Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury
2013; Elsevier BV; Volume: 84; Issue: 5 Linguagem: Inglês
10.1038/ki.2013.175
ISSN1523-1755
AutoresSimone M. Blattner, Jeffrey B. Hodgin, Masashi Nishio, Stephanie A. Wylie, Jharna Saha, Abdul Soofi, C. W. Vining, Ann Randolph, Nadja Herbach, Rüdiger Wanke, Kevin B. Atkins, Hee Gyung Kang, Anna Henger, Cord Brakebusch, Lawrence B. Holzman, Matthias Kretzler,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoPodocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury. Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury. The podocyte is a highly differentiated epithelial cell essential for a functional glomerular filtration barrier. Located on the outside of the glomerulus, covering the capillary wall and in the urinary space, the podocyte adopts an intricate and polarized cellular organization consisting of a cell body, major processes, and foot processes that interdigitate with foot processes from neighboring podocytes. The unique shape derives from an abundantly rich actin cytoskeleton that is key to podocyte morphology and function, and crucial for establishing stability between the cell–cell and the cell–matrix contacts.1.Faul C. Asanuma K. Yanagida-Asanuma E. et al.Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.Trends Cell Biol. 2007; 17: 428-437Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar,2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1203) Google Scholar Regulation of the podocyte cytoskeleton is dynamic, and dysregulation, morphologically identified as foot process effacement, is closely associated with proteinuria, the clinical signature of podocyte injury.1.Faul C. Asanuma K. Yanagida-Asanuma E. et al.Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.Trends Cell Biol. 2007; 17: 428-437Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar Mammalian Rho GTPases comprise a family of more than 20 intracellular signaling molecules that regulate diverse biological processes, but are best known for their important roles in regulating the actin cytoskeleton.3.Heasman S.J. Ridley A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies.Nat Rev Mol Cell Biol. 2008; 9: 690-701Crossref PubMed Scopus (1433) Google Scholar,4.Wennerberg K. Der C.J. Rho-family GTPases: it's not only Rac and Rho (and I like it).J Cell Sci. 2004; 117: 1301-1312Crossref PubMed Scopus (475) Google Scholar The GTPases of the Rho subfamily, of which Rac1 and Cdc42 are two of the best studied, are likely to have key roles in regulation of the podocyte cytoskeleton. Each GTPase acts as a molecular switch, cycling between an active GTP-bound state and an inactive GDP-bound state. Once activated, Rho GTPases bind to a range of effectors to regulate downstream signaling pathways in addition to those linked to the actin cytoskeleton, including cell polarity, cell–extracellular matrix adhesion, microtubule dynamics, membrane trafficking, and gene transcription.5.Etienne-Manneville S. Hall A. Rho GTPases in cell biology.Nature. 2002; 420: 629-635Crossref PubMed Scopus (3841) Google Scholar, 6.Jaffe A.B. Hall A. Rho GTPases in transformation and metastasis.Adv Cancer Res. 2002; 84: 57-80Crossref PubMed Scopus (255) Google Scholar, 7.Schwartz M. Rho signalling at a glance.J Cell Sci. 2004; 117: 5457-5458Crossref PubMed Scopus (157) Google Scholar In this study, we demonstrate that mice with podocyte-specific deletion of Rac1 show no kidney dysfunction and have morphologically normal podocytes well into adulthood. When acutely injured by protamine sulfate perfusion, Rac1 deletion prevents foot process effacement in podocytes. However, mice with podocyte-specific Rac1 deletion display exacerbated albuminuria and glomerulosclerosis in a chronic model of progressive glomerular failure secondary to uninephrectomy and deoxycorticosterone acetate—high salt (UNX/DOCA-salt)–induced hypertension. In sharp contrast, podocyte-specific deletion of Cdc42 results in heavy proteinuria, kidney failure, and death. This was accompanied by foot process effacement, glomerulosclerosis, and eventually end-stage kidneys. Our findings demonstrate Cdc42 has a crucial role in podocyte cell maintenance. Rac1, however, is dispensable for preservation of the glomerular filtration barrier in the unchallenged setting, but has complex and divergent roles in acute and chronic podocyte injury. To define the function of