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

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ARHGEF7 (B-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function BASIC RESEARCH www.jasn.org 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 protein. 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 gene 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. proteins 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 guanosine triphosphate 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
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