Congenital biliary atresia is correlated with disrupted cell junctions and polarity caused by Cdc42 insufficiency in the liver.

Rationale: Congenital biliary atresia (BA) is a destructive obliterative cholangiopathy of neonates that affects both intrahepatic and extrahepatic bile ducts. However, the cause of BA is largely unknown. Methods: We explored the cell junctions and polarity complexes in early biopsy BA livers by immunofluorescence staining and western blot. Cdc42, as a key cell junction and polarity regulator, was found dramatically decreased in BA livers. Therefore, in order to investigate the role of Cdc42 in BA development, we constructed liver-specific and tamoxifen induced cholangiocyte-specific Cdc42 deleted transgenic mice. We further evaluated the role of bile acid in aggravating biliary damage in Cdc42 insufficient mouse liver. Results: We found a dramatic defect in the assembly of cell junctions and polarity complexes in both cholangiocytes and hepatocytes in BA livers. This defect was characterized by the disordered location of cell junction proteins, including ZO1, ß-catenin, E-cadherin and claudin-3. Cdc42 and its active form, Cdc42-GTP, which serves as a small Rho GTPase to orchestrate the assembly of polarity complexes with Par6/Par3/αPKC, were substantially reduced in BA livers. Selective Cdc42 deficiency in fetal mouse cholangiocytes resulted in histological changes similar to those found in human BA livers, including obstruction in both the intra- and extrahepatic bile ducts, epithelial atrophy, and the disruption of cell junction and polarity complexes. A reduction in bile acids notably improved the histology and serological indices in Cdc42-mutant mice. Conclusion: Our results illustrate that BA is closely correlated with the impaired assembly of cell junction and polarity complexes in liver cells, which is likely caused by Cdc42 insufficiency and aggravated by bile acid corrosion.

Pancytopenia, Recurrent Infection, Poor Wound Healing, Heterotopia of the Brain Probably Associated with A Candidate Novel de Novo CDC42 Gene Defect: Expanding the Molecular and Phenotypic Spectrum.

CDC42 (cell division cycle protein 42) belongs to the Rho GTPase family that is known to control the signaling axis that regulates several cellular functions, including cell cycle progression, migration, and proliferation. However, the functional characterization of the CDC42 gene in mammalian physiology remains largely unclear. Here, we report the genetic and functional characterization of a non-consanguineous Saudi family with a single affected individual. Clinical examinations revealed poor wound healing, heterotopia of the brain, pancytopenia, and recurrent infections. Whole exome sequencing revealed a de novo missense variant (c.101C > A, p.Pro34Gln) in the CDC42 gene. The functional assays revealed a substantial reduction in the growth and motility of the patient cells as compared to the normal cells control. Homology three-dimensional (3-D) modeling of CDC42 revealed that the Pro34 is important for the proper protein secondary structure. In conclusion, we report a candidate disease-causing variant, which requires further confirmation for the etiology of CDC42 pathogenesis. This represents the first case from the Saudi population. The current study adds to the spectrum of mutations in the CDC42 gene that might help in genetic counseling and contributes to the CDC42-related genetic and functional characterization. However, further studies into the molecular mechanisms that are involved are needed in order to determine the role of the CDC42 gene associated with aberrant cell migration and immune response.

Aplasia cutis congenita in a CDC42-related developmental phenotype.

