AJP - Renal AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F148-F156, 2007. First published March 20, 2007; doi:10.1152/ajprenal.00294.2006
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F148    most recent
00294.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, H.
Right arrow Articles by Takano, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, H.
Right arrow Articles by Takano, T.

Role of Rho-GTPases in complement-mediated glomerular epithelial cell injury

Hui Zhang,1 Andrey V. Cybulsky,1 Lamine Aoudjit,1 Jianxin Zhu,1 Hongping Li,1 Nathalie Lamarche-Vane,2 and Tomoko Takano1

Departments of 1Medicine and 2Anatomy and Cell Biology, McGill University, Montreal, Canada

Submitted 28 July 2006 ; accepted in final form 13 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Visceral glomerular epithelial cells (GEC) are essential for maintenance of normal glomerular permselectivity. The actin cytoskeleton is a key determinant of GEC morphology and function. In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces nonlytic GEC injury associated with morphological changes of GEC and proteinuria. The current study addresses the role of Rho family of small GTPases in complement-mediated GEC injury. When cultured rat GEC were stimulated with complement C5b-9 for 18 h, RhoA activity increased, whereas Rac1/Cdc42 activities decreased, compared with control cells. Similar changes in Rho-GTPase activities were observed in glomeruli from rats with PHN. The amount of active p190RhoGAP, a negative upstream regulator of RhoA, was decreased in complement-stimulated GEC, potentially contributing to increased RhoA activity. To address the functional effects of Rho-GTPases, GEC were transfected with constitutively active (CA) or dominant negative (DN) Rho-GTPase mutants. GEC transfected with CA-RhoA showed a smaller and round contour and prominent cortical F-actin. In contrast, GEC transfected with CA-Rac1 demonstrated morphological changes that resembled process formation. In addition, expression of CA-RhoA attenuated complement-mediated cytotoxicity, whereas cytotoxicity was augmented by DN-RhoA. Thus exposure of GEC to complement alters the balance of RhoA, Rac1, and Cdc42 activities. The activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement. Activation of RhoA increases the resistance of GEC to complement-mediated injury.

passive Heymann nephritis; RhoA/Rac1/Cdc42; actin cytoskeleton

VISCERAL GLOMERULAR EPITHELIAL cells (GEC), also known as podocytes, are terminally differentiated cells and have highly specialized morphology (24). The foot processes of adjacent GEC interdigitate and encircle the outer aspect of the glomerular basement membrane (GBM), forming the final barrier to protein loss. Among the three major cytoskeletal components, i.e., microtubules, intermediate filaments, and actin filaments, the latter represent the core structural component of foot processes (19). Recently, a number of proteins, which are expressed in podocyte foot processes, such as nephrin, podocin, CD2AP, and {alpha}-actinin-4, have been identified (24). Mutations of these foot process proteins lead to impaired glomerular permselectivity and proteinuria either in human or in mouse (24). It is believed that the foot process molecules bind to and regulate the actin cytoskeleton, either directly or indirectly, thereby contributing to normal morphology and function of podocytes (24). The proteins that regulate the actin cytoskeleton of podocytes are therefore of critical importance for the maintenance of glomerular permselectivity (19, 24).

An example of GEC injury in vivo is membranous nephropathy, a common cause of nephrotic syndrome in adults. The rat model of passive Heymann nephritis (PHN) has been used extensively to study the pathophysiology of membranous nephropathy and its validity has been discussed in detail recently (35). In PHN, an antibody binds to GEC antigens, leading to in situ formation of subepithelial immune complexes, activation of complement, and assembly of the membrane attack complex C5b-9 in the plasma membrane of GEC (6). Nucleated cells require multiple C5b-9 lesions for lysis but, at lower doses, C5b-9 induces sublytic injury and various metabolic effects (6). In GEC, C5b-9 can activate cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase pathways, cell cycle proteins, transcription factors, endoplasmic reticulum stress, Hsp27, and others. Many of these pathways are able to modulate complement-mediated GEC injury (8). C5b-9 causes reorganization of the actin microfilaments in cultured GEC (37). Reorganization of the actin cytoskeleton may be one of the mechanisms by which complement-mediated GEC injury leads to morphological changes and impaired permselectivity (37).

Many cytoskeletal responses are mediated by the Rho family of small GTPases (14). The mammalian Rho family consists of at least 20 distinct members, among which, Rho/Rac/Cdc42 proteins have been most extensively studied. In fibroblasts, Rho and one of its downstream effectors, Rho-associated coiled-coil forming kinase (ROCK), regulate stress fiber assembly as well as focal adhesion formation, while Rac and Cdc42 regulate formation of lamellipodia and filopodia, respectively (14). Rho-GTPases also impact on the F-actin pattern in epithelial cells, although cytoskeletal changes induced by Rho-GTPases are different from those in fibroblasts (18). In addition, Rho-GTPases are involved in a variety of cellular processes, such as adhesion, cell motility, transcriptional regulation, cell cycle progression, and cell survival (14, 16, 38). Rho-GTPases cycle between GTP-bound (active) and GDP-bound (inactive) forms and only the GTP-bound forms interact with downstream effectors and initiate signaling events. These two forms are tightly regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (14). One of the well-characterized RhoGAP family members, p190RhoGAP, interacts preferentially with RhoA, compared with Rac1 and Cdc42. p190RhoGAP is expressed ubiquitously and is activated by tyrosine phosphorylation (12, 22). The coordinated regulation of signal transduction pathways by Rho family GTPases is essential for many normal cell activities, and inappropriate activation of these molecular switches contributes to the pathogenesis of several diseases, including cancers (21).

