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ARHGEF7 (b-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function

Jun Matsuda, Mirela Maier, Lamine Aoudjit, Cindy Baldwin, and Tomoko Takano

Division of Nephrology, McGill University Health Centre, Montreal, Quebec, Canada

ABSTRACT Background Previous studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown. Methods We used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange fac- tors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specificARHGEF7 (commonly known as b-PIX) knockout mice by crossing b-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with b-PIX knockdown and their controls. Results We identified b-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific b-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific b-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific b-PIX knockout mice and cultured mouse podocytes with b-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of b-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated . Conclusions These findings indicate that b-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho–guanine nucleotide exchange factor plays a critical role in podocytes.

JASN 31: 996–1008, 2020. doi: https://doi.org/10.1681/ASN.2019090982

Podocytes are the specialized epithelial cells in the Rho GTPases, represented by RhoA, Rac1, and kidney glomerulus and form the kidney filtration Cdc42, are regulators of the actin cytoskeleton.3 barrier together with the endothelium and the glo- Using podocyte-specific manipulations in merular basement membrane. Podocytes are post- mice, several studies highlighted that Rho GTPases mitotic cells with a limited capacity of self-renewal. have important roles in podocyte morphology Their detachment from the glomerular basement and efficient barrier function.4–7 Deletion of membrane and loss in the urine lead to proteinuria RhoA or Rac1 in podocytes in mice caused no basal and glomerulosclerosis, and contribute to the pro- gression of CKD.1 Although accumulating evidence has shown that podocyte depletion occurs in nearly Received September 28, 2019. Accepted February 9, 2020. all human glomerulopathies, the precise mecha- Published online ahead of print. Publication date available at nisms of podocyte injury/loss is incompletely www.jasn.org. 2 understood. Correspondence: Dr. Tomoko Takano, Division of Nephrology, Podocytes have numerous actin-based projec- McGill University Health Centre, 1001 Decarie EM13244, Mon- tions called foot processes. The Rho family of small treal, QC H4A 3J1, Canada. Email: [email protected] guanosine 5ʹ-triphophatases (GTPases) known as Copyright © 2020 by the American Society of Nephrology

996 ISSN : 1046-6673/3105-996 JASN 31: 996–1008, 2020 www.jasn.org BASIC RESEARCH phenotype. However, podocyte-specific, Rac1-deficient mice Significance Statement were protected in the acute protamine sulfate model, while showing a worsened disease in a chronic hypertensive glomer- Dysregulation of Cdc42 and other members of the Rho family of ular injury model, suggesting that Rac1 is likely pathogenic or small GTPases in podocytes contributes to the pathogenesis of adaptive, depending on the situation.5 The most striking phe- proteinuria. However, the upstream regulatory mechanisms for fi fi Cdc42 activity in podocytes are largely unknown. The authors notype was observed in podocyte-speci c, Cdc42-de cient identified ARHGEF7 (commonly known as b-PIX) as a predominant mice that developed congenital nephrotic syndrome and guanine nucleotide exchange factor and activator of Cdc42 in po- died at around 3 weeks of age, suggesting that Cdc42 plays a docytes. They also demonstrated that b-PIX is required for the critical role in podocyte development.4,5 However, the mech- maintenance of podocyte architecture and glomerular function via anism by which Cdc42 activity is regulated in podocytes is Cdc42 and its downstream effects on Yes-associated protein (YAP) activity. Elucidating the precise details of how numerous regulatory not known. maintain the delicate balance of Rho GTPases in podocytes Rho GTPases exist in either inactive guanosine diphos- will be essential in understanding the pathogenesis of proteinuric phate (GDP)–bound or active glomerular diseases and identifying therapeutic targets. (GTP)–bound conformation. The GDP/GTP cycle is regu- lated by the three families of proteins: guanine nucleotide ex- the indicated time points. Mice were genotyped by PCR of tail change factors (GEFs), GTPase-activating proteins (GAPs), DNA. The genotyping primer sequences used were as follows: and GDP dissociation inhibitors (GDIs).3 Whereas GAPs and GDIs act as negative regulators of Rho GTPases, GEFs Cre forward, 59-gcttctgtccgtttgccg-39; promote exchange of GDP for GTP, thereby activating Rho GTPases in response to stimuli. To date, 82 GEFs,8,9 69 Cre reverse, 59-actgtgtccagaccaggc-39; GAPs,10,11 and three GDIs12 have been identified in hu- mans. The regulatory proteins far outnumbers their sub- b-PIX flox forward, 59-aaggcgcataacgataccac-39; strate Rho GTPases (22 members) and act in concert to achieve a cell-/context-dependent dynamic balance of Rho b-PIX flox reverse, 59-ccgcctactgcgactatagaga-39; GTPases.8,10 Although we and others have demonstrated the b 9 9 importance of RhoGDIa in the normal development of -PIX wild type forward, 5 -tgctaaaacagtggcaggtg-3 ;and kidneys, very little is known regarding how GEFs and b-PIX wild type reverse, 59-acagaacactgctgcttcca-39. GAPs function in podocytes. In this study, using proximity-based ligation assay BioID Cell Culture fi and proteomics, we identi ed ARHGEF7, also known as The original immortalized mouse podocytes were obtained b -PIX, as the predominant Cdc42 GEF in human podocytes. from Dr. Shankland (University of Washington, Seattle, WA) fi b – fi We showed that podocyte-speci c, -PIX de cient mice de- and maintained at 33°C (permissive temperature) as velop heavy proteinuria and glomerulosclerosis and that this is described.15 mediated by podocyte apoptosis and loss via reduced Cdc42 b-PIX knockdown (KD) mouse podocytes were estab- activity and consequent inactivation of the prosurvival tran- lished using MISSION shRNA (short hairpin RNA) Lentiviral scriptional regulator, Yes-associated protein (YAP). This is the Transduction Particles (TRCN0000110029; Sigma-Aldrich) fi fi rst demonstration that a speci c Rho GEF, among 82 mem- with puromycin (Wisent Inc.) selection. Mouse podocytes bers, plays a critical role in podocyte function. were transduced using the lentivirus packaged in HEK293T cells. Virus-containing supernatants were added to mouse podocytes in permissive conditions for 16 hours. METHODS Puromycin was added 48 hours later and puromycin- resistant cells were pooled for further experiments. Animals All procedures involving mice were performed in accor- Antibodies and Reagents dance with the Animal Care Committee, McGill University. The antibodies and reagents used are as follows: antibodies fl fl b-PIX ox/ ox mice (mixed C3H/C57BL/6 background), in for myc (sc-40; Santa Cruz Biotechnology), Flag (F1804; which exon 6 is flanked by loxP sites, were obtained from Sigma-Aldrich), streptavidin horseradish peroxidase Dr. Anderson and Dr. Omelchenko (Memorial Sloan Ketter- (ab7403; Abcam), tubulin (T5168; Sigma-Aldrich), nephrin ingCancerCenter,NewYork,NY).13 Podocin-Cre mice (generated in the laboratory),16 b-PIX (07-1450-I for im- (mixed ICR/129/B6 background) have been described previ- munoblotting; Millipore; ab92657 for immunofluorescence ously.14 Offspring that were heterozygous for the floxed b-PIX staining; Abcam), synaptopodin (65194; Progen), normal fl 1 allele and carried a Podocin-Cre allele (b-PIX ox/ ;Pod-Cre) rabbit IgG (sc-3888; Santa Cruz Biotechnology), WT1 (sc- fl fl fl fl were bred with b-PIX ox/ ox mice. b-PIX ox/ ox;Pod-Cre mice 192; Santa Cruz Biotechnology), cleaved caspase 3 (PC679; 2 2 (b-PIXPod / ) were used for experiments. Cre-negative mice Millipore), Rac (2465; CST), Cdc42 (05-542; Millipore), Alexa were used as controls. Spot urine samples were collected at 555–conjugated secondary antibodies (4409 [anti-mouse