Cell division cycle 42 (CDC42) is a small Rho GTPase, which serves as a fundamental intracellular signal node regulating actin cytoskeletal dynamics and several other integral cellular processes. CDC42-associated disorders encompass a broad clinical spectrum including Takenouchi-Kosaki syndrome, autoinflammatory syndromes and neurodevelopmental phenotypes mimicking RASopathies. Dysregulation of CDC42 signaling by genetic defects in either DOCK6 or ARHGAP31 is also considered to play a role in the pathogenesis of Adams-Oliver syndrome (AOS). Here, we report a mother and her child carrying the previously reported pathogenic CDC42 variant c.511G>A (p.Glu171Lys). Both affected individuals presented with short stature, distinctive craniofacial features, pectus deformity as well as heart and eye anomalies, similar to the recently described Noonan syndrome-like phenotype associated with this variant. Remarkably, one of the patients additionally exhibited aplasia cutis congenita of the scalp. Multi-gene panel sequencing of the known AOS-causative genes and whole exome sequencing revealed no second pathogenic variant in any disease-associated gene explaining the aplasia cutis phenotype in our patient. This observation further expands the phenotypic spectrum of CDC42-associated disorders and underscores the role of CDC42 dysregulation in the pathogenesis of aplasia cutis congenita.

Cdc42 Deficiency Leads To Epidermal Barrier Dysfunction by Regulating Intercellular Junctions and Keratinization of Epidermal Cells during Mouse Skin Development.

Rationale: Cdc42 is a Rho GTPase that regulates diverse cellular functions. Here, we used genetic techniques to investigate the role of Cdc42 in epidermal development and epidermal barrier formation. Methods: Keratinocyte-restricted Cdc42 knockout mice were generated with the Cre-LoxP system under the keratin 14 (K14) promoter. The skin and other tissues were collected from mutant and wild-type mice, and their cellular, molecular, morphological, and physiological features were analyzed. Results: Loss of Cdc42 in the epidermis in vivo resulted in neonatal lethality and impairment of epidermal barrier formation. Cdc42 deficiency led to the loss of epidermal stem cells. The absence of Cdc42 led to increased thickening of the epidermis, which was associated with increased proliferation and reduced apoptosis of keratinocytes. In addition, Cdc42 deficiency damaged tight junctions, adherens junctions and desmosomes. RNA sequencing results showed that the most significantly altered genes were enriched by the terms of "keratinization" and "cornified envelope" (CE). Among the differentially expressed genes in the CE term, several members of the small proline-rich protein (SPRR) family were upregulated. Further study revealed that there may be a Cdc42-SPRR pathway, which may correlate with epidermal barrier function. Conclusions: Our study indicates that Cdc42 is essential for epidermal development and epidermal barrier formation. Defects in Cdc42-SPRR signaling may be associated with skin barrier dysfunction and a variety of skin diseases.

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development.

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Automated profiling of growth cone heterogeneity defines relations between morphology and motility.

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.

Defective endothelial cell migration in the absence of Cdc42 leads to capillary-venous malformations.

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigated the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We found that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. Although the loss of Cdc42 seems generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42-depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network.

Endothelial Cdc42 deficiency impairs endothelial regeneration and vascular repair after inflammatory vascular injury.

BACKGROUND: Endothelial cell (EC) regeneration is essential for inflammation resolution and vascular integrity recovery after inflammatory vascular injury. Cdc42 is a central regulator of cell survival and vessel formation in EC development. However, it is unknown that whether Cdc42 could be a regulating role of EC repair following the inflammatory injury in the lung. The study sought to test the hypothesis that Cdc42 is required for endothelial regeneration and vascular integrity recovery after LPS-induced inflammatory injury. METHODS AND RESULTS: The role of Cdc42 for the regulation of pulmonary vascular endothelial repair was tested in vitro and in vivo. In LPS-induced acute lung injury (ALI) mouse models, knockout of the Cdc42 gene in ECs increased inflammatory cell infiltration and pulmonary vascular leakage and inhibited vascular EC proliferation, which eventually resulted in more severe inflammatory lung injury. In addition, siRNA-mediated knockdown of Cdc42 protein on ECs disrupted cell proliferation and migration and tube formation, which are necessary processes for recovery after inflammatory vascular injury, resulting in inflammatory vascular injury recovery defects. CONCLUSION: We found that Cdc42 deficiency impairs EC function and regeneration, which are crucial in the post-inflammatory vascular injury repair process. These findings indicate that Cdc42 is a potential target for novel treatments designed to facilitate endothelial regeneration and vascular repair in inflammatory pulmonary vascular diseases, such as ALI/ARDS.