The current study addresses the changes in activity of RhoA, Rac1, and Cdc42, three well-characterized Rho-GTPases, in complement-mediated GEC injury in vitro and in vivo. We also studied the impact of these changes on cell morphology and complement-mediated cytolysis in GEC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Tissue culture media and Lipofectamine 2000 were from Invitrogen-Life Technologies (Burlington, ON). Electrophoresis reagents were from Bio-Rad Laboratories (Mississauga, ON). Anti-Rho, anti-p190RhoGAP, and anti-Rac antibodies were from Upstate Biotechnology (Lake Placid, NY). Antibodies for phospho-myosin light chain 2 (Ser19) and myosin light chain were from Cell Signaling (Beverly, MA). Anti-Cdc42 antibody and protein G-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody (PY69) was from BD Biosciences (Missisauga, ON). Enhanced chemiluminescence (ECL) detection reagents and glutathione-Sepharose beads were from Amersham Bioscience (Baie d'Urfé, PQ). Rhodamine-phalloidin was from Molecular Probes (Eugene, OR). Male Sprague-Dawley rats were from Charles River Canada (St. Constant, PQ). The plasmids pRK5-RhoA(L63)-Myc [constitutively active (CA)], pRK5-RhoA(N19)-Myc [dominant negative (DN)], pRK5-Rac1(L61)-Myc (CA), pRK5-Rac1(N17)-Myc (DN), pRK5-Cdc42(L61)-Myc (CA), pRK5-Cdc42(N17)-Myc (DN), and Cdc42/Rac interactive binding domain fused to GST (GST-CRIB) were described previously (26, 29). Rhotekin Rho-binding domain fused to GST (GST-RBD) was a gift from Dr. M. A. Schwartz (Scripps Research Institute, La Jolla, CA) (28). Human C8-deficient serum, purified human C8, and other chemicals were from Sigma (Mississauga, ON).

GEC culture. Rat GEC culture and characterization were described previously (5, 27). Briefly, GEC were cultured in K1 medium (50% DMEM, 50% Ham F-12, 10% NuSerum, hormone mix) and studies were carried out between passages 10 and 60. For transient transfection morphological studies (see GoGoGoGoFig. 5), a subclone of GEC that grows on plastic substratum (GEC-pl) was used. For complement stimulation (Figs. 1 and 2 and see Figs. 4 and Go7), a subclone of GEC, which stably overexpress cPLA2 (GEC-cPLA2), was used (25). GEC-cPLA2 generally have augmented responses to complement stimulation (25). Since GEC in vivo express enzymatically active cPLA2 (11), we believe that GEC-cPLA2 are more representative of GEC in vivo. GEC subclones that overexpress CA-RhoA or DN-RhoA in an inducible manner were established using a method analogous to one we described previously (1). CA-RhoA or DN-RhoA (both with a Myc epitope tag) was subcloned into the inducible vector, pIND-hygro (Invitrogen-Life Technologies), and its expression was induced by an insect hormone, ponasterone A (Invitrogen-Life Technologies), which otherwise has no known effects in mammalian cells.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 1. Complement alters the activities of RhoA, Rac1, and Cdc42 in cultured rat GEC (short-term incubation). A: expression of RhoA, Rac1, and Cdc42 in cultured rat GEC was determined by immunoblotting. B and C: cultured rat GEC (see MATERIALS AND METHODS) were incubated with antibody and complement (NS, 1.5% vol/vol) or HIS in control incubations (40 min at 37°C). Cell lysates were subjected to pull-down assays with GST-Rhotekin and GST-CRIB to assay for active (GTP-bound) RhoA (B) and Rac1/Cdc42 (C and not shown), respectively (see MATERIALS AND METHODS). Pull-down samples and total lysates were analyzed by immunoblotting using antibodies for RhoA (B), Rac1 (C), and Cdc42 (not shown). B and C: top is a representative immunoblot and bottom is densitometric analysis. RhoA: *P < 0.01 vs. HIS, n = 4; Rac1: +P < 0.05 vs. HIS, n = 4.

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. Complement alters the activities of RhoA, Rac1, and Cdc42 in cultured rat GEC (long-term incubation). Antibody-sensitized GEC were incubated with 1.5% (vol/vol) NS or HIS (control) for 18 h at 37°C. GTP-bound (active) RhoA (A), Rac1 (B), and Cdc42 (C) were quantified as in Fig. 1. *P < 0.05 vs. HIS, n = 4 (RhoA) or 5 (Rac1 and Cdc42). D: GEC were incubated with HIS, NS, C8-deficient serum alone (to form C5b-7), or C8-deficient serum reconstituted with purified C8 (C8D+C8; to form sublytic C5b-9) for 18 h at 37°C, and GTP-bound RhoA was quantified by pull-down assay. Densitometric analysis is shown (average of 2 experiments).

