Introduction

Oxalate is an end product of glycolate metabolism that is primarily excreted by the kidney and is the most common constituent of kidney stones. Hyperoxaluria is one of the major risk factors for kidney stone formation and approximately 70–80% of kidney stones are composed of calcium oxalate crystals [1]. Since, cell injury is the predisposing factor for calcium oxalate crystal nucleation, aggregation, and stone formation, several studies have shown that oxalate-induced free radical generation leads to oxidative cell injury in renal epithelial cells in culture and in the kidney of hyperoxaluria-induced rats [2–4]. In addition, oxalate exposure alters DNA synthesis, changes cell morphology, induces immediate early gene, redistributes phosphatidylserine to the surface of the cell membrane, lowers viability, reduces antioxidative enzymes and induces apoptosis in renal cells [5–10].

Although a variety of cellular sources of reactive oxygen species (ROS) have been demonstrated, NADPH oxidase has been shown to modulate redox status of the kidney during renal diseases [11]. However, the potential role of NADPH oxidase in hyperoxaluria-induced kidney stone formation is not well known until recently. We were the first to demonstrate in 2004 that oxalate induces ROS generation through the activation of NADPH oxidase, which plays a major role in renal proximal tubular injury [12]. Following completion of our study, Umekawa et al [13] demonstrated in 2005 that involvement of NADPH oxidase in oxalate and calcium oxalate monohydrate crystal induced ROS generation in rat kidney epithelial cells. Since then, research has been focused on controlling the NADPH oxidase-mediated cell injury to prevent hyperoxaluria-induced kidney stone formation [14–18]. The NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which contains gp91phox and p22phox, the cytosolic regulatory subunits p47phox and p67phox, and the small guanosine triphosphate-binding protein Rac. On stimulation, the cytosolic subunits translocate to the membrane and associate with cytochrome b558, resulting in activation of the NADPH oxidase [19]. Formation and activation of NADPH oxidase allow electrons to be passed from the cofactor NADPH to molecular oxygen, producing superoxide radicals [20]. In view of the fact that, NADPH oxidase activity is noticeably increased in renal cells exposed to oxalate, focusing on mechanisms leading to NADPH oxidase activation could unveil further molecular details involved in oxalate-induced renal injury.

Rac1, a small G protein, is a signaling molecule that coordinates the intracellular transduction pathways which activate NADPH oxidase [21]. Once activated, Rac1 migrates from the cytosol to the plasma membrane where its attachment favors assembly of the various NADPH oxidase subunits [22, 23]. While many investigations, including recent animal models, have implicated Rac1 as a central mediator in cardiac and vascular hypertrophy and leukocyte migration [24–27], its role in oxalate-induced renal cell injury is not known. We previously showed that oxalate induces oxidative injury via PKC alpha and delta-mediated activation of NADPH oxidase in renal proximal tubular epithelial cells [15]. However, no direct evidence is available on how NADPH oxidase is activated by oxalate in renal tubular epithelial cells. To determine the signaling component downstream of PKC that regulate NADPH oxidase activation, we focused on Rac1. We determined the impact of Rac1 on oxalate-induced NADPH oxidase activation, ROS generation; and investigated the role of Rac1 in oxalate-induced cell injury in renal epithelial cells.

Materials and methods

Materials

DMEM was purchased from Invitrogen (Gaithersburg, MD) Lucigenin, NADPH, and the anti-Na/K-ATPase antibody was obtained from Sigma (St. Louis, MO). NSC23766 and rottlerin from EMD (Gibbstown, NJ). PKC α inhibitor peptide and anti-Rac1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture

Cultures of LLC-PK1 cells, an epithelial cell line from pig kidney with properties of proximal tubular cells (CRL 1392, ATCC, Rockville, MD) were maintained as sub confluent monolayers in 75-cm2 Falcon T-flasks in DMEM containing 10% fetal bovine serum, streptomycin (0.20 mg/ml) and penicillin (1.0 × 102 IU/ml), pH 7.4, at 37°C in a 5% CO2–95% air atmosphere. Experiments were carried out with serum- and pyruvate-free MEM. Oxalate was prepared as a stock solution of 10 mM sodium oxalate in normal sterile PBS and diluting it to 0.75 mM in the medium [15].

