Role of Rho GDP Dissociation Inhibitor α in Control of Epithelial Sodium Channel (ENaC)-mediated Sodium Reabsorption*

Background: RhoGDIα regulates activity of Rho family small GTPases, which are recognized as important modulators of ENaC. Results: RhoGDIα modulates Rac1 activity and partially participates in the effects of EGF on ENaC in the cortical collecting ducts. Conclusion: RhoGDIα controls ENaC expression and activity via Rac1. Significance: A novel mechanism for RhoGDIα-dependent regulation of ENaC is presented. The epithelial sodium channel (ENaC) is expressed in the aldosterone-sensitive distal nephron where it performs sodium reabsorption from the lumen. We have recently shown that ENaC activity contributes to the development of salt-induced hypertension as a result of deficiency of EGF level. Previous studies revealed that Rho GDP-dissociation inhibitor α (RhoGDIα) is involved in the control of salt-sensitive hypertension and renal injury via Rac1, which is one of the small GTPases activating ENaC. Here we investigated the intracellular mechanism mediating the involvement of the RhoGDIα/Rac1 axis in the control of ENaC and the effect of EGF on ENaC in this pathway. We demonstrated that RhoGDIα is highly expressed in the cortical collecting ducts of mice and rats, and its expression is down-regulated in Dahl salt-sensitive rats fed a high salt diet. Knockdown of RhoGDIα in cultured cortical collecting duct principal cells increased ENaC subunits expression and ENaC-mediated sodium reabsorption. Furthermore, RhoGDIα deficiency causes enhanced response to EGF treatment. Patch clamp analysis reveals that RhoGDIα significantly decreases ENaC current density and prevents its up-regulation by RhoA and Rac1. Inhibition of Rho kinase with Y27632 had no effects on ENaC response to EGF either in control or RhoGDIα knocked down cells. However, EGF treatment increased levels of active Rac1, which was further enhanced in RhoGDIα-deficient cells. We conclude that changes in the RhoGDIα-dependent pathway have a permissive role in the Rac1-mediated enhancement of ENaC activity observed in salt-induced hypertension.

Epithelial sodium channels (ENaC) 3 are responsible for the fine-tuning of Na ϩ reabsorption in the kidney and are regulated by various stimuli including hormones and growth factors. Among these, epidermal growth factor (EGF) and its family members are recognized as important mediators of ENaC activity involved in maintaining sodium homeostasis in the kidney and particularly in the collecting ducts (1). For instance it was shown that EGF reduces Na ϩ transport in the isolated and perfused rabbit cortical ducts (2)(3)(4)(5) and immortalized renal epithelial cells (6 -9).
Previously we demonstrated the biphasic effect of EGF, TGF-␣, heparin-binding EGF and amphiregulin on activity of ENaC in the cultured mouse cortical collecting duct (CCD) principal cell line, mpkCCD c14 . Basolateral application of these endocrine factors acutely produced a short term increase in ENaC-mediated transepithelial current and then caused long term current reduction (10). A similar observation was reported in A6 amphibian kidney cells; acute treatment with EGF and TGF-␣ enhanced ENaC activity by increasing channel open probability, whereas continuous treatment resulted in a decrease of the number of channels in the plasma membrane and, therefore, inhibited the current (11). Importantly, we recently demonstrated that the deficiency of renal cortical EGF promotes ENaC-mediated Na ϩ reabsorption in the CCD and contributes to the development of hypertension in Dahl saltsensitive (SS) rats (12). However, specific molecular mechanisms mediating effects of EGF on ENaC are not clear yet.