 

Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Complement alters the activities of RhoA, Rac1, and Cdc42 in GEC in vivo. Glomeruli were isolated from normal rats (control) and rats with passive Heymann nephritis (PHN; day 14). Glomerular lysates were subjected to pull-down assays as in Fig. 1 for RhoA (A), Rac1 (B), and Cdc42 (C). *P < 0.05 vs. control, n = 6 rats in each group.

 

Figure 4
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 4. Complement decreases p190RhoGAP activity in cultured rat GEC. GEC were incubated with complement as in Fig. 2. Cell lysates were immunoprecipitated with anti-p190RhoGAP antibody or mouse IgG (control). Immunoprecipitates were analyzed by immunoblotting with anti-p190RhoGAP or anti-phosphotyrosine (PY69) antibodies. Top: representative blot. Bottom: densitometric analysis. *P < 0.05 vs. HIS, n = 4.

 

Figure 5
View larger version (109K):
[in this window]
[in a new window]

  		Fig. 5. Impact of Rho-GTPase activities on GEC morphology. GFP-conjugated Rho-GTPase mutants were transiently transfected into cultured GEC. After fixation and permeabilization, cells were stained with rhodamine-phalloidin. Cells were examined by confocal microscopy and successfully transfected cells were identified by green fluorescence. Transfection of empty vector did not affect morphology (GFP). CA-RhoA predominantly localized in the plasma membrane and made cells smaller and rounder with a strong cortical F-actin pattern (arrows). CA-Rac1 appeared to distribute equally in the plasma membrane and cytoplasm and induced cell elongation (arrowhead: bottom) and protrusion (arrowhead: top). CA-Cdc42 localized mainly in the plasma membrane and enhanced cortical F-actin, similar to CA-RhoA (double arrows); however, unlike CA-RhoA, cells had more "angular" contour and were not notably smaller than nontransfected cells. DN mutants did not affect morphology significantly (not shown).

 

Figure 6
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 6. Effects of RhoA activation on complement-mediated cytolysis in GEC. A: GEC, which express CA-RhoA in an inducible manner, were stimulated with ponasterone A (to induce CA-RhoA) for 7.5 and 18 h and RhoA activity was quantified by pull-down assay as in Fig. 1B. Note that CA-RhoA is tagged with the myc-epitope and runs at a higher molecular size than the endogenous RhoA. Endogenous active RhoA was not detected because of a short exposure time. B: CA-RhoA was induced in the presence or absence of Y27632 (10 µM) for 18 h. Cells were stained with rhodamine-phalloidin and studied by confocal microscopy. Control cells were treated with vehicle (ethanol) for 18 h. C: cells were treated as in B and stimulated with a lytic concentration of complement (NS: 3%). In controls, cells were treated with ethanol in the place of ponasterone A. Cytotoxicity was quantified by specific LDH release (see MATERIALS AND METHODS). *P < 0.05 vs. Ethanol + Vehicle, n = 5.

 

Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 7. Effects of dominant negative (DN)-RhoA on complement-mediated cytolysis in GEC. GEC, which express DN-RhoA in an inducible manner, were stimulated with ponasterone A (to induce DN-RhoA expression) for 18 h. A: immunoblotting with anti-Myc antibody demonstrated optimal induction of DN-RhoA by ponasterone A at 8 µM. B: cells were treated with ponasterone A (8 µM) or vehicle (ethanol) for 18 h and were stimulated with complement (NS: 1 and 2.5%). Cytotoxicity was quantified by specific LDH release (see MATERIALS AND METHODS). Significant differences were present among groups (*P < 0.05, ethanol vs. ponasterone, 2-way ANOVA, n = 3 experiments performed in duplicate).

 
Incubation of GEC with complement. Complement stimulation of GEC was carried out as described previously (7, 27). Briefly, GEC-cPLA2 were incubated with rabbit anti-GEC antiserum or sheep anti-Fx1A antiserum (5% vol/vol) for 40 min at 22°C, followed by incubation with normal human serum (NS; 1.5% vol/vol) for 40 min (short-term or acute) or 18 h (long-term or chronic) to assemble C5b-9. In control, incubation with decomplemented, heat-inactivated serum (HIS; 56°C, 30 min) was used. In some experiments, antibody-sensitized GEC were incubated with C8-deficient human serum (C8D; 1.5% vol/vol) with or without reconstitution with purified human C8 (2 µg/ml in undiluted serum).

Induction of PHN. PHN was induced in male Sprague-Dawley rats (150–175 g) by a single intravenous injection (400 µl/rat) of sheep anti-Fx1A antiserum as described previously (34). On day 14, rats were killed and glomeruli were isolated by differential sieving (33). All studies were approved by the McGill University Animal Care Committee.

Immunoblotting. Cells or glomeruli were lysed in IP buffer [1% Triton X-100, 125 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 10 mM sodium pyrophosphate, 25 mM NaF] and protease inhibitor cocktail (Roche Diagnostics). After insoluble components were removed by centrifugation (14,000 rpm, 10 min, 4°C), the protein concentration of supernatants was quantified using a commercial reagent (Bio-Rad). Proteins were separated by SDS-PAGE under reducing conditions and were transferred electrophoretically to nitrocellulose membranes. Membranes were blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight. After three washes, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase. Immunoreactive proteins were identified by the ECL system. Densitometric analysis was carried out by Image J software.