Inhibitor and oxalate treatments

Thirty minutes before the addition of 0.75 mM oxalate, confluent monolayers of LLC-PK1 cells were exposed to a PKCα-selective inhibitor (2.5 μg/ml inhibitor peptide), a PKCδ-selective inhibitor (7.5 μM rottlerin), a Rac1 inhibitor (50 μM NSC23766). Control cells were treated with vehicle (0.1% DMSO). The cells treated with or without oxalate along with inhibitors for various time periods were used for the assays as described below.

LDH assay

At the end of the experimental period, lactate dehydrogenase (LDH) was measured in the medium using a kit from Roche Diagnostics (Indianapolis, IN) [15]. All determinations were made against appropriate reagent blanks. The reaction product was read at 490 nm and expressed as percent release. The values of treated samples were normalized to the untreated controls.

H2O2 assay

Hydrogen peroxide in the medium was measured with a kit from Assay Designs (Ann Arbor, MI) [15]. This assay is based on the reaction of xylenol orange with sorbitol and ammonium iron sulfate in an acidic solution, producing a purple color proportional to the concentration of H2O2 in the medium. The reaction product was quantified at 550 nm and expressed as micromolar H2O2 released. The values of treated cells were normalized to control.

NADPH oxidase assay

NADPH oxidase activity was determined using an assay based on the chemiluminescence of lucigenin (bis-N-methylacridinium nitrate; CL) as described in our previous studies [15]. Briefly, control cultures or cultures exposed to oxalate with or without inhibitors were washed with 5 ml ice-cold PBS and scraped from the plate into 5 ml of the same solution. Samples were transferred to a 50-ml tube and centrifuged at 750g for 10 min at 4°C. The pellet was resuspended in lysis buffer containing protease inhibitors (20 mM monobasic potassium phosphate, pH 7.0, 1 mM EGTA, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride). The cell suspension was then disrupted using a dounce homogenizer on ice, and the homogenate was stored on ice until use. Protein content was measured in a homogenate aliquot by Lowry’s method [28], and NADPH oxidase activity was assessed by luminescence assay in 50 mM phosphate buffer (pH 7.0) containing 1 mM EGTA, 150 mM sucrose, 500 μM lucigenin as the electron acceptor, and 100 μM NADPH as the substrate. Enzyme activity was expressed as nanomoles superoxide produced per minute per milligram protein, and the data were normalized to control.

Sub cellular fractionation and Western blot

At the end of the experimental period, cells were resuspended in hypotonic lysis buffer with 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin, incubated for 30 min on ice, and cytosolic and membrane fractions isolated as we described previously [15]. Equal amounts of membrane protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk and incubated with an anti-Rac1 antibody followed by a horseradish peroxidase-conjugated secondary antibody at room temperature. The blots were washed with Tris-buffered saline and 0.1% Tween-20. Immunoreactive bands were visualized with an enhanced chemiluminescence Western blot kit (GE Healthcare Bio-Sciences, Piscataway, NJ) and analyzed with a densitometer using Kodak imaging software. Finally, the membranes were reprobed for Na+/K+-ATPase as a loading control for the membrane fraction.

Statistical analysis

Results are expressed as mean ± SE. Student’s t test was used to evaluate differences between treated and untreated cells using Sigma-Stat software, taking p < 0.05 as significant.

Results

Oxalate induces NADPH oxidase activity in renal epithelial cells

As NADPH oxidases are a major source of ROS in renal epithelial cells, we examined the effect of oxalate on NADPH oxidase activity in LLC-PK1, a renal epithelial cells. Oxalate (0.75 mM) significantly increased NADPH oxidase activity in renal epithelial cells in a time-dependent manner (15–180 min) (Fig. 1; n = 6; p < 0.05). A significant increase was observed as early as 15 min and sustained for 180 min compared with control cells.