Inhibition of Cdc42 and Rac1 activities in pheochromocytoma, the adrenal medulla tumor.

Altered Rho GTPase signaling has been linked to many types of cancer. As many small G proteins, Rho GTPases cycle between an active and inactive state thanks to specific regulators that catalyze exchange of GDP into GTP (Rho-GEF) or hydrolysis of GTP into GDP (Rho-GAP). Recent studies have shown that alteration takes place either at the level of Rho proteins themselves (expression levels, point mutations) or at the level of their regulators, mostly RhoGEFs and RhoGAPs. Most reports describe Rho GTPases gain of function that may participate to the tumorigenesis processes. In contrast, we have recently reported that decreased activities of Cdc42 and Rac1 as well as decreased expression of 2 Rho-GEFs, FARP1 and ARHGEF1, correlate with pheochromocytomas, a tumor developing in the medulla of the adrenal gland (Croisé et al., Endocrine Related Cancer, 2016). Here we highlight the major evidence and further study the correlation between Rho GTPases activities and expression levels of ARHGEF1 and FARP1. Finally we also discuss how the decrease of Cdc42 and Rac1 activities may help human pheochromocytomas to develop and comment the possible relationship between FARP1, ARHGEF1 and the 2 Rho GTPases Cdc42 and Rac1 in tumorigenesis.

Low Gene Dosage of Cdc42 Is Not Associated with Protein Dysfunction in Patients with Colorectal Cancer.

High incidence of Rho Cdc42-GTPase overexpression has been found in Colorectal Cancer (CRC) samples, suggesting its potential role in tumor development. However, no conclusive studies have shown the lack of mutations and/or copy number of Cdc42 gene in this type of samples. To understand mutation/deletion and copy number status of Cdc42 gene, CRC patients were evaluated for both parameters. More than Cdc42 mutants, single-nucleotide variants were found. Analysis of regions flanking the Cdc42 gene showed allelic imbalance; 58.7% were loss of heterozygosity (LOH) positive and 14.8% presented microsatellite instability. The highest LOH percentage was located between microsatellite markers D1S199 and D1S2674, where the Cdc42 gene is located. No association between gender, age, and tumor stage was found. LOH validation through gene dosage analysis showed most CRC patients with allelic imbalance also presented a low gene dosage of Cdc42, although equal amounts of Cdc42 mRNA were detected in all samples. Although changes in Cdc42 expression were not found in any condition, Cdc42 activation was different between high and normal gene dosage samples, but not between samples with normal and low copy number. Low dosage of Cdc42 was also not related to changes in methylation status at the Cdc42 promoter region. Results suggest that low copy of Cdc42 gene is not associated with Cdc42 protein dysfunction in CRC patients.

Cdc42 deficiency induces podocyte apoptosis by inhibiting the Nwasp/stress fibers/YAP pathway.

Podocyte apoptosis is a major mechanism that leads to proteinuria in many chronic kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. The Rho family of small GTPases has been shown to be required in maintaining podocyte structure and function. Recent studies have indicated that podocyte-specific deletion of Cdc42 in vivo, but not of RhoA or Rac1, leads to congenital nephrotic syndrome and glomerulosclerosis. However, the underlying cellular events in podocyte controlled by Cdc42 remain unclear. Here, we assessed the cellular mechanisms by which Cdc42 regulates podocyte apoptosis. We found that the expression of Cdc42 and its activity were significantly decreased in high glucose-, lipopolysaccharide- or adriamycin-injured podocytes. Reduced Cdc42 expression in vitro and in vivo by small interfering RNA and selective Cdc42 inhibitor ML-141, respectively, caused podocyte apoptosis and proteinuria. Our results further demonstrated that insufficient Cdc42 or Nwasp, its downstream effector, could decrease the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Moreover, our data indicated that the loss of stress fibers caused by Cdc42/Nwasp deficiency also decreased Yes-associated protein (YAP) mRNA and protein expression, and induced podocyte apoptosis. Podocyte apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited by overexpressing-active YAP. Thus, the Cdc42/Nwasp/stress fibers/YAP signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases.