Small GTPases-molecular switches that control the activity of cellular and membrane proteins, including ion channels (13), are well known regulators of ENaC. It was previously reported that several small G proteins in Rho, Ras, and Rab families are able to regulate ENaC activity via different mechanisms (14 -21). Small GTPases cycle between inactive GDP-bound and active GTP-bound states; the exchange between G-protein-bound GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs), whereas termination of signaling is performed by GTPase-activating proteins which induce GTP hydrolysis (22). Another group of proteins regulating activity of small G proteins is guanine dissociation inhibitors (GDIs), which bind certain G proteins and keep them in an inactive form preventing dissociation of GDP (23). RhoGDI␣ is one of such factors that binds to the Rho family members RhoA, Rac1, and Cdc42 and keeps them in an inactive state, preventing their activation by GEFs (24,25). The importance of RhoGDI␣ for the kidney was demonstrated in mice lacking this protein (Arhgdia Ϫ/Ϫ mice), where renal abnormalities, including heavy albuminuria and podocyte damage, were associated with increased Rac1 activity and mineralocorticoid receptor signaling (26,27). Further studies demonstrated the key role of Rac1 in modulating salt susceptibility in mice lacking RhoGDI␣. Using SS rats the authors found that a high salt diet caused Rac1 up-regulation in the kidney, whereas Rac1 inhibition prevented hypertension and renal damage (28).
Our previous studies revealed that among Rho family members, Cdc42 has no effect on ENaC current density (29), whereas RhoA and Rac1 were able to increase the channel activity (14, 16, 29 -31). Furthermore, our recent findings indicate that EGF had no effect in Rac1-deficient principal cells (32). Thus, it appears that the small GTPase Rac1, which has been shown to be implicated in many renal and cardiovascular diseases (27,28,33), is a critical protein in transmission of the signal from EGF to ENaC. However, the mechanistic link between EGF, RhoGDI␣, small GTPases in Rho family, and ENaC was unclear.
In this study we investigated if RhoGD⌱␣ is involved in the control of ENaC activity and EGF-dependent regulation of this channel. This hypothesis might explain intracellular processes underlying abnormal ENaC regulation in some pathological conditions such as salt-sensitive hypertension.

EXPERIMENTAL PROCEDURES
Animals-Experiments were performed on C57BL/6J mice and Rapp Dahl salt-sensitive rats (SS/JrHsdMcwi). The mice were obtained from Harlan Laboratories and kept on a standard Harlan Teklad TD.96208 diet until the age of 8 weeks. SS rats have been inbred for Ͼ50 generations at the Medical College of Wisconsin. SS rats are a salt-sensitive strain that develops hypertension when fed a high salt diet. To investigate the effect of high salt consumption in these rats, chronic blood pressure monitoring in 8-week-old animals was performed using Data Sciences International (New Brighton, MN) telemetry as previously described (34). After transmitter implantation the rats were allowed to recover for at least 3 days. After an additional 3 days on a 0.4% NaCl diet the salt content of the chow was either maintained at 0.4% NaCl in one group or increased to 4.0% NaCl, and the rats were maintained on these diets for an additional 3 weeks. Both chows were obtained from Dyets Inc. (Bethlehem, PA). Animal use and welfare procedures adhered to the NIH Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.
Chinese hamster ovarian (CHO) cells (ATCC) were maintained under standard culture conditions (DMEM, 10% FBS, 1ϫ penicillin-streptomycin, 37°C, 5% CO 2 ) and transfected using the Polyfect reagent (Qiagen, Valencia, CA) as previously described (38,39). For expression of mouse ENaC in CHO cells, subunit cDNA transfection ratios of 1:1:1 were used with 0.3 g of each cDNA/35-mm culture dish. The plasmids encoding ␣-, ␤-, and mouse ␥-ENaC have been previously described (15). To define successfully transfected cells, 0.5 g of a cDNA encoding green fluorescent protein (GFP) was added to the cDNA mix. RhoA, Rac1, and Cdc42 plasmids were obtained from the University of Missouri-Rolla cDNA Resource Center. Myc-tagged RhoGDI␣ plasmid was kindly provided by Dr. Mark Shapiro (University of Texas Health Science Center, San Antonio, TX).
Generation of Stable RhoGDI␣-deficient Cell Lines-To generate a RhoGDI␣ knockdown in mCCD cl1 and M-1 cells, RhoGDI␣ short hairpin shRNA pLKO.1 plasmids (catalog no. TRCN0000106160 and TRCN0000106161, respectively) were purchased from OpenBiosystems, Thermo Scientific. After transduction with jetPRIME reagent (Polyplus-transfection SA, Illkirch, France), a stable cell line expressing the shRNA was isolated via puromycin selection. In short, cells were seeded in 6-well cluster plates (Corning) and grown to ϳ90% confluence. 3 g of plasmid DNA was mixed with 200 l of jetPRIME buffer and 4 l of jetPRIME reagent. Cells were overlaid with this DNA mix for another 48 h. Selection of successfully transfected cells was performed by applying 10 g/ml puromycin to the media.
