were taken by (C) laser scanning and (D) structured illumination microscopy. There is no palladin signal in podocytes (P) but a strong signal in vascular smooth muscle cells (V), which can be taken as a positive control. The presence of podocytes is confirmed by synaptopodin (Synpo) staining. Scale bars represent (C) 20 mmand(D)5mm, respectively. qRT-PCR, quantitative reverse transcription PCR; WB, Western blot.

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Figure 6. PodoPalld2/2 mice develop glomeruli and podocytes with a disturbed morphology. (A) Staining of mouse kidney cryosections for F-actin did not show any difference in the cytoskeleton (arrowheads). However, hematoxylin and eosin (H&E) staining of kidney paraffin sections revealed highly dilated capillaries (asterisk) in PodoPalld2/2 mice. Furthermore, podocytes with enlarged subpodocyte space (dotted line) were found in Richardson’s-stained semithin sections as illustrated in the higher magnification. Scale bars represent 10 mm. Additionally, electron microscopy (EM) revealed a mild effacement of podocyte foot processes (arrows) at the glomerular basement membrane (GBM). Scale bar represents 1 mm. (B) PodoPalld2/2 mice had significantly more glomeruli with dilated capillaries and affected podocytes (podocytes with enlarged subpodocyte space and cyst) than PodoPalld+/+ mice.

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Figure 7. Palladin KO in podocytes leads to reduced nephrin expression in vivo and wider foot processes. (A) Immunofluorescence staining of kidney paraffin sections. There is no obvious difference in synaptopodin and a-actinin-4 expression in PodoPalld2/2 mice compared with control mice, but a reduction of nephrin was observed. Scale bar represents 20 mm. (B) The significant downregulation of vinculin, synaptopodin, and nephrin mRNA in PodoPalld2/2 mice by qRT-PCR (mean6SEM, n=7 per group, *P,0.05, **P,0.001). (C) The graphic shows the measurements of the slit diaphragm density of nephrin-stained paraffin sections. The slit diaphragm density was significantly lower in PodoPalld2/2 mice compared with PodoPalld+/+ mice (mean6SEM, n=58 and n=60 glomeruli of three mice per group, ****P,0.001). (D) PodoPalld2/2 mice showed significantly more aggravated proteinuria compared with control mice after NTS-injection at day 8 and day 12. No proteinuria was observed in mice treated with PBS (mean6SEM, PodoPalld+/+ and PodoPalld2/2 mice+PBS n=4 each, PodoPalld+/+ and PodoPalld2/2 mice+NTS n=8 each, *P,0.05). qRT-PCR, quantitative reverse transcription PCR; Synpo, Synaptopodin; Vinc, Vinculin. glomerulopathies such as DN and FSGS, we studied the effect Quantitative analysis of vinculin-positive focal adhesions of palladin with regard to the expression of focal adhesion revealed that the reduction of palladin led to a reduction of proteins such as vinculin, talin-1, and b1-integrin, all ex- the focal adhesion area compared with the controls. This pressed in podocytes in vitro and in vivo.14,38–40 We found observation is in agreement with recent findings by Azatov that vinculin was significantly downregulated in palladin- et al.,12 who measured shorter focal adhesions in palladin- knockdown podocytes, whereas talin-1, a protein which is deficient tumor-associated fibroblasts. They found that described to be a mechanosensor,41 and b1-integrin, one es- a reduction of the focal adhesion size also influenced sential integrin for proper podocyte adhesion in vivo,42 were cellular traction forces and mechanosensitivity of the not affected. cells.12 Whether palladin influences the traction forces or

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Figure 8. Palladin expression is downregulated in glomeruli of patients suffering from FSGS and DN. Human renal biopsy samples were stained for palladin and synaptopodin. In control kidneys both proteins colocalized in podocytes. In biopsy samples from patients suffering from FSGS or DN, palladin expression showed a signiﬁcant reduction, whereas synaptopodin expression was unchanged. Scale bar represents 50 mm. mechanosensitivity of podocytes needs to be investigated in was described for other cell types.45 Therefore, palladin most the future. likely modulates the adhesion and outside-in signaling in po- Although vinculin is such an important component of the docytes via vinculin as was reported for neuronal N1E-115 integrin-mediated linkage of actin ﬁlaments to the extracellu- cells and NIH3T3 cells.46,47 In this context, Miao et al.48 lar matrix as well as a force-transducing and regulating pro- have recently shown that puromycin aminonucleoside tein,43 only a few studies have investigated the role of vinculin treatment of rats is associated with podocyte foot process efface- in podocytes to date.44 It was suggested that vinculin is able to ment together with a downregulation of vinculin. Furthermore, recruit proteins such as a-actinin and Arp2/3 in podocytes as Babayeva et al.49 observed a dispersion of vinculin-positive focal