A chimeric embryoid body model to study vascular morphogenesis.

Embryonic stem cell (ESC)-derived embryoid body (EB) is a unique model for studying vascular development, in that it provides a three-dimensional microenvironment that mimics an in vivo milieu. When using gene-targeting EBs to study certain defects in vascular morphogenesis, it is necessary to determine whether the defect is due to the intrinsic loss of the gene in endothelial cells (EC) or rather due to the lack of surrounding factors that would typically promote vascular development. Here we describe a chimeric EB vessel development model, in which the utilization of the PECAM-GFP reporter gene in wild-type ESCs allows for the introduction of "normal" extracellular factors formed by its parallel differentiation to the gene-deletion EC that might otherwise be devoid of these factors.

A crucial role for CDC42 in senescence-associated inflammation and atherosclerosis.

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.

Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

Deletion of Cdc42 enhances ADAM17-mediated vascular endothelial growth factor receptor 2 shedding and impairs vascular endothelial cell survival and vasculogenesis.

Cdc42 is a Ras-related GTPase that plays an important role in the regulation of a range of cellular functions, including cell migration, proliferation, and survival. Consistent with its critical functions in vitro, the inactivation of Cdc42 in mice has been shown to result in embryonic lethality at embryonic day 6.5 (E6.5) before blood vessel formation. To determine the role of Cdc42 in new blood vessel formation, we have generated vascular endothelial cell (EC)-specific Cdc42 knockout mice by crossing Cdc42(flox/flox) mice with Tie2-Cre mice. The deletion of Cdc42 in ECs caused embryonic lethality with vasculogenesis and angiogenesis defects. We observed that Cdc42 is critical for EC migration and survival but not for cell cycle progression. Moreover, we found that the inactivation of Cdc42 in ECs decreased the level of vascular endothelial growth factor receptor 2 (VEGFR2) protein on the EC surface and promoted the production of a 75-kDa membrane-associated C-terminal VEGFR2 fragment. Using cultured primary mouse ECs and human umbilical vein ECs, we have demonstrated that the deletion of Cdc42 increased ADAM17-mediated VEGFR2 shedding. Notably, inhibition of ADAM17 or overexpression of VEGFR2 can partially reverse Cdc42 deletion-induced EC apoptosis. These data indicate that Cdc42 is essential for VEGFR2-mediated signal transduction in blood vessel formation.

RhoA and Cdc42 are required in pre-migratory progenitors of the medial ganglionic eminence ventricular zone for proper cortical interneuron migration.

Cortical interneurons arise from the ganglionic eminences in the ventral telencephalon and migrate tangentially to the cortex. Although RhoA and Cdc42, members of the Rho family of small GTPases, have been implicated in regulating neuronal migration, their respective roles in the tangential migration of cortical interneurons remain unknown. Here we show that loss of RhoA and Cdc42 in the ventricular zone (VZ) of the medial ganglionic eminence (MGE) using Olig2-Cre mice causes moderate or severe defects in the migration of cortical interneurons, respectively. Furthermore, RhoA- or Cdc42-deleted MGE cells exhibit impaired migration in vitro. To determine whether RhoA and Cdc42 directly regulate the motility of cortical interneurons during migration, we deleted RhoA and Cdc42 in the subventricular zone (SVZ), where more fate-restricted progenitors are located within the ganglionic eminences, using Dlx5/6-Cre-ires-EGFP (Dlx5/6-CIE) mice. Deletion of either gene within the SVZ does not cause any obvious defects in cortical interneuron migration, indicating that cell motility is not dependent upon RhoA or Cdc42. These findings provide genetic evidence that RhoA and Cdc42 are required in progenitors of the MGE in the VZ, but not the SVZ, for proper cortical interneuron migration.

Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42.

Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.

Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte 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.