Transepithelial Current Measurements-mCCD cl1 cells were seeded in the confluence to ϳ10 6 on permeable membranes (Transwell, Costar; 0.4-m pore, 24-mm diameter) and grown in the media described above. In 7-10 days the cells polarize and form a monolayer with high resistance and avid Na ϩ flux. Transepithelial Na ϩ current across the mCCD cl1 cell monolayer was measured by a Millicell Electrical Resistance System with dual Ag ϩ /AgCl pellet electrodes (Millipore Corp., Billerica, MA) and calculated using Ohm's law. 18 h before the experiments growth media was changed to FBS-and supplements-free media. To confirm the net Na ϩ transport through ENaC, 10 M amiloride was added to the apical surface of the monolayer at the end of each experiment. EGF (E4127) was obtained from Sigma, and Y27632 (#1254) was from Tocris Bioscience (Bristol, UK).
Patch Clamp Analysis-Whole cell current recordings of mouse ENaC expressed in CHO cells were made under voltage clamp conditions using standard protocols (15,29). Cells were clamped to a 40-mV holding potential with voltage ramps (500 ms) from 60 mV down to Ϫ100 mV used to elicit current. Pipette solution for whole-cell configuration was 120 mM CsCl, 5 mM NaCl, 2 mM MgCl 2 , 5 mM EGTA, 2 mM Mg-ATP, 0.1 mM GTP, and 10 mM HEPES (pH 7.4). Bath solution was 150 mM NaCl, 1 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES (pH 7.4). ENaC activity was assessed as the amiloride-sensitive current density at Ϫ80 mV. Whole-cell capacitance, on average 6 -10 picofarads, and series resistances, on average 2-5 megaohms, were compensated.
Cell-attached measurements of single channel ENaC activity were performed on wild type and RhoGDI␣-overexpressing mCCD cl1 cells. Cells were transfected with RhoGDI␣ and GFP encoding plasmids as described above for CHO cells. Individual GFP-positive cells were considered as successfully transfected and used for patch clamp analysis as described earlier (38). Briefly, single-channel signal was registered with an Axopatch amplifier (Molecular Devices, Sunnyvale, CA), filtered with an 8-pole Bessel filter LPF-8 (Warner Inst., Hamden, CT) at 0.2 kHz, and acquired with a Digidata 1440A to a PC running the pClamp 10.2 suite of software (Molecular Devices). Typical bath solution was 150 mM NaCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES (pH 7.4). Pipette solution for cell attached configuration was 140 mM LiCl, 2 mM MgCl 2 , and 10 mM HEPES (pH 7.4).
Western Blotting-mCCD cl1 or M-1 cells were washed twice in PBS and lysed in a buffer as described previously (29,32). Equal amounts (20 g) of proteins were separated by using 10 -20% SDS-PAGE and were then electrophoretically transferred onto nitrocellulose membrane (Millipore), immunoblotted with the appropriate antibody, and visualized by ECL (Amersham Biosciences). Antibodies for RhoGDI␣ were from Santa Cruz (sc-359), and antibodies for ␣-, ␤-, and ␥-ENaC (catalog nos. SPC-403D, SPC-404D, and SPC-405D, respectively) were from StressMarq Biosciences Inc. (Victoria, BC, Canada). Active Rac1 detection was performed using the kit purchased from Enzo Life Science (ADI-EKS-450) according to the manufacturer's protocol. The assay uses a GST fusion protein containing the p21 binding domain of human p21-activated kinase 1 (Pak1) to affinity-precipitate active Rac1 (GTP-Rac1) from cell lysates. The GST-Pak binding domain fusion protein (ϳ35 kDa) was incubated with cell lysate and an immobilized glutathione disc. The pulled down active or GTP-Rac1 was detected by Western blot analysis using a provided specific monoclonal Rac1 antibody.