Rac1 and Cdc42 in podocytes in vivo, we used mice that express Cre-recombinase under control of a podocyte-specific promoter and crossed them with mice with floxed exon 3 of the Rac1 gene, or mice with floxed exon 2 of the Cdc42 gene, resulting in targeted inactivation of either Rac1 (podoRac1–/–) or Cdc42 (podoCdc42–/–) (Figure 1a). Mouse genotypes containing Cre-recombinase–expressing construct and homozygous for either floxed Rac1 or Cdc42 were confirmed by PCR analysis of tail genomic DNA (Figure 1b). Western blot analysis of protein lysates obtained from glomeruli isolated from podoRac1–/– and podoCdc42–/– mice demonstrated profound reduction of Rac1 and Cdc42 protein expression compared with glomerular lysates from floxed controls (Rac1-fl/fl and Cdc42-fl/fl) (Figure 1c). As podocytes constitute a fraction of the glomerular cell population, endothelial and mesangial cells contribute to the remaining signals in podoRac1–/– and podoCdc42–/– glomeruli. Survival was severely limited in podoCdc42–/– animals, whereas podoRac1–/– mice demonstrated no difference in lifespan compared with floxed controls (Figure 1d). The majority of podoCdc42–/– mice died by the age of 4 weeks, and no podoCdc42–/– mice survived past day 60 of life. Though born at a normal Mendelian distribution and normal in appearance at birth, podoCdc42–/– animals began to display significant morbidity with growth retardation at ~2 weeks postnatal (body weight 10.4±0.8 vs. 18.0±1.6 of controls at killing, P<0.01, n=5 per group), most likely the result of heavy proteinuria (Figure 1e). By 10 days of age, SDS–polyacrylamide gel electrophoresis analysis of urine samples revealed significant selective proteinuria (albuminuria) in some podoCdc42–/– mice. By 16 days of age, the proteinuria had progressed dramatically and was nonselective in nature, as evidenced by the presence of proteins of varying molecular weight (Figure 1e). Quantitation of the albumin-to-creatinine ratio revealed an increase of several orders of magnitude in podoCdc42–/– mice compared with controls (Figure 1f, n=5 per group). Mice heterozygous for podocyte-specific deletion of Cdc42 (podocinCre/+, Cdc42fl/+) displayed no phenotype up to 12 months of age (data not shown). Kidneys from 3–4-week-old podoCdc42–/– mice were grossly pale yellow, firm, and with a granular surface, consistent with end-stage kidneys (Figure 1g). In contrast, podoRac1–/– mice remained alive and healthy up to 1 year (Figure 1d), demonstrated no proteinuria at 6 months of age (Figure 1e), and showed no gross renal pathology. Kidney morphology at 4 weeks of age was examined by light microscopy (Figure 2). Compared with floxed Cdc42 or Rac1 controls (Figure 2a and b), podoRac1–/– kidneys (Figure 2c and d) showed no alteration in glomerular or tubulointerstitial morphology for up to 12 months of age. In contrast, podoCdc42–/– mice displayed progressive focal and global glomerulosclerosis accompanied by diffuse tubular dilatation with protein casts and tubular injury (Figure 2e and f). Podocytes often appeared prominent and vacuolated. Segmentally sclerotic portions contained abundant extracellular matrix and adhered to Bowman’s capsule. Additional tubulointerstitial lesions (not shown in Figure 2) included focal atrophy and fibrosis. Transmission electron microscopy (Figure 3a) and scanning electron microscopy (Figure 3b) were performed on 4-week-old podoRac1–/–, podoCdc42–/–, and floxed controls. Control mice displayed a normal, intact arrangement of interdigitating foot processes and preserved filtration slits (Figure 3a and b). Ultrastructural examination of podoRac1–/– podocytes revealed morphology indistinguishable from control podocytes (Figure 3c and d). In contrast, well-formed foot processes were replaced by broad cellular extensions (effacement) covering glomerular capillaries in podoCdc42–/– mice (Figure 3e and f). To define molecular alterations of the podocyte slit diaphragm, we measured glomerular gene expression and examined the cellular distribution of nephrin, podocin, and synaptopodin by quantitative reverse transcriptase PCR (Figure 4a) and confocal laser microscopy in 4-week-old mice (Figure 4b and c). Analysis of mRNA obtained from isolated glomeruli revealed a reduction of nephrin and podocin transcript levels in podoCdc42–/– mice (P<0.05), but no significant change in the expression of synaptopodin mRNA, compared with