J Am Soc Nephrol 29: 1662–1678, 2018 Palladin Regulates Podocyte Function 1675 BASIC RESEARCH www.jasn.org contacts in cultured podocytes after incubation with NTS-induced GN that PodoPalld2/2 mice developed a sig- plasma of patients suffering from FSGS. These findings in- nificantly increased proteinuria compared with NTS-treated dicate an essential role of vinculin in the maintenance of controls, demonstrating an essential role of palladin for a properly formed foot processes which might be influenced proper glomerular filtration barrier.56,57 by palladin. To find out whether palladin is differentially expressed in Because of the observation that actin plays a key role in cell podocytes of patients suffering from chronic glomerulopa- motility,50 we investigated the influenceofpalladinonpo- thies, we stained human biopsy samples from patients with docyte migration and dynamics. Migration studies of DN and FSGS for palladin. Interestingly, the palladin expres- cultured podocytes revealed that palladin-knockdown po- sion was significantly reduced in these biopsy samples com- docytes showed a lateral as well as a stationary motility,17 pared with control tissue, indicating a regulation of the protein exhibiting highly dynamic structures such as ring-like struc- in these kidney diseases. tures in contrast to the controls. These results are in agree- Taken together, this study demonstrates that palladin plays ment with former studies presented by Chin and Toker6 and an important role in the morphology and dynamic behavior of Asano et al.13 However, these results obtained in cell culture podocytes in vivo and in vitro. are not in agreement with the results obtained in animal models. Here, the palladin-KO cells neither migrate in lateral nor in basal directions.4 To study the influence of palladin in a living organism and to overcome the problem that ACKNOWLEDGMENTS palladin KO in mice is lethal at a very early time point in utero,4 we used the zebrafish larvae as a model organism for The authors thank Regina Maciejewski and Henny Wegner for in vivo observation studies. Similar to palladin-KO mice, the technical assistance. The 2.5P-Cre mice and primers for genotyping KO in zebrafishembryosisalsolethalatanearlytimepoint. were kindly provided by Dr. Marcus J. Moeller (Department of In- By the injection of palladin-specific MOs at later develop- ternal Medicine II, Nephrology and Clinical Immunology, Rheinisch- mental time points, we induced a viable mosaic knockdown in Westfälische Technische Hochschule (RWTH) Aachen University the zebrafish embryos. Surprisingly, we observed that the epi- Hospital, Aachen, Germany). thelial cells of the embryos have a different cell morphology. Part of the work was supported by an Emerging Fields Initiative for Instead of a flat epithelial morphology observed in palladin- Cell Cycle in Disease and Regeneration from the Friedrich-Alexander- expressing cells, we found a rounded cell shape after palladin Universität Erlangen-Nürnberg (Germany). This study was supported knockdown. Additionally, in vivo observation of palladin- by a grant from the German Research Foundation (DFG, grant INST knockdown cells showed that these cells were highly dynamic 2026/131, FUGG) to K.E. and N.E. and by a grant from the Federal without lateral migration (stationary motility), suggesting that Ministry of Education and Research (BMBF, grant 01GM1518B, STOP- the palladin-knockdown cells were unable to migrate in a spe- FSGS) to N.E. This work was also partially supported by grants from the cificdirection.Thesefindings are in agreement with prior stud- Italian Space Agency (ASI) (grant number: DC-DTE-2011-2013) and ies where a palladin KO reduced the capability for invasion and the Italian Ministry of Education, Universities and Research (PRIN migration.31,51,52 2010–2011 grant number 2010R8JK2X_006) to M.-L.B. The work was To study the role of palladin in podocytes in mice, we funded by the Forschungsverbund Molekulare Medizin, University generated podocyte-specific palladin-KO mice (Podo- Medicine Greifswald, Greifswald, Germany, to N.E. Palld2/2). Morphologic analysis of these mice revealed The study was designed by N.E., K.E., and N.A.; cell culture exper- that beside highly dilated capillaries, podocytes with an en- iments and morphologic analysis of mouse kidneys were conducted by larged subpodocyte space were present, a phenotype previ- N.A.; biopsy samples were handled and analyzed by N.A.; T.A.L. per- ously described by Kriz and colleagues53,54 in a variety of formed the zebrafish experiments; induction of GN by nephrotoxic models. Ultrastructural analysis further showed that beside serum injection and analysis of mouse urine samples were performed by areas with well developed podocyte foot processes we also P.K., C.E.C., and C.C.; H.R. programmed the software F_Seg and the found a higher number of effaced foot processes. That palla- focal adhesion analysis software; F.S. performed super-resolution din KO affects podocyte foot processes was further validated analysis of the slit diaphragm density; electron microscopy was con- by super-resolution microscopy, revealing a significant re- ducted by A.B.; M.-L.B. generated floxed Palld mice; J.v.d.B. provided duction of the slit diaphragm density, a marker inversely cor- PodoPalld mice; C.A.O. and K.A. generated the palladin antibody and related with foot process width,26 in PodoPalld2/2 mice. handled human kidney biopsy samples, respectively; all other experi- Moreover, the slit diaphragm protein nephrin was signifi- ments were performed by N.A.; experimental data were analyzed cantly downregulated in these mice. Surprisingly, despite by N.A.; N.A., N.E., and K.E. wrote the main manuscript text. N.A. these clear changes of podocyte and glomerular morphology, prepared figures. All authors reviewed the manuscript. we did not detect albumin in the urine of PodoPalld2/2 mice. This might be due to an effective reabsorption of fil- tered albumin by the tubules as was postulated by Dickson DISCLOSURES et al.55 However, we observed in the established model of None.

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1678 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1662–1678, 2018