Pull-down assay for active Rho and Rac/Cdc42. Preparation of GST-CRIB and GST-RBD was described previously (29, 30). Cells or glomeruli were lysed in lysis buffer [25 mM HEPES (pH 7.5), 1% NP-40, 10 mM MgCl2, 100 mM NaCl, 5% glycerol, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin]. Equal amounts of protein (250–1,000 µg) were incubated for 1 h at 4°C with purified GST-CRIB or GST-RBD (10–15 µg) bound to glutathione-Sepharose beads. The beads and proteins bound to the fusion protein were washed twice and were subjected to SDS-PAGE (12%) and immunoblotting.

Transient transfection of GEC with Rho-GTPase mutants and F-actin staining. CA and DN mutants of Rho-GTPases were subcloned into pEGFP-C1 (BD Sciences). GEC-pl grown on glass coverslips (40–50% confluency) were transiently transfected with lipofectamine 2000 transfection reagent, using 3 µl of reagent per microgram of plasmid. After 24 h, cells were fixed with 3% formaldehyde and permeabilized with 0.5% Triton X-100. After being blocked with 3% BSA, GEC were stained with rhodamine-phalloidin (0.05 µg/ml) to label F-actin and examined with a confocal microscope (Zeiss LSM 510 META).

Measurement of complement-dependent cytotoxicity. Complement-induced cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH), as described previously (2). The specific release of LDH was calculated by the formula, (NS – HIS)/(100 – HIS) x 100, where NS and HIS represent percent LDH release with NS and HIS, respectively.

Statistics. Data are presented as means ± SE. The t statistic was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t statistic and adjusting the critical value according to the Bonferroni method. Two-way ANOVA was used to determine significant differences in multiple measurements among groups.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activities of RhoA, Rac1, and Cdc42 are altered by complement in GEC in vitro. In lysates of cultured rat GEC, RhoA, Rac1, and Cdc42 were detected as bands of 22, 21, and 25 kDa, respectively, by immunoblotting, confirming the endogenous expression of these proteins (Fig. 1A). To address the role of Rho-GTPases in GEC injury, we studied the effect of the sublytic concentration of complement on RhoA, Rac1, and Cdc42 activities in cultured rat GEC. We first determined the optimal concentration of complement to induce GEC sublytic injury. We reported previously that sublytic complement stimulation leads to phosphorylation of ERK. When cells were stimulated with various concentrations of complement (NS), 1.5% NS induced most prominent and consistent phosphorylation of ERK; thus this concentration was chosen for further experiments.

In the first series of experiments, GEC were stimulated with complement for 40 min (short-term or acute) and Rho-GTPase activities were determined by pull-down assays. Control cells (incubated with HIS) showed a small basal amount of active RhoA, whereas active Rac1 and Cdc42 were easily detectable (Fig. 1, B and C, and not shown). Complement stimulation increased the amount of active RhoA and Rac1 by 2.6- and 1.7-fold, respectively, compared with control (RhoA: HIS 13 ± 5, NS 37 ± 9, arbitrary units, n = 4, P < 0.01; Rac1: HIS 24 ± 8, NS 43 ± 7, arbitrary units, n = 4, P < 0.05; Fig. 1, B and C). The amount of active Cdc42 did not change significantly (not shown). In the next series of experiments, we studied the impact of long-term (or chronic, 18 h) complement stimulation on Rho-GTPase activities. The amount of active RhoA continued to be increased by complement, albeit the increase was much more modest, compared with the acute stimulation (HIS 31 ± 10, NS 38 ± 9, n = 4, P < 0.05). The amounts of active Rac1 and Cdc42 were decreased by 26 and 46%, respectively (Rac1: 55 ± 10, NS 41 ± 8, n = 4, P < 0.05; Cdc42: HIS 35 ± 8, NS 19 ± 5, n = 4, P < 0.05; Fig. 2, A–C). Therefore, complement-induced RhoA activation occurs early and persists chronically. In contrast, Rac1 was activated initially, but together with Cdc42, was suppressed during chronic incubation with complement.

To verify that the complement-mediated change in RhoA activity was due to C5b-9 assembly, we compared C8D, which assembles C5b-7, with C8DS reconstituted with purified C8. When antibody-sensitized GEC were incubated with C8D alone, the amount of active RhoA was not different from control (HIS). However, when C8D was reconstituted with purified C8, active RhoA increased, similar to cells stimulated with NS (Fig. 2D). These results indicate that activation of RhoA is dependent on C5b-9 assembly.