floxed controls. In contrast, nephrin and podocin expression in podoRac1–/– mice did not change (Figure 4a). Through an analysis of nephrin, podocin, and synaptopodin distribution in podocytes at the protein level by immunofluorescence, we observed continuous distribution along the glomerular capillary wall for each marker in wild-type animals. In contrast, nephrin and podocin immunofluorescence staining appeared discontinuous and granular in glomeruli from podoCdc42–/– mice, whereas synaptopodin remained unchanged. The intensity and pattern of these markers in podoRac1–/– mice, however, was identical to floxed controls (Figure 4b and c). These observations were consistent with the morphological findings and lack of proteinuria described above. Cofilin is a tightly regulated effector molecule for both Cdc42 and Rac1 that severs actin filaments and promotes disassembly, which are essential for actin remodeling and productive membrane protrusions.8.Bamburg J.R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics.Annu Rev Cell Dev Biol. 1999; 15: 185-230Crossref PubMed Scopus (839) Google Scholar,9.Garg P. Verma R. Cook L. et al.Actin-depolymerizing factor cofilin-1 is necessary in maintaining mature podocyte architecture.J Biol Chem. 2010; 285: 22676-22688Crossref PubMed Scopus (92) Google Scholar Cofilin inactivation occurs through phosphorylation by LIM kinases, which are activated by the PAK family of Rac/Cdc42-dependent kinases. Western blot analysis of phospho-cofilin to cofilin ratio from isolated glomeruli revealed a near total loss of phospho-cofilin in podoCdc42–/– mice compared with all floxed controls (0.005±0.005 vs. 0.62±0.14, P<0.05), but no significant change in the phosphorylation status in podoRac1–/– mice (0.56±0.16 vs. 0.62±0.14, P=0.78) (Figure 5). Thus, loss of Cdc42 in podocytes results in a significant imbalance in cofilin activation. At 10 days of age, some, but not all, podoCdc42–/– mice display albuminuria (Figure 1e). In order to determine if dysfunction of the glomerular filtration barrier can be detected earlier, 1-week-old podoCdc42–/– and floxed controls were examined. Semiquantitative SDS–polyacrylamide gel electrophoresis analysis of urine samples revealed no significant albuminuria in any animal (Figure 6a). Both control and podoCdc42–/– kidneys displayed an immature nephrogenic zone and primitive tubules at low power (Figure 6b, left). Closer inspection revealed immature appearing glomeruli with prominent podocytes in all animals as expected at this age, with no evidence of glomerulosclerosis (Figure 6b, right). Equally well-formed podocyte foot processes and slit diaphragms could be identified in control podoCdc42–/– and glomeruli by transmission electron microscopy (Figure 6c, 6d, and insets). Thus, podoCdc42–/– mice appear to form a functional glomerular filtration barrier in utero and through the perinatal period. Protamine sulfate in rodent models results in alterations in podocyte shape characterized by foot process effacement within minutes of perfusion. This is thought to be an actin-dependent process triggered through neutralization of anionic charge and/or disruption of podocyte–basement membrane interactions.10.Pippin J.W. Brinkkoetter P.T. Cormack-Aboud F.C. et al.Inducible rodent models of acquired podocyte diseases.Am J Physiol Renal Physiol. 2009; 296: F213-F229Crossref PubMed Scopus (211) Google Scholar Infusing control mice with protamine sulfate resulted in morphologically distinct foot process effacement (Figure 7a, upper panels). Calculation of the podocyte filtration slit frequency revealed a significant reduction by ~27% (Figure 7b). In contrast, the podocyte foot process morphology appeared unchanged by protamine sulfate perfusion in podoRac1–/– mice (Figure 7a, lower panels), and filtration slit frequency was not significantly reduced after 15min of perfusion (Figure 7b). Albuminuria and progressive glomerulosclerosis are recognized as hallmarks of podocyte injury in the UNX/DOCA-salt–hypertensive rodent model.11.Kretzler M. Koeppen-Hagemann I. Kriz W. Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat.Virchows Arch. 1994; 425: 181-193Crossref PubMed Scopus (147) Google Scholar To investigate the effect of podocyte-specific Rac1 deletion in a chronic model of podocyte injury, we employed this injury model in podoRac1–/– and Rac1-fl/fl controls, and compared them to sham treatment. Four weeks after UNX/DOCA-salt treatment, the average body weights of all four groups were not significantly different (Figure 8a). As expected, systolic blood pressure, whole kidney weight, and left ventricular heart weight were elevated by treatment in both Rac1-fl/fl and podoRac1–/– mice compared with sham control, with no difference observed between UNX/DOCA-salt–treated Rac1-fl/fl and podoRac1–/– (Figure 8b–d). UNX/DOCA-salt–treated mice of either group displayed elevated albuminuria at 2 and 4 weeks; however, urine albumin in UNX/DOCA-salt–treated podoRac1–/– mice was nearly twice that of Rac1-fl/fl at 2 weeks (P<0.05) and 4 weeks (Figure 9a), although the difference was not significant at the latter time point (P=0.19). The percentage of glomeruli with segmental and global sclerosis was likewise doubled in treated podoRac1–/– mice at 4 weeks versus treated controls, (Figure 9b–d) indicating an exacerbation of podocyte injury, rather than protection, in podoRac1–/– mice. Though increased in frequency, the morphological appearance of segmental sclerosis in UNX/DOCA-salt–treated podoRac1–/– mice was indistinguishable from Rac1-fl/fl controls. A qualitative ultrastructural examination by transmission electron microscopy revealed focal and segmental podocyte foot process effacement in both UNX/DOCA-salt–treated podoRac1–/– and Rac1-fl/fl mice (Figure 10a and b, and Supplementary Figure S1 online), which is consistent with the focal nature of effacement in glomerular hyperfiltration injury akin to secondary focal segmental glomerulosclerosis (FSGS), such as seen in hypertensive or obese humans.12.D'Agati V.D. Kaskel F.J. Falk R.J. Focal segmental glomerulosclerosis.N Engl J Med. 2011; 365: 2398-2411Crossref PubMed Scopus (549) Google Scholar Interestingly, we observed focal foot process effacement in UNX/DOCA-salt–treated podoRac1–/– mice, indicating that Rac1-independent mechanisms of effacement exist.Figure 10Transmission electron microscopy (TEM). Glomerular capillary walls of UNX/deoxycorticosterone acetate (DOCA)-salt–treated Rac1-fl/fl (a) and podoRac1–/– (b) mice display focal and segmental foot process effacement. Sham-treated podoRac1–/– and Rac1-fl/fl (c) exhibit only normal appearing, regularly interdigitating podocyte foot processes.View Large Image Figure ViewerDownload (PPT) Download .jpg (.46 MB) Help with files Supplementary Figure S1 Podocytes are polarized cells with an abundantly rich and highly dynamic actin-based cytoskeleton vital to proper podocyte function and glomerular filtration.1.Faul C. Asanuma K. Yanagida-Asanuma E. et al.Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.Trends Cell Biol. 2007; 17: 428-437Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar,13.Shankland S.J. The podocyte's response to injury: role in proteinuria and glomerulosclerosis.Kidney Int. 2006; 69: 2131-2147Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar Furthermore, dysregulation of the podocyte cytoskeleton, seen as foot process effacement, is invariably seen in podocyte injury. Each cell membrane domain of the foot process (slit diaphragm, basal, and apical) has the ability to regulate actin dynamics through Rho GTPase activation.1.Faul C. Asanuma K. Yanagida-Asanuma E. et al.Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.Trends Cell Biol. 2007; 17: 428-437Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar Nephrin phosphorylation increases Rac1 activity through phosphoinositide 3-kinase14.Zhu J. Sun N. Aoudjit L. et al.Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes.Kidney Int. 2008; 73: 556-566Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar and nephrin directly interacts with IQGAP1, an effector protein that binds and maintains Rac1 and Cdc42 in an active state.15.Liu X.L. Kilpelainen P. Hellman U. et al.Characterization of the interactions of the nephrin intracellular domain.FEBS J. 2005; 272: 228-243Crossref PubMed Scopus (51) Google Scholar Synaptopodin, an actin-associated protein, induces RhoA stabilization and cell migration while preventing Cdc42-mediated filopodia formation in mouse podocytes.16.Yanagida-Asanuma E. Asanuma K. Kim K. et al.Synaptopodin protects against proteinuria by disrupting Cdc42:IRSp53:Mena signaling complexes in kidney podocytes.Am J Pathol. 