JASN 31: 996–1008, 2020 b-PIX in Podocytes 997 BASIC RESEARCH www.jasn.org

IgG]), 4413 [anti-rabbit IgG]; CST), Alexa 488–conjugated sec- were obtained using a Zeiss LSM780 laser scanning confocal ondary antibodies (4408 [anti-mouse IgG], 4412 [anti-rabbit microscope. Podocyte density was analyzed in at least 20 glo- IgG]; CST), horseradish peroxidase–conjugated secondary anti- meruli for each sample as previously described.6,22 Transmis- bodies (ab6789 [anti-mouse IgG], ab6721 [anti-rabbit IgG]; sion electron micrographs were captured using an FEI Tecnai Abcam), IRDye 680LT (926-68050 [anti-mouse], 926- 12 BioTwin 120 kV transmission electron microscope. Analysis 68021 [anti-rabbit]; LI-COR), and 800CW (926-32210 [anti- for foot process width has been described previously.14 mouse], 926-32211 [anti-rabbit]; LI-COR) secondary antibodies, YAP (sc-101199; Santa Cruz Biotechnology), PromoFluor-647- Isolation of Glomeruli phalloidin (PK-PF647P-7-01; PromoKine), Rho/Rac/Cdc42 acti- The isolation of glomeruli was conducted as described pre- vator I (CN04; Cytoskelton, Inc.), ML141 (217708; Millipore), viously.23 Briefly, mice were perfused through the heart with and TAZ (ab110239; Abcam). magnetic Dynabeads (4.5 mm in diameter; Invitrogen). Kid- neys were minced into small pieces, and digested by collage- BioID/Proteomics nase type II (Gibco BRL) in Hanks’ balanced salt solution. The Cdc42G15A (from Dr. Jean-Francois Cote, Montreal Clinical digested tissue was filtered through a 100-mmcellstrainer Research Institute) was subcloned into pSTV6 (tetracycline- (FALCON) and collected using a magnetic particle concentra- inducible lentivirus vector that contains BirA; from Dr. Anne- tor (Invitrogen). Claude Gingras, Lunenfeld-Tanenbaum Research Institute).17 Lentivirus was packaged in HEK293T and expressed in im- Immunoblot Analysis mortalized human podocytes.18 As control, human podocytes Isolated glomeruli or cultured podocytes were lysed as de- that express myc-BirA alone were generated similarly. BioID scribed previously.24 Protein concentrations were deter- experiment was performed as described previously.19 Briefly, mined using Pierce BCA Protein Assay Reagent (Thermo human podocytes were incubated for 16 hours with doxycy- Scientific). Quantitative densitometry was performed using cline (D9891; Sigma) (myc-BirA, 5 mg/ml; Cdc42G15A, 1 mg/ml) a digital image-analyzing software, ImageJ (available at and 50 mM biotin (BIO302; BioShop). Cells were collected by http://rsbweb.nih.gov/ij/index.html; National Institutes of scraping in PBS, pelleted at 800 rpm for 5 minutes at 4°C, and Health, Bethesda, MD). snap frozen on dry ice. The pellets were incubated for 1 hour at 4°C with radioimmunoprecipitation assay buffer (50 mM Tris Biochemical Measurements [pH 7.4], 150 mM sodium chloride, 1% NP-40, 0.5% sodium The urinary albumin level was measured by ELISA as de- deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with scribed previously,14 and normalized to the urinary creatinine 1 mM PMSF and protease inhibitor cocktail. Lysates were then level which was measured using the creatinine colorimetric sonicated and centrifuged. Biotinylated proteins in the superna- assay kit (Cayman Chemical) to obtain the albumin- tant were pulled down with Dynabeads MyOne Streptavidin C1 creatinine ratio (ACR). Serum creatinine and BUN were mea- (Thermo Scientific) for 3 hours at 4°C. Finally, the beads were sured at the Comparative Medicine and Animal Resources rinsed five times in radioimmunoprecipitation assay buffer fol- Centre of McGill University. lowed by four washes in low detergent buffer (25 mM Tris [pH 7.4], 100 mM sodium chloride, 0.025% SDS). Samples were LPS-Induced Proteinuria Model 2 2 then analyzed by mass spectrometry and the results were ana- Female 5-week-old b-PIXPod / and control mice were in- lyzed by the Scaffold Q1 Scaffold_4.9.0 software (Proteome traperitoneally injected with 200 mg LPS (L2630; Sigma- Sciences). For statistics, t test within Scaffold was used after Aldrich) in 200 ml of PBS. Urine was collected at 0, 6, and normalizing to total spectral counts. All proteins with a P value 24 hours after LPS treatment, and analyzed for ACR. Kidneys of ,0.05 and a fold change of two over the BirA controls were were harvested at 24 hours and were then subjected to electron considered as interactors of BirA-Cdc42G15A. microscopy.