Activities of RhoA, Rac1, and Cdc42 are altered by complement in GEC in vivo. We next studied the amount of active RhoA, Rac1, and Cdc42 in glomeruli from rats with PHN, where GEC injury and proteinuria are dependent on assembly of C5b-9. Fourteen days after induction of PHN, rats showed significant proteinuria (325 ± 125 mg/day, n = 6) compared with normal rats (15 ± 3 mg/day, n = 6, P < 0.01 vs. PHN). By analogy to GEC in culture, the amount of active RhoA in normal rat glomeruli was small, while active Rac1 and Cdc42 were easily detectable (Fig. 3). In glomeruli from rats with PHN, the amount of active RhoA was increased by 2.3-fold, compared with normal rat glomeruli (control 100 ± 17, PHN 228 ± 50, arbitrary units, n = 6, P < 0.05; Fig. 3A). In contrast, the amounts of active Rac1 and Cdc42 were decreased by 38 and 55%, respectively, in glomeruli from rats with PHN (Rac1: control 100 ± 1, PHN 62 ± 12, arbitrary units, n = 6, P < 0.05; Cdc42: control 100 ± 2, PHN 45 ± 19, arbitrary units, n = 6, P < 0.05; Fig. 3, B and C). There was no significant difference in the total amount of Rho-GTPases (Fig. 3, A-C). Since the GEC is the target of C5b-9 in PHN (35), it is reasonable to conclude that C5b-9 induced changes in Rho-GTPase activities in GEC. Moreover, the changes in the glomerular activities of Rho-GTPases in PHN paralleled changes during chronic incubation with complement in cultured rat GEC.

Activity of p190RhoGAP is decreased by complement in GEC. To address the mechanism of complement-mediated RhoA activation in GEC, we next studied the effect of complement on the negative regulator of RhoA, p190RhoGAP (22). Upon undergoing tyrosine phosphorylation, p190RhoGAP becomes active and inactivates RhoA (22). Thus we assessed the level of tyrosine phosphorylation of p190RhoGAP as a surrogate marker of its activity. p190RhoGAP was clearly tyrosine phosphorylated in control GEC indicating an active status, consistent with the low level of RhoA activity in unstimulated GEC. When GEC were stimulated with complement for 40 min, tyrosine phosphorylation of p190RhoGAP decreased by 41% (HIS 21 ± 3, NS 12 ± 1, arbitrary units, n = 4, P < 0.05; Fig. 4). These results suggest that the complement-induced decrease of p190RhoGAP activity may, at least in part, contribute to complement-mediated RhoA activation.

Impact of Rho-GTPase activities on morphology of GEC in culture. Since Rho-GTPases can mediate various cytoskeletal responses, and the activities of Rho-GTPases were differentially altered in complement-mediated GEC injury, we first addressed the impact of a sublytic concentration of complement on the actin cytoskeleton in GEC. Phalloidin staining of control GEC showed a clear cortical F-actin pattern, as well as some stress fibers, as described previously (10). After stimulation of GEC with a sublytic concentration of complement acutely (40 min), changes in F-actin were not remarkable. Furthermore, attempts to induce more marked changes in the actin cytoskeleton with chronic complement stimulation, either by increasing the complement concentration or by repeated addition of fresh complement to the cells resulted in cell death, were not successful. Therefore, we adopted an alternate approach, which focused on identifying the effects of individual Rho-GTPases on the GEC cytoskeleton and morphology. To facilitate these studies, we employed transient transfection of constitutively active (CA) or dominant negative (DN) mutants of Rho-GTPases. All mutants were fused with GFP to identify successfully transfected cells. GEC transfected with GFP alone did not demonstrate any morphological changes, compared with untransfected cells (Fig. 5, GFP). Since complement stimulated RhoA, and reduced Rac1 and Cdc42 activities in culture and in vivo, our primary focus was on the effects of CA-RhoA, as well as DN-Rac1 and DN-Cdc42. CA-RhoA localized predominantly at the plasma membrane. Transfected cells were smaller and rounder, and staining with rhodamine-phalloidin demonstrated a strong cortical F-actin pattern (Fig. 5, CA-RhoA). DN-Rac1 and DN-Cdc42 transfections did not, however, affect GEC morphology significantly (not shown).

Effects of other Rho-GTPase mutants were examined for comparison. CA-Rac1 appeared to localize both at the plasma membrane and cytoplasm, and transfected cells demonstrated elongation and protrusions that resembled process formation (Fig. 5, CA-Rac1). CA-Cdc42 localized mainly in the plasma membrane and enhanced cortical F-actin, similar to CA-RhoA; however, unlike CA-RhoA, cells had more "angular" contour and were not notably smaller than nontransfected cells (Fig. 5, CA-Cdc42). DN-RhoA did not affect GEC morphology significantly (not shown). These results indicate that each Rho family member has a distinct effect on the F-actin pattern and morphology in GEC. In particular, the results suggest that the activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement.

Impact of RhoA activity on complement-mediated injury in GEC. RhoA activity was increased in complement-stimulated GEC and in PHN. To determine whether activation of the RhoA pathway is functionally important in the development of GEC injury, we adopted an overexpression approach. Thus we established a subclone of GEC, which overexpress CA-RhoA in an inducible manner. When these cells were stimulated with ponasterone A for 7.5 h, a small amount of active RhoA was detected by pulldown assay, while stimulation for 18 h markedly increased the amount of active RhoA (Fig. 6A). Induction of CA-RhoA for 18 h induced morphological changes similar to Fig. 5, i.e., prominent cortical F-actin and round/small cell contour (Fig. 6B). ROCK is one of the major downstream effectors of RhoA and mediates a variety of Rho-induced changes in the actin cytoskeleton (4). The effects of CA-RhoA in GEC were not, however, attenuated by the ROCK inhibitor, Y27632, suggesting involvement of Rho effector(s) other than ROCK (Fig. 6B). Control cells stimulated with vehicle (ethanol) were not distinguishable from unstimulated GEC (Fig. 6B).