For renal tissue analysis, rat kidneys were flushed and isolated under isoflurane anesthesia. The approximate apical kidney sections corresponding to cortex were carved (ϳ1 g) and then diced into small pieces with a razor blade. Samples were pulse-sonicated in gentle lysis buffer with protease inhibitor mixture (Roche Applied Science) for 10 s and spin-cleared at 10,000 ϫ g for 10 min.
Immunohistochemistry-The C57BL/6J mouse and SS rat kidneys were fixed in zinc formalin and processed for paraffin embedding. The kidney sections were cut at 4 m, dried, and deparaffinized for subsequent labeled streptavidin-biotin immunohistochemistry. After deparaffinization, the slides were treated with a citrate buffer (pH 6) for a total of 35 min. The slides were blocked with a peroxidase block (DAKO), avi-din block (Vector Labs), biotin block (Vector Labs), and serumfree protein block (DAKO). Tissue sections were incubated for 90 min in a 1:200 concentration of anti-RhoGDI␣ (sc-359, Santa Cruz). Mouse kidney sections were also stained with nonimmune IgG as a negative control and anti-aquaporin-2 antibodies to demonstrate RhoGDI␣ localization in the CCDs (sc-2027 and sc-9882, Santa Cruz, respectively). Secondary detection was performed with goat anti-rabbit biotinylated IgG (Biocare) followed by streptavidin horseradish peroxidase (Biocare) and visualized with diaminobenzidine (DAKO). All slides were counterstained with a Mayer hematoxylin (DAKO), dehydrated, and mounted with permanent mounting media (Sakura). RhoGDI␣ signal intensity was determined in CCDs using ImageJ 1.84D software package. Mean signal intensities in each CCD (background level was subtracted) were averaged for the experimental groups.
Statistics-All summarized data are reported as the mean Ϯ S.E.; statistical analyses were performed using the Mann-Whitney test with Bonferroni correction. Differences were considered statistically significant at p Ͻ 0.05 (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001).

RhoGDI␣ Abundance in Murine
CCDs-RhoGDI␣ is a ubiquitous protein expressed in many cell types (24). Initial experiments tested whether RhoGDI␣ is expressed in mouse and rat CCDs, a nephron segment with high activity of ENaC (40). To study RhoGDI␣ abundance we used kidneys isolated from 8-week-old C57BL/6J mice. Shown in Fig. 1A are images taken from two consecutively cut slices of a kidney immunohistochemically stained for RhoGDI␣ (shown in brown) at 20ϫ and 40ϫ magnifications. Negative control (control; stained with secondary antibodies in the absence of primary antibodies) is also shown. Additional negative control experiments (stained without primary or secondary antibodies) also did not reveal any staining (data not shown). As seen from Fig. 1A, RhoGDI␣ is highly expressed in the CCDs of C57BL/6J mice compared with other nephron segments. Fig. 1B represents additional negative control stained with non-immune IgG and secondary antibodies and shows the absence of any significant signal in the cortex. To ensure that the RhoGDI␣ signal observed in Fig. 1A was localized in CCD, we performed double staining of RhoGDI␣ and aquaporin-2, a marker for collecting duct principal cells. As shown in Fig. 2C both proteins are co-localized in CCDs.
Further experiments tested RhoGDI␣ expression in Dahl SS rats. SS rats develop severe hypertension and renal damage when fed a high salt diet (41)(42)(43), and Rac1 hyperactivation was found to be critical in these processes (28). Similar to previously published data (34,44,45), changing of the diet from 0.4% to 4% resulted in the development of hypertension. As assessed by telemetry, the mean arterial pressure after 3 weeks on diets was 148.8 Ϯ 5.3 and 117.9 Ϯ 6.7 mm Hg in rats fed high and low salt diets, respectively ( Fig. 2A). Immunohistochemistry analysis revealed that SS rats after 3 weeks on a high salt diet (4%) exhibit lower RhoGDI␣ abundance than on a normal salt diet (Figs. 2, B  and C). Fig. 2D represents a Western blot analysis of RhoGDI␣ signal in the cortical lysate collected from three SS rats fed a 0.4% diet. The image demonstrates specificity of the used antibodies, which detect only one significant band at ϳ25 kDa.