Histologic Analysis Cell Sorting fl fl Histologic analysis was performed as previously described Podocytes were isolated from b-PIX ox/ ox mice and immor- with some modifications.6,20 Human kidney sections were ob- talized by lentiviral transduction of SV40-T-tsa58 (ABM). tained from a kidney transplantation donor. Mice were trans- After selection by puromycin, cells were transduced with len- cardially perfused with PBS. Tissues were postfixed with 4% tiviral EF1a-Cre-mCherry (SignaGen). After 72 hours, cells paraformaldehyde (PFA) and embedded in paraffin. Paraffin were trypsinized and resuspended in PBS supplemented with sections were stained with Periodic acid–Schiff or immuno- 1% FBS and 2.5 mM EDTA. Cell sorting was performed with a stained with the respective antibodies. Histologic changes BD FACSAria Fusion (BD Biosciences). in Periodic acid–Schiff staining were quantified as previously described.21 Citrate treatment was performed for antigen re- Attachment Assay trieval. Tissue sections were counterstained with 49,6-diamidino- Mouse podocytes were differentiated under nonpermissive 2-phenylindole (Invitrogen). Immunofluorescence images conditions for 7 days. Then, 5000 or 10,000 cells per well

998 JASN JASN 31: 996–1008, 2020 www.jasn.org BASIC RESEARCH were seeded on a 96-well plate with 0.25 mg/cm2 laminin 521 RNA Sequence 2 2 (CORNING) and allowed to attach for 4 hours at 37°C. After Isolated glomeruli from 3-week-old b-PIXPod / and control three washes with PBS, adherent cells were fixed with 4% PFA male mice were subjected to RNA extraction using RNeasy for 15 minutes. Fixed cells were incubated with 0.1% crystal Mini Kit (QIAGEN). RNA sequencing was performed at the violet (Sigma-Aldrich) dissolved in 200 mM 3-(N-morpho- McGill University and Genome Quebec Innovation Centre. lino)propanesulfonic acid (BioShop) for 15 minutes at room For all samples, Illumina 100-bp paired-end reads were trim- temperature. After three washes with PBS, 10% acetic acid med to remove sequencing adapters and low quality bases (Fisher Scientific) was added for 15 minutes. Dissolved crystal using the fastp software with default parameters.27 From violet was quantified by the absorbance at 550 nm and nor- there, we followed the expression mRNA pipeline from the malized to the protein level. Genomics Data Commons documentation. Trimmed reads were aligned on the GRCm38 genome with the STAR aligner Detachment Assay with the two-pass method.28 With the alignment files as input, Mouse podocytes were cultured under nonpermissive condi- read-count tables were generated using the htseq-count func- tionsfor7daystoconfluency using 35-mm culture dishes tion from the HTSeq Python library.29 The DESeq2 R package coated with laminin 521. Cells were treated with 250 ng/ml was used for the differential expression analysis.30 Adriamycin (ADR) (Sigma). Photographs were taken at 0, 6, 24, and 48 hours after ADR treatment and used to measure the Statistical Analysis percentage of adherent cells. Five areas per dish were used to All results are expressed as the mean6SE. Statistical analyses calculate the average. were conducted using JMP software (SAS Institute, Cary, NC). Multiple-group comparisons were performed using ANOVA Immunocytochemistry with post-testing using the Tukey–Kramer test. Differences Mouse podocytes plated on glass coverslips coated with 1 mg/cm2 between two experimental values were assessed by the t test. collagen type I (Sigma) and were fixed in 4% PFA before being A P,0.05 was considered to be statistically significant. permeabilized with 0.1% Triton (Sigma-Aldrich). After blocking with 3% BSA in PBS, cells were immunostained with the re- spective antibodies or phalloidin. RESULTS

Terminal Deoxynucleotidyl Transferase–Mediated b-PIX is a Predominant Cdc42 Interactor in Podocytes Deoxyuridine Triphosphate Nick-End Labeling Staining Among the three prototypical Rho GTPases, RhoA, Rac1, and The terminal deoxynucleotidyl transferase–mediated deoxy- Cdc42, only the podocyte-specific gene deletion of Cdc42 uridine triphosphate nick-end labeling staining (TUNEL) as- resulted in a strong renal phenotype, suggesting a critical say was performed with the in situ apoptosis detection kit role of the Cdc42 activity in podocyte health. To identify (Takara) according to the manufacturer’s instructions. At least GEFs that activate Cdc42 in podocytes, we used the ten high-power fields (3630) were analyzed by confocal proximity-based ligation assay, BioID.19 Cdc42G15A is a microscopy. nucleotide-free mutant of Cdc42 that binds to active Cdc42-GEFs with high affinity.31 Cdc42G15A was fused Rho-GTPase Pull-Down Assay with a promiscuous Escherichia coli biotin protein ligase Mouse podocytes were serum starved in RPMI containing (BirA) and expressed in immortalized human podocytes. 1% FBS for 16 hours before the experiment. Active Rac1 Control cells expressed BirA alone (Figure 1, A and B). In and Cdc42 were pulled down using the Cdc42-Rac1 inter- the presence of exogenous biotin, active GEFs that came in active binding domain fused to GST beads (GST-CRIB; close proximity to Cdc42G15A were biotinylated by BirA and GE Healthcare Bio-Sciences), as described previously.25 these were captured by streptavidin beads and analyzed by Isolated glomeruli and mouse podocytes were lysed and mass spectrometry.19 A total of 188 proteins were significantly mixed with the beads for 1 hour at 4°C. After washing three enriched in BirA-Cdc42G15A–expressing podocytes, as com- times, beads were subjected to SDS-PAGE followed by pared with control cells expressing BirA alone. Cross- immunoblotting. referencing of these 188 proteins to the list of 82 known Rho GEFs8 identified six GEFs namely ARHGEF7, ITSN2, Luciferase Reporter Assay DOCK9, DNMBP, ARHGEF26, and DOCK7. Among them, The TEA domain (TEAD)–luciferase reporter (from Dr. Andras ARHGEF7 (commonly known as b-PIX) was consistently by Kapus, University of Toronto)26 and pRL-TKwere transfected to far the most abundant (Figure 1C). mouse podocytes in a 24-well plate using Lipofectamine 2000 b-PIX is known to be expressed in a wide variety of human (Invitrogen). Luciferase activities in cell lysates were measured tissues including the kidney.32 b-PIX protein expression was using the dual luciferase reporter assay system (Promega). The confirmed by immunoblotting in isolated mouse/rat glomeruli RL/FL ratio was calculated for each sample, and normalized to and immortalized human/mouse podocytes (Supplemental control mouse podocytes. Figure 1, A and B). By immunostaining of human, mouse,