2007; 171: 415-427Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar In addition, podocalyxin, an apical membrane domain protein, has been shown to activate RhoA and induce actin reorganization in MDCK cells.17.Schmieder S. Nagai M. Orlando R.A. et al.Podocalyxin activates RhoA and induces actin reorganization through NHERF1 and Ezrin in MDCK cells.J Am Soc Nephrol. 2004; 15: 2289-2298Crossref PubMed Scopus (102) Google Scholar Bidirectional signaling between Rho GTPases and integrins to modulate cell–basement membrane adhesion has also been described.3.Heasman S.J. Ridley A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies.Nat Rev Mol Cell Biol. 2008; 9: 690-701Crossref PubMed Scopus (1433) Google Scholar Evidence suggests current immunosuppressive strategies to reduce proteinuria and treat FSGS, such as calcineurin inhibitors and glucocorticoids, have the ability to directly target the podocyte cytoskeleton,18.Faul C. Donnelly M. Merscher-Gomez S. et al.The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A.Nat Med. 2008; 14: 931-938Crossref PubMed Scopus (755) Google Scholar, 19.Ransom R.F. Lam N.G. Hallett M.A. et al.Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization.Kidney Int. 2005; 68: 2473-2483Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20.Schonenberger E. Ehrich J.H. Haller H. et al.The podocyte as a direct target of immunosuppressive agents.Nephrol Dial Transplant. 2011; 26: 18-24Crossref PubMed Scopus (93) Google Scholar implicating cytoskeleton as a therapeutic goal to abrogate podocyte injury. Thus, elucidation of Rho GTPase contribution in podocyte function is important. Rho GTPases, including Rac1, are important for proper neuron21.Govek E.E. Newey S.E. Van Aelst L. The role of the Rho GTPases in neuronal development.Genes Dev. 2005; 19: 1-49Crossref PubMed Scopus (764) Google Scholar and vascular development.22.Tan W. Palmby T.R. Gavard J. et al.An essential role for Rac1 in endothelial cell function and vascular development.FASEB J. 2008; 22: 1829-1838Crossref PubMed Scopus (179) Google Scholar However, mice with podocytes lacking Rac1 develop normal glomeruli and display no renal dysfunction well into adulthood. Thus, it appears Rac1 is not required for the development or maintenance of podocyte architecture and the glomerular filtration barrier. It should be noted that Rac1 and its isoforms Rac2 and Rac3 share a high degree of homology. Studies have revealed a redundant role between Rac1 and Rac223.Guo F. Cancelas J.A. Hildeman D. et al.Rac GTPase isoforms Rac1 and Rac2 play a redundant and crucial role in T-cell development.Blood. 2008; 112: 1767-1775Crossref PubMed Scopus (83) Google Scholar and Rac1 and Rac324.Corbetta S. Gualdoni S. Ciceri G. et al.Essential role of Rac1 and Rac3 GTPases in neuronal development.FASEB J. 2009; 23: 1347-1357Crossref PubMed Scopus (73) Google Scholar in T cell and neuronal development, respectively. Therefore, we cannot yet rule out a compensatory effect of Rac2 and Rac3 in podoRac1–/– mice as an explanation for the normal phenotype. However, we believe this to be unlikely because microarray analysis of normal mouse glomeruli reveal levels of Rac2 and Rac3 transcripts to be ~1% of Rac1 transcript abundance (data available at Gene Expression Omnibus under GEO no. GSE33744). Additional experiments will be needed to test our hypothesis. Interestingly, we find that podoRac1–/– mice do not develop protamine sulfate–induced foot process effacement, a model of acute podocyte injury that alters anionic charge and results in the disruption of podocyte foot process architecture.10.Pippin J.W. Brinkkoetter P.T. Cormack-Aboud F.C. et al.Inducible rodent models of acquired podocyte diseases.Am J Physiol Renal Physiol. 2009; 296: F213-F229Crossref PubMed Scopus (211) Google Scholar Alterations in podocyte motility in response to injury are considered to underlie foot process effacement, and recent studies have highlighted the importance of podocyte actin cytoskeleton dynamics and reorganization in these processes.25.Mundel P. Reiser J. Proteinuria: an enzymatic disease of the podocyte?.Kidney Int. 2010; 77: 571-580Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar,26.Welsh G.I. Saleem M.A. The podocyte cytoskeleton-key to a functioning glomerulus in health and disease.Nat Rev Nephrol. 2011; 8: 14-21Crossref PubMed Scopus (196) Google Scholar As Rac1 is known to induce lamellipodia formation at the leading edge of motile cells,27.Burridge K. Wennerberg K. Rho and Rac take center stage.Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1509) Google Scholar it would be expected to have a central role in podocyte motility, which is supported by several recent studies. In vitro, angiotensin II induces a phenotypic shift in human podocytes from being dynamically stable to adaptively migratory through regulation of the cytoskeleton involving signaling pathways dominated by Rac1.28.Hsu H.H. Hoffmann S. Endlich N. et al.Mechanisms of angiotensin II signaling on cytoskeleton of podocytes.J Mol Med (Berl). 2008; 86: 1379-1394Crossref PubMed Scopus (125) Google Scholar In addition, the urokinase receptor uPAR is activated in murine podocytes in response to lipopolysaccharide and puromycin aminoglycoside treatment in vitro and in vivo, which leads to foot process effacement and proteinuria. uPAR complexes with and activates β3-integrin in podocytes, which activates Rac1 and Cdc42, and promotes cell motility.29.Wei C. Moller C.C. Altintas M.M. et al.Modification of kidney barrier function by the urokinase receptor.Nat Med. 2008; 14: 55-63Crossref PubMed Scopus (431) Google Scholar Surprisingly, we found that in long-term injury by UNX/DOCA-salt treatment podoctye-specific loss of Rac1 exacerbates proteinuria and glomerulosclerosis. Podocyte injury is a critical step in the development of glomerulosclerosis in models of DOCA-salt–induced hypertension, likely owing to an inability of the podocyte to adapt to cover increased glomerular capillary surfaces of hypertrophic glomeruli.11.Kretzler M. Koeppen-Hagemann I. Kriz W. Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat.Virchows Arch. 1994; 425: 181-193Crossref PubMed Scopus (147) Google Scholar Recently, Fukuda et al.30.Fukuda A. Chowdhury M.A. Venkatareddy M.P. et al.Growth-dependent podocyte failure causes glomerulosclerosis.J Am Soc Nephrol. 2012; 23: 1351-1363Crossref PubMed Scopus (132) Google Scholar demonstrated that a failure of podocytes to match glomerular tuft growth triggers proteinuria and glomerulosclerosis. This suggests podocyte-specific deletion of Rac1 impairs the podocyte’s ability to respond to hypertrophic stress in the UNX/DOCA-salt model. This could be owing to an impairment of hypertrophic signaling pathways, or a direct consequence of impaired mobility of podocyte foot processes. In fact, a role for Rac1-dependent hypertrophic signaling pathways has been known for some time in the myocardium through enhancement of hypertrophic gene expression via reactive oxygen species signaling and direct effects on transcription factors.31.Brown J.H. Del Re D.P. Sussman M.A. The Rac and Rho hall of fame: a decade of hypertrophic signaling hits.Circ Res. 2006; 98: 730-742Crossref PubMed Scopus (289) Google Scholar In addition, the balance between activation of GTPases Rac1 and RhoA may be important in the podocyte. It is known that Rac1 stimulation results in downmodulation of RhoA activity32.Sander E.E. ten Klooster J.P. van Delft S. et al.Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.J Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (737) Google Scholar,33.Wu Y.I. Frey D. Lungu O.I. et al.A genetically encoded photoactivatable Rac controls the motility of living cells.Nature. 2009; 461: 104-108Crossref PubMed Scopus (819) Google Scholar and that excessive RhoA activation in podocytes induces FSGS.34.Zhu L. Jiang R. Aoudjit L. et al.Activation of RhoA in podocytes induces focal segmental glomerulosclerosis.J Am Soc Nephrol. 2011; 22: 1621-1630Crossref PubMed Scopus (104) Google Scholar Thus, loss of Rac1 may allow excessive RhoA signaling and induce significant podocyte dysfunction and loss. Understanding the mechanisms driving, or abrogating, these process may lead to new opportunities for targeted therapeutic interventions in glomerular disease. Additional evidence indicates excess Rac1 activity is harmful to the mature podocyte. Recently, a mutant form of the ARHGAP24 gene that impairs Arhgap24 Rac1–GAP activity was found to be associated with FSGS in humans.35.Akilesh S. Suleiman H. Yu H. et al.Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis.J Clin Invest. 2011; 121: 4127-4137Crossref PubMed Scopus (202) Google Scholar Ahgap24 inactivates Rac1 and suppresses lamellipodia formation downstream of RhoA signaling. Arhgap24-knockdown studies in mouse podocytes revealed increased motility and
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