The role of RhoA in GEC injury was tested by monitoring complement-mediated cytotoxicity, using a lytic concentration of complement (NS 3%). This protocol allows for complement to initially induce biochemical/metabolic changes in GEC, but eventually a portion of the cells will undergo lysis, which can be quantified by release of LDH. After induction of CA-RhoA by ponasterone A (18 h), complement-mediated cytotoxicity was markedly attenuated (Fig. 6C). The cytoprotective effect of CA-RhoA was not reversed by the ROCK inhibitor, Y27632, indicating that Rho effector(s) other than ROCK most likely mediated the cytoprotection (Fig. 6C, right 2 columns). When CA-RhoA was induced for only 7.5 h, expression of CA-RhoA was small, and there was no cytoprotection against complement-mediated cytotoxicity (not shown), suggesting that strong/sustained activation of RhoA is required for cytoprotection.

To substantiate the above results, we established a subclone of GEC, which express DN-RhoA in an inducible manner. After blocking the entire RhoA pathway by inducing expression of DN-RhoA (Fig. 7A), complement-mediated cytotoxicity was augmented (Fig. 7B). Taken together, these results support the view that the predominant effect of RhoA activation in GEC is cytoprotection, i.e., restriction of complement-mediated injury, which is most likely mediated via effector(s) other than ROCK.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To date, there is limited information on the role of Rho-GTPases in GEC/podocytes. Rho GDI{alpha}–/– mice developed massive proteinuria (36). In this model, podocytes were severely injured and foot processes were disrupted, suggesting a critical role of Rho-GTPases in podocyte morphology and function. However, since Rho GDI{alpha} regulates various Rho-GTPases nonselectively, specific roles of individual Rho-GTPases could not be identified in this study. In immortalized mouse podocytes in culture, the ROCK inhibitor, Y27632, blocked actin cytoskeleton redistribution caused by mechanical stretch (13). In the same cells, Y27632 also inhibited redistribution of F-actin and upregulation of prepro-endothelin (ET)-1 induced by a protein load (23). Recently, Shibata et al. (32) reported that another ROCK inhibitor, fasudil, ameliorated proteinuria in rats with puromycin aminonucleoside nephrosis. These results support a pathological role for RhoA in certain proteinuric disorders involving podocytes. Alternatively, a recent report by Mundel and colleagues (3) presented the possibility that RhoA may be essential for podocyte migration. It is likely that the roles of Rho-GTPases are complex and vary depending on the developmental stage of the kidney/podocytes and pathophysiological conditions.

GEC and neurons may share some cell biological characteristics, including a highly arborized morphology. In both cells, actin filaments are thought to play a central role in maintaining structure and function (19). Activation of RhoA in neuronal cells induced neurite retraction and cell rounding (17), while activation of Rac1 promoted the formation of lamellipodia, along the extension of neurite-like structures (20). By analogy, Kobayashi and colleagues (15) recently reported that in immortalized mouse podocytes, Y27632 promoted elongation of microtubule-based thick processes and Y27632 in combination with forskolin further induced actin filament-based thin projections elongating from thick processes. These results suggest that Rac1 activation promotes process formation in neural cells and podocytes, while RhoA activation has an opposite effect.

In the present study, we showed that in complement-stimulated cultured GEC and in PHN, RhoA activity increased, while Rac1 activity decreased (Figs. 1–3). The complement-mediated increases in RhoA activity ranged from 1.2-fold (long-term incubation in culture) to 2.3-fold (in vivo) or 2.6-fold (short-term incubation in culture). These increases are relatively small, but they are in keeping with Swiss 3T3 cells adherent to fibronectin in serum-free conditions, where a comparable degree of RhoA activation (~1.5-fold) was biologically significant (28). Notably, lysophosphatidic acid, a well-known potent stimulator of RhoA, induced only a modest increase in RhoA activity in cultured GEC (~1.3-fold; not shown). To address the functional role of RhoA, GEC were transfected with CA-RhoA. These cells had a smaller and round contour with prominent cortical F-actin. Moreover, the RhoA-transfected GEC were markedly different from GEC that had been transfected with CA-Rac1, which demonstrated elongation and protrusions (Fig. 5). Based on these observations, as well as those of earlier studies, it is reasonable to speculate that increased RhoA activity with or without decreased Rac1 activity would disturb the intricate structure of podocyte foot processes, leading to proteinuria. It should be noted that none of the cultured GEC lines available to date display the precise morphological features of podocytes in vivo, including formation of foot processes and the slit diaphragm, in a reliable manner. Therefore, morphological studies using cultured GEC have certain limitations.