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A B C BirA BirA -Cdc42G15A BirA Biotin – + – +

BirA -Cdc42G15A BirA- Normalized 75kDa BirA Cdc42G15A peptide count

50kDa ARHGEF7 >20 ITSN2 15~20 37kDa 75kDa DOCK9 10~15 Tubulin 50kDa DNMBP 5~10 ARHGEF26 1~5 37kDa DOCK7 0

Figure 1. BioID identifies b-PIX (ARHGEF7) as a predominant Cdc42-interactor. (A) Doxycycline-inducible myc-BirA (control, 37 kDa) or Flag-BirA-Cdc42G15A (60 kDa) were stably expressed in human podocytes. Both cells were treated with doxycycline for 16 hours as in Methods. Representative immunoblots for myc (for BirA), Flag (for BirA-Cdc42G15A), and tubulin (for loading) are shown. (B) After 16 hours of incubation with biotin and doxycycline, presence of biotinylated proteins was verified by immunoblotting with streptavidin. (C) Heatmap represents normalized peptide counts toward each bait of the six GEFs, which are significantly enriched in BirA- Cdc42G15A–expressing podocytes, compared with control cells expressing BirA alone. and rat kidneys, we confirmed that b-PIX is expressed in podo- 8 weeks, which became heavy by 13 weeks (Figure 2G). Serum 2 2 cytes as well as in endothelial, mesangial, and tubular epithelial creatinine and BUN were normal at 5 weeks in b-PIXPod / cells (Supplemental Figure 1C). Thus, we postulated that b-PIX mice, but were significantly elevated at 13 weeks (Figure 3, A 2 2 could be an important GEF and activator of Cdc42 in podocytes and B). Consistently, b-PIXPod / mouse kidneys appeared nor- and proceeded to study its function in podocytes. mal by light microscopy up to 5 weeks but started to show FSGS, as well as some tubular casts at 8 weeks, which exacerbated at 1 2 b-PIX Deficiency in Podocytes Leads to Proteinuria, 13 weeks (Figure 3, C–E). Heterozygous b-PIX (b-PIXPod / ) Glomerulosclerosis, and Renal Dysfunction in Adult mutants had neither proteinuria nor histologic abnormalities Mice during the observed period (Figures 2G and 3, D and E). By Systemic b-PIX deletion in mice causes early embryonic le- electron microscopy, podocyte foot-process effacement (quan- thality due to defective anterior visceral endoderm cell migra- tified by foot process width) was not observed at 5 weeks, but 2 2 tion.13 Thus, to investigate the function of b-PIX in podocytes, became evident at 8 weeks in b-PIXPod / mouse (Figure 4, A we generated podocyte-specific, b-PIX–deficient mice and B). Thus, b-PIX deficiency in podocytes results in progres- 2 2 (b-PIXPod / mice hereafter) by breeding Podocin-Cre mice sive proteinuria, glomerulosclerosis, and renal dysfunction in fl fl 2 2 with b-PIX ox/ ox mice (Methods). b-PIXPod / were born at adulthood. the expected Mendelian frequency. By immunostaining of the To evaluate the b-PIX protein expression after glomerular kidney, colocalization of b-PIX and the podocyte marker, injury, we injected LPS to 7-week-old control mice. At 6 hours 2 2 synaptopodin, was lost in b-PIXPod / mice, confirming after LPS injection, b-PIX expression in glomeruli tended to podocyte-specificlossofb-PIX (Figure 2A). By immunoblot- increase compared with vehicle-treated control mice by im- ting, the expression of b-PIX protein in the isolated glomeruli munoblotting (Supplemental Figure 2A), suggesting a possi- 2 2 from b-PIXPod / mice was significantly reduced compared ble adaptive/protective role of b-PIX against injury. Under with control mice (36% reduction; Figure 2, B and C). These basal conditions, podocyte abnormality was not evident in 2 2 findings indicate successful elimination of b-PIX from podo- b-PIXPod / mice before 8 weeks. Thus, to study if the young 2 2 2 2 cytes in b-PIXPod / mice. b-PIXPod / mice are more susceptible to injury, we chal- 2 2 2 2 b-PIXPod / mice appeared normal at birth, but their body lenged 5-week-old b-PIXPod / and control mice with LPS. weight was significantly lower than control mice at 13 weeks Both groups demonstrated acute rise of urine ACR peaking at 2 2 2 2 (Figure 2D). Kidneys from 13-week-old b-PIXPod / mice 24 hours after LPS injection, but b-PIXPod / mice showed were smaller than control, with pale yellow color and granular significantly higher ACR than control mice at 6 and 24 hours 2 2 surface, consistent with advanced kidney injury (Figure 2, E and (Supplemental Figure 2B). Consistently, b-PIXPod / mice 2 2 F). Quantitation of the urine ACR revealed that b-PIXPod / had significantly more podocyte foot-process effacement at mice developed progressive proteinuria starting at around 24 hours (Supplemental Figure 2, C and D). The results