Effect of RhoGDI␣ Deficiency on ENaC-dependent Current Formation-To investigate the physiological significance of RhoGDI␣, we generated a stable knockdown of this protein in mCCD cl1 cells. These cells were derived from mouse CCD principal cells and retain its characteristics, including morphology and CCD antigens (35,36,46,47). We used short hairpin RNA (shRNA)-mediated silencing of RhoGDI␣ mRNA in the cells, and one successful clone was used for further experiments. Western blot analysis confirmed that utilization of control shRNA did not affect RhoGDI␣ level, whereas anti-RhoGDI␣ significantly reduced the abundance of the protein to ϳ13% compared with the control cells (Figs. 3, A and B).
Control cells, when seeded onto permeable membranes, form an epithelium-like monolayer and develop amiloride-sensitive sodium current of ϳ10 A/cm 2 within 12-14 days after seeding. These basal values were similar to those reported previously (10,48,49). RhoGDI␣-deficient cells generated a significantly higher current of ϳ20 A/cm 2 (Fig. 3C). These data demonstrate that RhoGDI␣ exhibits a suppressive role in formation of ENaC-mediated transepithelial flux.

Effect of RhoGDI␣ Deficiency on ENaC Subunits
Abundance in M-1 Cells-M-1 is a commercially available cell culture line retaining many characteristics of cortical collecting duct cells including morphology and CCD antigens (50). The line was also used to generate RhoGDI␣-knocked down immortalized cells for independent study on ENaC subunit abundance.    illustrates Western blot analysis of protein levels in wild type, RhoGDI␣-deficient, and control shRNA-expressing cells. As summarized in Fig. 4B, RhoGDI␣ knockdown causes significant increases in expression of ␣-, ␤-, and ␥-ENaC subunits. These data are in accord with the assessment of transepithelial currents shown in mCCDc l1 cells (Fig. 3).
Effect of EGF Application on ENaC Activity in the RhoGDI␣deficient Cells-Our previous studies show that basolateral application of EGF on polarized mouse-collecting duct cells up-regulates ENaC activity (10), and this effect was blunted in Rac1 knockdown cells (32). We tested the effect of EGF in the mCCD cl1 cells lacking RhoGDI␣ (cell line described above). Application of EGF (10 ng/ml) to the basolateral side increased amiloride-sensitive flux in the control cells. However, in the RhoGDI␣-deficient cells response to EGF application was significantly enhanced compared with control cells (Fig. 5A). These findings reveal that RhoGDI␣ participates in the regulation of ENaC activity by EGF. However, a 2-h treatment with EGF did not change the abundance of RhoGDI␣ proteins in control mCCD cl1 cells (Fig. 5B).
Effect of EGF Application on ENaC Activity in the RhoGDI␣overexpressing Cells-As the transepithelial current measurements indicate that RhoGDI␣ deficiency produces an enhanced response to EGF in mCCD cl1 cells, we tested the effect of EGF on RhoGDI␣-overexpressing cells. For this aim we transfected mCCD cl1 cells with RhoGDI␣-and GFP-encoding cDNA plasmids. Because the transfection was performed on the formed epithelial monolayer, its efficiency was low for assessment of transepithelial current but allowed the use of individual transfected cells for patch clamp analysis. Fig. 6A illustrates representative ENaC activity traces recorded in cell attached configuration from apical membranes of the cells transfected with GFP only or with RhoGDI␣ and treated with vehicle or EGF (50 ng/ml; 2 h). As summarized on Fig. 6B, cotransfection of mCCD cl1 cells with RhoGDI␣ significantly decreased ENaC activity, which represents direct evidence of the critical role of this protein in the control of ENaC activity. Treatment with EGF increased the activity of ENaC in both control and RhoGDI␣-overexpressing cells.
Effect of RhoGDI␣ on RhoA-and Rac1-dependent Up-regulation of ENaC Activity-RhoGDI␣ controls the Rho family proteins by keeping them in an inactive state. Our previous studies in the CHO expression system revealed that neither wild type nor constitutively active Cdc42 overexpression affects ENaC activity (29). Wild type and constitutively active forms of RhoA and Rac1 were demonstrated to increase ENaC current density (29,30).