Exposure of GEC to complement induces injury, which is associated with derangements in multiple metabolic pathways in culture and in vivo, and is associated with proteinuria in vivo (8). Overexpression of CA-RhoA was cytoprotective against complement attack, although this effect was not blocked by the ROCK inhibitor, Y27632, suggesting that another RhoA effector was involved. The are numerous potential effectors of RhoA, including serine/threonine kinases (e.g., ROCK, Citron kinase), lipid kinases (e.g., phosphatidylinositol-4-phosphate 5-kinase), scaffold proteins (e.g., Rhotekin, Dia1 and 2), and others (4). It has been reported that some Rho effector proteins have opposing actions on cells. For example, the density and appearance of stress fibers could be experimentally varied by titrating different levels of active ROCK and mDia (39). Notably, when the entire RhoA pathway was blocked by the expression of DN-RhoA in GEC, complement-mediated cytolysis was augmented significantly (Fig. 7), indicating that the overall effect of RhoA activation in GEC is cytoprotection against complement attack. Given that the most likely target of RhoA is the actin cytoskeleton, this result is in keeping with an earlier study, which showed that disassembly of the actin cytoskeleton with drugs including latrunculin B or cytochalasin D augmented complement-mediated cytotoxicity (9). In an attempt to identify downstream effectors of RhoA in complement-mediated cell injury, we studied phosphorylation of myosin light chain; however, complement had no consistent impact on myosin light chain phosphorylation (not shown). Other potential downstream effectors of RhoA, which include cofilin/actin-dissociation factor or ezrin-radixin-moesin family proteins, will require further study (31).

In summary, the present study supports an important role for Rho-GTPases in the morphology and function of GEC. In particular, activation of RhoA appears to be protective against complement-mediated injury. Concurrently, activation of RhoA with or without inactivation of Rac1 may disfavor the maintenance of well-differentiated foot process structure, contributing to morphological changes of GEC and proteinuria in experimental membranous nephropathy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the Canadian Institute of Health Research (A. V. Cybulsky and T. Takano) and the Kidney Foundation of Canada (A. V. Cybulsky and T. Takano). A. V. Cybulsky and T. Takano hold scholarships from the Fonds de la Recherche en Santé du Québec. N. Lamarche-Vane is a Canadian Institute of Health Research New Investigator.