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β-PIX Synaptopodin merge A B KOCTRL

Nephrin 180kDa

β

CTRL -PIX 75kDa

Tubulin 50kDa

C 1.5 *

KO 1.0

0.5 β -PIX/Tubulin (Arbitrary Unit) 0.0 CTRL KO

D E F G ** 180 * 50,000 40 ** ** CTRL 160 40,000 30 30,000 140 20 KO 20,000 10 120 10,000 100 0 Body Weight (g)

0 Urine ACR (µg/mg) Kidney Weight (mg) 3wk 8wk 13wk 5wk 8wk 13wk 8wk 13wk CTRL Hetero CTRL KO KO

Figure 2. b-PIX deficiency in podocytes leads to heavy proteinuria in adult mice. (A) Representative images of the immunofluo- rescence staining for b-PIX (red), synaptopodin (green), and 49,6-diamidino-2-phenylindole (blue) of the kidney from 3-week-old 2 2 control (CTRL) and b-PIXPod / (KO) mice. The right panels show a magnification of the indicated areas (white squares) in the left panels. Bars, 20 mm. (B) Representative immunoblots for nephrin, b-PIX, and tubulin of glomerular lysates from 5-week-old CTRL and KO mice. (C) Densitometric quantification of the protein levels in (B) (n54). (D) Body weight (n53–8), (E) appearance of the kidney, (F) kidney weight (n53or4),and(G)urineACR(n52–8). Statistically significant differences are indicated (*P,0.05, **P,0.01).

2 2 showed worsened phenotype in podocytes of b-PIXPod / and proliferate (Supplemental Figure 3). The results suggest that mice, indicating that b-PIX has a protective role against po- loss of b-PIX causes podocyte detachment from the matrix. docyte injury even at a young age. Because b-PIX–deficient mouse podocytes could not be maintained in culture, we generated b-PIX KD podocytes b-PIX Deficiency Causes Podocyte Apoptosis and Loss for further study, using immortalized mouse podocytes and Reduced podocyte number and density is a known predictor short hairpin RNA (Methods). Immunoblotting confirmed a of the progression of glomerular diseases.22 The podocyte KD efficiency of approximately 80% (Figure 5, C and D). 2 2 density in b-PIXPod / mice was comparable with control When plated on laminin, there was no difference in cell at- at 5 weeks, started to decrease at 8 weeks, and was significantly tachment between b-PIX KD and control podocytes up to decreased at 13 weeks (Figure 5, A and B). 4 hours (Supplemental Figure 4). When cells were treated We next questioned if b-PIX deficiency affects the adher- with ADR, a known podocyte toxin,33 fewer b-PIX KD podo- ence properties using cultured podocytes. Podocytes were iso- cytes remained adherent at 8, 24, and 48 hours compared with fl fl lated from b-PIX ox/ ox mice, transduced with mCherry-Cre, control cells (Figure 5E). The results support that b-PIX de- and mCherry-positive podocytes were selected by cell sort- ficiency facilitates podocyte detachment. ing. When mCherry-positive (b-PIX-deficient) podocytes Cell detachment could be secondary to cell death. Thus, we were plated onto the laminin-coated plates, they attached next studied apoptosis. When unstimulated, neither b-PIX but the attachment was unstable; most cells were detached KD nor control podocytes showed positive staining for cleaved and floating by 48 hours after plating, whereas mCherry- (active) caspase 3. When stimulated with ADR, cleaved cas- negative (b-PIX-expressing) podocytes continued to attach pase 3–positive apoptotic cells increased in both cells at

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A B D E 4 40 ** 30 ** ** 5 ** 3 30 4 20 2 3 20 10 2

1 damage 10 1 BUN (mmol/L)

0 0 0 Tubulointerstitial 0 Glomerulosclerosis Creatinine (µmol/L) 5wk 13wk 5wk 13wk 5wk 8wk 13wk 5wk 8wk 13wk CTRL KO CTRL Hetero KO

C CTRL KO 5wk 8wk 13wk

Figure 3. b-PIX deficiency in podocytes leads to glomerulosclerosis and renal dysfunction in adult mice. (A) Serum creatinine and (B) BUN. (C) Representative images of Periodic acid–Schiff staining of the kidney from 5, 8, and 13-week-old control (CTRL) and 2 2 b-PIXPod / (KO) mice. The right panels show a magnification of the indicated areas (black squares) in the left panels. Tubular casts (arrowhead) and segmental glomerulosclerosis (arrow) are shown. Bars, 50 mm. (D and E) Histologic changes were quantified by blinded scoring of (D) glomerulosclerosis and (E) tubulointerstitial damage. n54 or 5 in (A and B); n52–8 in (D and E) in each group. Statistically significant differences are indicated (**P,0.01).

24 hours in a dose-dependent manner; the increase was sig- be secondary to reduced activities of Rac1 and/or Cdc42. nificantly more in b-PIX KD podocytes compared with con- Cdc42 activity, determined by pull-down assay, in isolated 2 2 trol cells (Figure 5, F and G). Similar results were obtained glomeruli from 5- to 6-week-old b-PIXPod / mice was sig- using TUNEL staining (Supplemental Figure 5). Cleaved caspase nificantly reduced by 34%, compared with control (Figure 6, A 3 staining also demonstrated that b-PIX KD podocytes had in- and B). In contrast, Rac1 activity was comparable between 2 2 creased apoptosis even with 24 hours of serum starvation alone, b-PIXPod / and control mice (Figure 6, A and C). In vitro, compared with controls (control, 0.63%60.20% versus b-PIX when control podocytes were stimulated with the Rho/Rac/ KD, 1.57%60.19%; P50.027; n53 in each group). These results Cdc42 activator, both Rac1 and Cdc42 were activated. In suggest that loss of b-PIX promotes podocyte apoptosis and this b-PIX KD podocytes, however, Cdc42 activation was signifi- is, at least in part, responsible for cell detachment and loss. cantly blunted, whereas Rac1 activation was not affected (Fig- ure 6, D–F). Thus, in podocytes, b-PIX deficiency reduces Loss of b-PIX Reduces Cdc42 Activity in Podocytes Cdc42 activity, but does not appear to have an effect on b-PIX is a GEF that is known to activate both Rac1 and Rac1 activity, in spite of the known dual activity of b-PIX Cdc42.34 Thus, podocyte apoptosis by b-PIX deletion could on both Rac1 and Cdc42.