Here we used CHO cells for transfection with RhoGDI␣ to investigate its effect on ENaC activity. Overexpression of RhoGDI␣ with all three mouse ␣-, ␤-, and ␥-ENaC subunits significantly decreased amiloride-sensitive ENaC current density (Fig. 7). Moreover, co-transfection of RhoGDI␣ with RhoA or Rac1 precluded ENaC up-regulation by these small G proteins. We assume that the high concentration of RhoGDI␣ in the cytosol keeps Rho proteins in inactive state and prevents augmentation of ENaC activity.
Rac1 Involvement in the Effects of EGF on ENaC Sodium Reabsorption in Native CCD Cell Monolayer-RhoGDI␣ has inhibitory effects on the small G proteins of the Rho family: RhoA, Rac1, and Cdc42. We have shown in the heterologous expression system that Cdc42 does not affect ENaC activity, whereas RhoA and Rac1 up-regulate it. We further tested the role of RhoA and Rac1 in the mCCD cl1 principal cell line. To determine if RhoA participated in the EGF-dependent regulation of ENaC activity, we used Rho kinase inhibitor Y27632 (51), which is a downstream target of RhoA (30,52). These experiments were designed similarly to data shown on Fig. 5, but control and RhoGDI␣-deficient cells were preincubated for 1 h with 2 M Y27632 (applied bilaterally). However, Y27632 had no effect on either EGF-treated control or RhoGDI␣-deficient cells (Fig. 8).
We further tested the levels of active Rac1 in mCCD cl1 cells before and after EGF treatment (10 ng/ml; 2 h). Detection of the GTP-bound form of Rac1 revealed a significantly increased  active Rac1/total Rac1 ratio after EGF application compared with vehicle-treated control cells. Moreover, cells lacking RhoGDI␣ demonstrate an increased active Rac1 level over control cells with similar levels in EGF-treated and -untreated cells (Fig. 9). Control experiments with the application of GDP and GTP␥s confirmed reduction and increase of Rac1 activity, respectively. Thus, these data demonstrate that in CCD cultured cells Rac1 activity is mediated by EGF, and RhoGDI␣ might be involved in this mechanism.

DISCUSSION
We demonstrate that RhoGDI␣ modulates Rho family protein Rac1 in the CCD. This interaction seems to play a permissive role in EGF-dependent regulation of Rac1 and ENaC. Fig.  10 provides schematic illustration of this regulation. It appears from our data that EGF affects Rac1 activity, whereas RhoGDI␣ modulates bioavailability of Rac1 for activation. Regulation of Rac1 seems to be critical for physiological regulation of ENaC. Rac1 might mediate its effects on ENaC either through the MAPK pathway (11,53) or WAVE (the WASP (Wiskott-Aldrich syndrome protein) family of verprolin homologous) proteins (14) and corresponding changes in the actin cytoskeleton (54 -58). In addition to its role in various intracellular pathways, Rac1 is one of the key subunits of the NADPH oxidase complex and is involved in the production of reactive oxygen species (32). At least the acute effects of EGF correlate with reactive oxygen species production as pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both generation of reactive oxygen species and an increase in ENaC-mediates current in response to EGF (32). An imbalance of NO, O 2 . , and H 2 O 2 within the kidney has important consequences on Na ϩ homeostasis and blood pressure (59 -61). Most likely, the increase in hydrogen peroxide levels mediates this effect as it was shown that H 2 O 2 is critical for ENaC activity (62)(63)(64)(65). The mechanism described in this manuscript is specific to Rac1. Our data show that two other members of Rho family  A, summary graph of amiloride-sensitive current density at Ϫ80 mV for CHO cells expressing mouse ENaC alone or co-expressed with RhoGDI␣ in the presence or absence of Rac1or RhoA. Whole-cell currents were evoked with a voltage ramp (60 to Ϫ100 mV from a holding potential of 40 mV). The number of observations for each group is shown. *, p Ͻ 0.05 versus the corresponding group without RhoGDI␣. pF, picofarads. B, Western blot demonstrates expression of myc-tagged RhoGDI␣ in CHO cells transiently expressed with corresponding plasmid. FIGURE 8. RhoA is not involved in EGF-dependent regulation of ENaC in mCCD cl1 cells. RhoA inhibition with Y27632 (1 h of pretreatment with 2 M Y27632) does not affect ENaC response to EGF application as measured in mCCD c11 monolayer. EGF (10 ng/ml) and vehicle (control) were added basolaterally at time 0, and current was normalized to the starting level (1 h before EGF). Amiloride (Amil; 10 M; arrow) was added to the apical membrane at the end of experiment. n ϭ 6 in each group.