   ACKNOWLEDGMENTS
 
The authors thank W. Zhou for contributions to pilot experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Takano, Nephrology Division, McGill Univ. Health Centre, 3775 Univ. St., Rm. 229, Montreal, Quebec H3A 2B4 (e-mail: tomoko.takano{at}mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aoudjit L, Potapov A, Takano T. Prostaglandin E2 promotes cell survival of glomerular epithelial cells via the EP4 receptor. Am J Physiol Renal Physiol 290: F1534–F1542, 2006.[Abstract/Free Full Text]
  2. Aoudjit L, Stanciu M, Li H, Lemay S, Takano T. p38 Mitogen-activated protein kinase protects glomerular epithelial cells from complement-mediated cell injury. Am J Physiol Renal Physiol 285: F765–F774, 2003.[Abstract/Free Full Text]
  3. Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P. Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling. Nat Cell Biol 8: 485–491, 2006.[CrossRef][Web of Science][Medline]
  4. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241–255, 2000.[CrossRef][Web of Science][Medline]
  5. Coers W, Reivinen J, Miettinen A, Huitema S, Vos JT, Salant DJ, Weening JJ. Characterization of a rat glomerular visceral epithelial cell line. Exp Nephrol 4: 184–192, 1996.[Web of Science][Medline]
  6. Cybulsky AV, Foster MH, Quigg RJ, Salant DJ. Immunologic Mechanisms of Glomerular Disease. Philadelphia: Lippincott-Raven Publishers, 2000.
  7. Cybulsky AV, Monge JC, Papillon J, McTavish AJ. Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 269: F739–F749, 1995.[Abstract/Free Full Text]
  8. Cybulsky AV, Quigg RJ, Salant DJ. Experimental membranous nephropathy redux. Am J Physiol Renal Physiol 289: F660–F671, 2005.[Abstract/Free Full Text]
  9. Cybulsky AV, Takano T, Papillon J, Bijian K, Guillemette J. Activation of the extracellular signal-regulated kinase by complement C5b-9. Am J Physiol Renal Physiol 289: F593–F603, 2005.[Abstract/Free Full Text]
  10. Cybulsky AV, Takano T, Papillon J, Khadir A, Bijian K, Le Berre L. The actin cytoskeleton facilitates complement-mediated activation of cytosolic phospholipase A2. Am J Physiol Renal Physiol 286: F466–F476, 2004.[Abstract/Free Full Text]
  11. Cybulsky AV, Takano T, Papillon J, McTavish AJ. Complement-induced phospholipase A2 activation in experimental membranous nephropathy. Kidney Int 57: 1052–1062, 2000.[CrossRef][Web of Science][Medline]
  12. Dib K, Melander F, Andersson T. Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils. J Immunol 166: 6311–6322, 2001.[Abstract/Free Full Text]
  13. Endlich N, Kress KR, Reiser J, Uttenweiler D, Kriz W, Mundel P, Endlich K. Podocytes respond to mechanical stress in vitro. J Am Soc Nephrol 12: 413–422, 2001.[Abstract/Free Full Text]
  14. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature 420: 629–635, 2002.[CrossRef][Medline]
  15. Gao SY, Li CY, Chen J, Pan L, Saito S, Terashita T, Saito K, Miyawaki K, Shigemoto K, Mominoki K, Matsuda S, Kobayashi N. Rho-ROCK signal pathway regulates microtubule-based process formation of cultured podocytes–inhibition of ROCK promoted process elongation. Nephron Exp Nephrol 97: e49–e61, 2004.[CrossRef][Medline]
  16. Hall A. Rho GTPases and the actin cytoskeleton. Science 279: 509–514, 1998.[Abstract/Free Full Text]
  17. Jalink K, van Corven EJ, Hengeveld T, Morii N, Narumiya S, Moolenaar WH. Inhibition of lysophosphatidate- and thrombin- induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J Cell Biol 126: 801–810, 1994.[Abstract/Free Full Text]
  18. Jou TS, Nelson WJ. Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity. J Cell Biol 142: 85–100, 1998.[Abstract/Free Full Text]
  19. Kobayashi N, Gao SY, Chen J, Saito K, Miyawaki K, Li CY, Pan L, Saito S, Terashita T, Matsuda S. Process formation of the renal glomerular podocyte: is there common molecular machinery for processes of podocytes and neurons? Anat Sci Int 79: 1–10, 2004.[CrossRef][Medline]
  20. Leeuwen FN, Kain HE, Kammen RA, Michiels F, Kranenburg OW, Collard JG. The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J Cell Biol 139: 797–807, 1997.[Abstract/Free Full Text]
  21. Li X, Lim B. RhoGTPases and their role in cancer. Oncol Res 13: 323–331, 2003.[Web of Science][Medline]
  22. Moon SY, Zheng Y. Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13: 13–22, 2003.[CrossRef][Web of Science][Medline]
  23. Morigi M, Buelli S, Angioletti S, Zanchi C, Longaretti L, Zoja C, Galbusera M, Gastoldi S, Mundel P, Remuzzi G, Benigni A. In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies. Am J Pathol 166: 1309–1320, 2005.[Abstract/Free Full Text]
  24. Mundel P, Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 13: 3005–3015, 2002.[Free Full Text]
  25. Panesar M, Papillon J, McTavish AJ, Cybulsky AV. Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells. J Immunol 159: 3584–3594, 1997.[Abstract]
  26. Pelletier S, Julien C, Popoff MR, Lamarche-Vane N, Meloche S. Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway. J Cell Physiol 204: 412–422, 2005.[CrossRef][Web of Science][Medline]
  27. Quigg RJ, Cybulsky AV, Jacobs JB, Salant DJ. Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells. Kidney Int 34: 43–52, 1988.[Web of Science][Medline]
  28. Ren XD, Kiosses WB, Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578–585, 1999.[CrossRef][Web of Science][Medline]
  29. Royal I, Lamarche-Vane N, Lamorte L, Kaibuchi K, Park M. Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol Biol Cell 11: 1709–1725, 2000.[Abstract/Free Full Text]
  30. Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol 147: 1009–1022, 1999.[Abstract/Free Full Text]
  31. Schwartz M. Rho signalling at a glance. J Cell Sci 117: 5457–5458, 2004.[Free Full Text]
  32. Shibata S, Nagase M, Fujita T. Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling. J Am Soc Nephrol 17: 754–764, 2006.[Abstract/Free Full Text]
  33. Takano T, Cybulsky AV. Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells: role of cyclooxygenase-1 and -2. Am J Pathol 156: 2091–2101, 2000.[Abstract/Free Full Text]
  34. Takano T, Cybulsky AV, Cupples WA, Ajikobi DO, Papillon J, Aoudjit L. Inhibition of cyclooxygenases reduces complement-induced glomerular epithelial cell injury and proteinuria in passive Heymann nephritis. J Pharmacol Exp Ther 305: 240–249, 2003.[Abstract/Free Full Text]
  35. Tischer DG, Couser WG. Milestones in nephrology. J Am Soc Nephrol 11: 183–188, 2000.[Web of Science][Medline]
  36. Togawa A, Miyoshi J, Ishizaki H, Tanaka M, Takakura A, Nishioka H, Yoshida H, Doi T, Mizoguchi A, Matsuura N, Niho Y, Nishimune Y, Nishikawa S, Takai Y. Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIalpha. Oncogene 18: 5373–5380, 1999.[CrossRef][Web of Science][Medline]
  37. Topham PS, Haydar SA, Kuphal R, Lightfoot JD, Salant DJ. Complement-mediated injury reversibly disrupts glomerular epithelial cell actin microfilaments and focal adhesions. Kidney Int 55: 1763–1775, 1999.[CrossRef][Web of Science][Medline]
  38. Van Aelst L, D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 11: 2295–2322, 1997.[Free Full Text]
  39. Watanabe N, Kato T, Fujita A, Ishizaki T, Narumiya S. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat Cell Biol 1: 136–143, 1999.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
K. Koop, M. Eikmans, M. Wehland, H. Baelde, D. Ijpelaar, R. Kreutz, H. Kawachi, D. Kerjaschki, E. de Heer, and J. A. Bruijn
Selective Loss of Podoplanin Protein Expression Accompanies Proteinuria and Precedes Alterations in Podocyte Morphology in a Spontaneous Proteinuric Rat Model
Am. J. Pathol., August 1, 2008; 173(2): 315 - 326.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F148    most recent
00294.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, H.
Right arrow Articles by Takano, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, H.
Right arrow Articles by Takano, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.