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A CTRL KO B

800 ** 600

5wk 400 200 Width (nm) Foot Process 0 5wk 8wk CTRL KO 8wk

Figure 4. b-PIX deficiency in podocytes leads to foot-process effacement in adult mice. (A) Representative electron microscopy of the 2 2 kidney from 5- and 8-week-old control (CTRL) and b-PIXPod / (KO) mice. The right panels show a magnification of the indicated areas (black squares) in the left panels. Bars, 1 mm. (B) Quantification of foot process width in (A). n53 in (B) in each group. Statistically significant differences are indicated (**P,0.01).

Loss of b-PIX Decreases YAP Activity in Podocytes via ML141 (Figure 7F). Taken together, these findings suggest Reduced Cdc42 Activity thatYAPactivityinpodocytesisdependentontheCdc42 Previous studies showed that Cdc42 activates YAP, which is a activity, which is downstream of b-PIX. transcriptional coactivator that prevents apoptosis and pro- To assess the YAP activity in podocytes in vivo,westudied motes cell proliferation.35,36 Dephosphorylated YAP translo- the expression of the well established YAP-TEAD target cates from the cytosol into the nucleus and functions mainly by RNA sequence using glomeruli isolated from 3-week-old 2 2 through interacting with the TEAD family of transcriptional b-PIXPod / and control mice. Among 344 genes that are factors, whereas YAP phosphorylation promotes its cytoplas- known to be regulated by YAP,41 expression of the majority 2 2 mic sequestration and inactivation.37 Because loss of b-PIX (199 genes) were decreased in b-PIXPod / mice, of which the reduced Cdc42 activity, we examined whether it also affects decrease of 84 was statistically significant. Examples of such YAP localization/activity in podocytes. In nonconfluent con- genes were Ctgf, Axl, Amotl2, Ajuba,andWtip (Figure 7G, trol podocytes, YAP was localized predominantly in the nu- Supplemental Table 1). Collectively, the findings indicate cleus, consistent with the previous reports.38 In contrast, that loss of b-PIX reduces Cdc42 activity, leading to cytoplas- b-PIX KD podocytes exhibited partial cytoplasmic retention mic retention and reduced transcriptional activity of the anti- of YAP at a similar cell density (Figure 7A); the proportion of apoptotic transcriptional cofactor, YAP, in podocytes and this YAP in the nucleus (nuclear/total YAP) was significantly lower may contribute to apoptosis. in b-PIX KD podocytes than in control (Figure 7B). The re- sults suggest that b-PIX KD leads to inactivation of YAP. We also evaluated the localization of transcriptional coactivator DISCUSSION with PDZ-binding motif (TAZ), a paralog of YAP, by immu- nostaining. In contrast to YAP, the distribution of TAZ was Although it has been recognized that a delicate and precise predominantly in the nucleus in both cells (Supplemental balance of Rho GTPase activities is critical for the function of Figure 6, A and B), suggesting that TAZ is not affected by podocytes, past studies have faced limitations in describing b-PIX KD. how such balance is achieved. One of the major challenges is We next studied the YAP activity using the TEAD-luciferase that, compared with a relatively small number of the Rho reporter, which reflects the transcriptional activity of YAP.26 GTPases (22 members), there are numerous Rho GTPase reg- The TEAD activity was significantly lower in b-PIX KD po- ulatory proteins: 82 GEFs, 69 GAPs, and three GDIs.8–12 Mu- docytes than in control (Figure 7C), consistent with the im- tations of ARHGDIA42–44 and ARHGAP24,45 which are one of munofluorescence staining. The reduced TEAD activity in the GDIs and GAPs, respectively, have been reported to alter b-PIX KD podocytes was rescued by b-PIX and the active Rho-GTPase activities in podocytes, leading to proteinuria mutant of Cdc42 (Cdc42L61) as well as by the active mutant and glomerulosclerosis in humans. But the roles of other reg- of YAP (S127A) (Figure 7, C–E).39,40 Reciprocally, the TEAD ulatory proteins in podocytes were largely unknown. In this activity was significantly decreased by the dominant negative study, we identified b-PIX as a predominant GEF that inter- mutant of Cdc42 (Cdc42N17) and the Cdc42 inhibitor, acts with Cdc42 in podocytes and demonstrated its critical role

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A CTRL KO DAPI Synaptopodin WT1 DAPI Synaptopodin WT1 5wk 13wk

B CDE p=0.054 β-PIX 1.5 1.2 250 ** ** CTRL KD 200 1.0 1.0 ) 3 150 β-PIX 0.8 µm 100 0.5 6 Tubulin *

50 β -PIX/Tubulin (Arbitrary Unit) 0.0 0.6 (/10 0 p=0.07 CTRL KD Podocyte Density 5wk 8wk 13wk 0.4

G Ratio of the N CTRL CTRL KO attached cells/0hr 0.2 * KD 0.08 * 0.0 F 0.06 CTRL KD 0hr 8hr 24hr 48hr 0.04 0.02 /Total cells - 0.00 0 250 500 Ratio of CC3-positive ADR ng/ml CTRL KD ADR 500ng/ml 250ng/ml