identified in CCD, RhoA and Cdc42, are not involved in EGFmediated regulation of ENaC activity. We previously reported that Cdc42 does not alter ENaC activity when overexpressed in the CHO expression system (29). Interestingly, although RhoA was able to increase ENaC current density in CHO cells, the protein does not affect flow-mediated activation of ENaC (52). Similar results were observed in whole animals under pathological conditions (27). RhoGDI␣ deficiency in Arhgdia Ϫ/Ϫ mice led to renal abnormalities that were accompanied with increased levels of active Rac1 but not RhoA. To investigate the physiological role of Rac1 and Rho kinase in the development of abnormalities in these mice, small GTPase activity was inhibited with NSC23766 and fasudil, respectively. Administration of NSC23766 (but not fasudil) significantly reduced mean blood pressure, albuminuria, and kidney damage concomitantly with repression of renal Rac1 activity (27). Studies by Fujita and coworkers (28) showed that high salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via a mineralocorticoid receptor (MR)-dependent pathway. A high salt diet caused renal Rac1 up-regulation in SS rats and down-regulation in salt-insensitive Dahl rats. Despite a reduction of serum aldosterone levels, salt-loaded SS rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hyper-tension and renal damage with MR repression. Shibata et al. (28) further demonstrated in aldosterone-infused rats as well as adrenalectomized SS rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR over-activity and hypertension . Recent review articles by Nagase and Fujita (66,67), who pioneered this area of research, nicely summarized the role of RhoGDI␣ and Rac1-MR signaling in cardiorenal diseases and, specifically, mechanisms implicated in the pathogenesis of salt-sensitive hypertension and kidney injury.
Recent studies also identified that mutations in ARHGDIA cause nephrotic syndrome in humans and this effect is mediated via Rac1 and Cdc42 but not RhoA (68). Another study using whole exome sequencing revealed that RhoGDI␣ is implicated in congenital nephrotic syndrome (69).
In addition to identifying a critical role of RhoGDI␣ in control of ENaC activity, our data also demonstrate that EGF at least partially mediates these effects. As followed from our experiments, RhoGDI␣ does not directly mediate the effect of EGF on ENaC but has a permissive influence. In addition there is a possibility that other GDIs controlling Rac1 activity might be involved in effects of EGF. Besides, the critical role of GEFs in the control of kidney function and specifically ENaC activity was demonstrated. For instance we demonstrated that Rac1 GEF ␤Pix is involved in the control of ENaC (29,70). Thus, precise balance between small GTPases in the Rho family and GDIs and GEFs controlling their activity might also GEF critical for EGF-mediated effects on ENaC.
The decreased RhoGDI␣ abundance discovered on a high salt diet (Figs. 2) could play a permissive role for Rac1 activation described is SS rats (28). As high salt consumption in SS rats leads to cortical EGF deficiency (12), this impairs the chronic FIGURE 9. EGF up-regulates Rac1 activity in cortical collecting duct principal cells. Western blot analysis of active and total Rac1 abundance in mCCD cl1 cell lysate. Treatments with GDP and GTP␥S were used as negative and positive controls, respectively. Below is a summary graph of the active Rac1/total Rac1 ratio in wild type and RhoGDI␣ knockdown (KD) cells after a 2-h treatment with EGF (10 ng/ml). *, p Ͻ 0.05; **, p Ͻ 0.05 versus control cells without EGF treatment.  OCTOBER 10, 2014 • VOLUME 289 • NUMBER 41 suppression of ENaC-mediated reabsorption in CCD (10) and results in increased fluid retention in the body and blood pressure. In summary, these findings provide a mechanistic insight on intracellular machinery underlying the contribution of RhoGDI␣ to the regulation of ENaC and the development of salt-sensitive hypertension.