Figure 5. b-PIX deficiency causes podocyte apoptosis and loss. (A) Representative images of the immunofluorescence staining for WT1 (red), synaptopodin (green), and 49,6-diamidino-2-phenylindole (DAPI; blue) of the kidney from 5- to 13-week-old control (CTRL) 2 2 and b-PIXPod / (KO) mice. (B) Podocyte density in (A) was analyzed. n53 or 4 in each group. (C) Representative immunoblots for b-PIX and tubulin of cultured mouse podocytes with b-PIX KD and CTRL. (D) Densitometric quantification of b-PIX in (C). (E) Detachment assay using CTRL and b-PIX KD podocytes. Cells were incubated with 250 ng/ml ADR for up to 48 hours on laminin 521–coated plates. The numbers of the attached cells are normalized to 0 hour. n53 in (D and E) in each group. (F) Representative images of the im- munofluorescence staining for cleaved caspase 3 (red) and DAPI (blue) of CTRL and b-PIX KD podocytes at 24 hours after ADR treatment. (G) Quantification of the ratio of cleaved caspase 3–positive cells to total cells at 24 hours after ADR treatment. n52–5in each group. Statistically significant differences are indicated (*P,0.05, **P,0.01). Bars, 20 mmin(AandF). in normal podocyte development and function. To the best of We found that loss of b-PIX in podocytes reduced Cdc42 our knowledge, this is the first report to demonstrate that a activity, suggesting that Cdc42 activation is largely dependent Rho GEF plays a critical role in podocyte function. on b-PIX in podocytes. Previous studies showed that

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A B C CTRL KO 2.0 * 2.0 ns Cdc42 1.5 1.5 PD: Rac 1.0 1.0 GST-CRIB /Tubulin Cdc42 /Tubulin 0.5 0.5 Active Rac Active Cdc42 (Arbitrary Unit) Rac (Arbitrary Unit) 0.0 0.0 β CTRL KO CTRL KO Input -PIX Tubulin

D E F CTRL KD 2.0 2.0 024240hr 1.5 * 1.5 ns Cdc42 1.0 1.0 PD: /Tubulin Rac /Tubulin 0.5 0.5 Active Rac Active Cdc42 GST-CRIB (Arbitrary Unit) (Arbitrary Unit) 0.0 0.0 0hr 2hr 4hr 0hr 2hr 4hr Cdc42 CTRL KD Rac Input Tubulin

Figure 6. b-PIX deficiency reduces Cdc42, but not Rac, activity. (A) Glomerular lysates from 5- to 6-week-old control (CTRL) and 2 2 b-PIXPod / (KO) mice were subjected to pull down with GST-CRIB for active Cdc42 and Rac. (B and C) Densitometric quantification of active (B) Cdc42 and (C) Rac in (A). (D) CTRL and b-PIX KD mouse podocytes were stimulated with 1 mg/ml Rho/Rac/Cdc42 activator. Lysates at the indicated times were subjected to pull down with GST-CRIB. (E and F) Densitometric quantification of active (E) Cdc42 and (F) Rac in (D). n57or10(BandC);n54or5(EandF)ineachgroup.Statisticallysignificant differences are indicated (*P,0.05). PD, pull-down.

2 2 podocyte-specific, Cdc42-deficient (Cdc42Pod / ) mice pre- decreased nuclear localization of YAP, leading to a reduction sent congenital nephropathy and die as a result of renal failure of YAP-dependent .35 Mouse podocytes with within 3 weeks after birth,4,5 indicating Cdc42 is essential for Cdc42 KD reduced both nuclear and cytoplasmic Yap expres- 2 2 podocyte maintenance and function. b-PIXPod / mice phe- sion.36 The important role of YAP in podocytes has been re- 2 2 nocopied Cdc42Pod / mice, but they presented milder and ported. Podocyte-specific YAP deletion in mice caused later-onset proteinuria. This can be explained by several other proteinuria and glomerulosclerosis,47 and activation of the GEFs that could activate Cdc42 in podocytes. One candidate Hippo pathway (which causes inactivation of YAP) induced could be ITSN2, which we identified as the second most abun- apoptosis of podocytes in vitro.48 It was also reported that YAP dant Cdc42-interacting GEF by BioID (Figure 1C). Indeed, phosphorylation is increased in glomeruli in human FSGS, loss-of-function mutations of ITSN2 and its isoform ITSN1 indicating YAP inactivation.49 Thus, reduced YAP activity in- have been reported as the cause of nephrotic syndrome in duced by inadequate Cdc42 activity likely leads to podocyte humans, suggesting its role in podocyte health.46 However, apoptosis and loss, eventually progressing to glomeruloscle- ITSN2-deficient mice showed only a mildly exacerbated pro- rosis. On the other hand, b-PIX was reported to promote the teinuric phenotype when challenged with LPS,46 thus it is cytoplasmic localization of YAP (which causes inactivation of likely that b-PIX has a predominant role in maintaining the YAP) in a breast cancer cell line,50 whereas our results clearly Cdc42 activity in podocytes. Because b-PIX is a known GEF indicate that b-PIX is required for the YAP activity in podo- for both Rac1 and Cdc42,34 we anticipated that Rac1 activity, cytes. The reason for this apparent discrepancy is unclear but as well as Cdc42 activity, would be decreased by b-PIX defi- could be due to different cell types. ciency. To our surprise, Rac1 activity was maintained in b-PIX is also known to have an important role in focal b-PIX–deficient podocytes. The result suggests that other adhesion (FA) dynamics. For example, b-PIX negatively GEFs likely compensate effectively for the loss of b-PIX in regulates FA maturation and promotes cell migration.51 Rac1 activation in podocytes. Thus, it is possible that b-PIX deficiency may contribute to Our results are consistent with the previous studies that podocyte detachment by affecting cell-matrix interaction, in reported the role of Cdc42 as an upstream regulator/activator addition to causing apoptosis. In fact, b-PIX KD podocytes of YAP. In mouse embryonic fibroblasts, ablation of Cdc42 showed abnormalities in cell behaviors linked to FA

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A CTRL KD B YAPPhalloidin DAPI YAPPhalloidin DAPI 1.0 ** 0.8 0.6 0.4 0.2 0.0 Nuclear/Total YAP CTRL KD

C 2.5 ## 2.0 ## 1.5 1.0 ** D E F 0.5 ## ## TEAD activity (Arbitrary Unit) 0.0 1.5 ## 2.0 ## ## 1.5 EV S127A S127A 1.5 1.0 1.0 +Dead ** TEAD 1.0 * ** ## CTRL 0.5 0.5 ## 0.5 ## ## KD TEAD activity TEAD activity TEAD activity (Arbitrary Unit) (Arbitrary Unit) (Arbitrary Unit) 0.0 0.0 0.0 EV β-PIX EV Cdc42 EV Cdc42 ML141 L61 N17 G 1.5 ns ns ** ** *** 1 ** CTRL 0.5 KO 0 mRNA levels (Arbitrary Unit)

Axl Ctgf Wtip Cyr61 Ajuba Ankrd1 Amotl2

Figure 7. b-PIX deficiency decreases YAP activity in podocytes via reduced Cdc42 activity. (A) Representative images of the immu- nofluorescence staining for YAP (red), phalloidin (gray), and 49,6-diamidino-2-phenylindole (DAPI; blue) of control (CTRL) and b-PIX KD podocytes. Bar, 10 mm. (B) Quantification of the ratio of nuclear to total YAP in (A). n512 or 15 in each group. (C–F) YAP-TEAD activity in CTRL and b-PIX KD podocytes which were transfected with (D) 0.5 ng b-PIX, (E) 1 ng Cdc42L61, (F) 50 ng Cdc42N17 or treated with 12.5 mM ML141 was measured by TEAD-luciferase assay. Active mutant of YAP (YAP S127A) and inactive mutant of TEAD (Dead TEAD) were used as positive and negative control, respectively, in (C). (G) The mRNA expression of representative YAP-TEAD target genes in 2 2 the isolated glomeruli from 3-week-old control (CTRL) and b-PIXPod / knockout (KO) mice were analyzed. n56 in (C and E), n59in(D), and n53 (F and G) in each group. Statistically significant differences are indicated (*P,0.05, **P,0.01 versus treatment-matched CTRL podocytes; ##P,0.01 versus vehicle-treated corresponding cells). EV, empty vector. dynamics, including impaired motility and reduced cell size ACKNOWLEDGMENTS (Supplemental Figure 7). However, we did not find notable differences in the number and size of the FA complex between We thank all of the investigators who provided the reagents, as de- b-PIX KD and control podocytes. It is possible that more scribed in Methods. We also thank the Proteomics, Molecular Im- detailed studies of live cells will uncover the alteration in aging, Histopathology, and Bioinformatics Platforms of McGill the FA dynamics and their contribution to detachment University Health Centre for expert technical assistance. from the matrix. Dr. Matsuda and Dr. Takano were responsible for conceptualiza- In summary, we established that b-PIX is required tion, methodology, and writing of the manuscript; Mr. Aoudjit, Ms. for the maintenance of podocyte architecture and glo- Baldwin, Ms. Maier, and Dr. Matsuda were responsible for in- merular function. Unraveling the precise manners of vestigation; Dr. Takano was responsible for supervision; all authors crosstalks, by which numerous regulatory proteins main- contributed to the discussion and approved the manuscript. tain the delicate balance of Rho GTPases in podocytes, will be essential in understanding the pathogenesis of, DISCLOSURES and identifying therapeutic targets for, proteinuric glo- merular diseases. None.

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FUNDING 11. Amin E, Jaiswal M, Derewenda U, Reis K, Nouri K, Koessmeier KT, et al.: Deciphering the molecular and functional basis of RHOGAP family proteins: A systematic approach toward selective inactivation of RHO Dr. Matsuda was supported by the Cell Science Research Foundation, Shi- family proteins. JBiolChem291: 20353–20371, 2016 nya Foundation, and the Uehara Memorial Foundation. Dr. Takano was sup- 12. Garcia-Mata R, Boulter E, Burridge K: The ‘invisible hand’: Regulation of ported by Canadian Institutes of Health Research grant PJT-399126 and – Kidney Foundation of Canada grant BRG-180016. RHO GTPases by RHOGDIs. Nat Rev Mol Cell Biol 12: 493 504, 2011 13. Omelchenko T, Rabadan MA, Hernández-Martínez R, Grego-Bessa J, Anderson KV, Hall A: b-Pix directs collective migration of anterior vis- ceral endoderm cells in the early mouse embryo. Genes Dev 28: SUPPLEMENTAL MATERIAL 2764–2777, 2014 14. Kumagai T, Baldwin C, Aoudjit L, Nezvitsky L, Robins R, Jiang R, et al.: This article contains the following supplemental material online at Protein tyrosine phosphatase 1B inhibition protects against podocyte injury and proteinuria. Am J Pathol 184: 2211– 2224, 2014 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019090982/-/ 15. Shankland SJ, Pippin JW, Reiser J, Mundel P: Podocytes in culture: DCSupplemental. Past, present, and future. Kidney Int 72: 26–36, 2007 Supplemental Figure 1. The expression of b-PIX in podocytes. 16. Li H, Lemay S, Aoudjit L, Kawachi H, Takano T: SRC-family kinase Fyn Supplemental Figure 2. b-PIX has a protective role against glo- phosphorylates the cytoplasmic domain of nephrin and modulates its merular injury. interaction with podocin. JAmSocNephrol15: 3006–3015, 2004 Supplemental Figure 3. b-PIX deficiency facilitates podocyte 17. Samavarchi-Tehrani P, Abdouni H, Samson R, Gingras AC: A versatile lentiviral delivery toolkit for proximity-dependent biotinylation in di- detachment. verse cell types. Mol Cell Proteomics 17: 2256–2269, 2018 b fi Supplemental Figure 4. -PIX de ciency does not affect short- 18. Saleem MA, O’Hare MJ, Reiser J, Coward RJ, Inward CD, Farren T, term podocyte attachment. et al.: A conditionally immortalized human podocyte cell line demon- Supplemental Figure 5. b-PIX deficiency facilitates podocyte strating nephrin and podocin expression. JAmSocNephrol13: – apoptosis. 630 638, 2002 19. Roux KJ, Kim DI, Raida M, Burke B: A promiscuous biotin ligase fusion Supplemental Figure 6. b-PIX deficiency does not affect TAZ lo- protein identifies proximal and interacting proteins in mammalian cells. calization in podocytes. JCellBiol196: 801–810, 2012 Supplemental Figure 7. b-PIX KD podocytes showed reduced 20. Auguste D, Maier M, Baldwin C, Aoudjit L, Robins R, Gupta IR, et al.: cell area. 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