Fig. 1
figure 1

Oxalate time dependently increases NADPH oxidase activity in LLC-PK1 cells. LLC-PK1 cells were treated with or without 0.75 mM oxalate for different time periods. NADPH oxidase activity was determined as described in “Materials and methods”. Data are normalized to control, and values are expressed as mean ± SE. Comparisons shown: a significant compared with control. *p < 0.05; n = 6

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Oxalate increases membrane-associated Rac1 protein expression

Since Rac1 regulates superoxide generation in many cell types, we first tested whether oxalate induces Rac1 activation in cultured renal proximal tubule cells. We have analyzed Rac1 protein expression levels in the membrane fraction, because studies have shown that Rac1 is normally found in the cytosolic compartment, and translocated to the plasma membrane upon activation [22, 23]. LLC-PK1 cells were exposed to 0.75 mM oxalate for 15 to 180 min, and a membrane-associated Rac1 was assessed by Western analysis. As shown in Fig. 2, oxalate increased membrane-associated Rac1 protein in a time-dependent manner (n = 3).

Fig. 2
figure 2

Oxalate time dependently activates Rac1 in LLC-PK1 cells. LLC-PK1 cells were treated with or without 0.75 mM oxalate for different time periods. Lysates of membrane factions were analyzed for Rac1 expression by Western blotting. A typical western blot from one of three experiments was shown. Na+/K+-ATPase was used as a membrane loading control

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Inhibition of Rac1 attenuates oxalate-induced ROS production and cell injury

To test whether Rac1 activation is required for oxalate-induced ROS generation, we measured hydrogen peroxide generation in the presence and absence of the Rac1 selective inhibitor, NSC23766 [29]. We found that, in the absence of a Rac1 inhibitor, oxalate exposure at a concentration of 0.75 mM for 3 h significantly increased hydrogen peroxide generation compared with vehicle-treated control cells. This oxalate induced increases in H2O2 generation was markedly attenuated in cells treated with the Rac1 specific inhibitor, NSC23766 (Fig. 3a; n = 6; p < 0.05).

Fig. 3
figure 3

Inhibition of Rac1 activation inhibits oxalate-induced reactive oxygen species production and cell injury in renal epithelial cells. LLC-PK1 cells were pretreated with a Rac1 inhibitor, NSC23766 (50 μM), for 30 min and then treated with or without 0.75 mM oxalate along with inhibitors for 3 h. Hydrogen peroxide production, and LDH release as a marker of cell injury were determined. DMSO was used as a vehicle control. Data are normalized to control, and values are expressed as mean ± SE. Comparisons shown: a significant compared with DMSO treated control, b significant compared with oxalate, c significant compared with inhibitor-treated control. *p < 0.05; n = 6

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To investigate the role of Rac1 in oxalate-induced cell injury in cultured proximal tubular epithelial cells, we assessed LDH release in the media. As shown in Fig. 3b oxalate exposure at a concentration of 0.75 mM for 3 h, significantly increased LDH release in LLC-PK1 cells compared with vehicle-treated control cells. In contrast, oxalate-induced increase in LDH release was significantly decreased in cells treated with the Rac1 inhibitor (n = 6; p < 0.05). The data indicate that activation of Rac1 is required for oxalate-induced cell injury via NADPH oxidase-mediated ROS generation in LLC-PK1 cells.

Effect of NSC23766 on Rac1 translocation

As Rac1 protein expression level was increased in the membrane fraction of oxalate exposed cells, we next demonstrated the specificity of NSC23766 on the inhibition of Rac1 translocation from cytosol to membrane in cells treated with oxalate. In the absence of Rac1 inhibitor, oxalate exposure at a concentration of 0.75 mM for 3 h, increased Rac1 protein expression levels in the membrane fraction. In contrast, in the presence of Rac1 inhibitor, oxalate exposure at a concentration of 0.75 mM for 3 h did not increase Rac1 protein expression levels in the membrane fraction. The data suggest that the membrane translocation of Rac1 was effectively blocked by the Rac1 inhibitor, NSC23766 (Fig. 4; n = 3).

Fig. 4
figure 4

NSC23766 inhibits oxalate-induced Rac1 translocation in renal epithelial cells. LLC-PK1 cells were pretreated with a Rac1 inhibitor, NSC23766 (50 μM) for 30 min and then treated with 0.75 mM oxalate along with inhibitor for 3 h. Lysates of membrane factions were analyzed for Rac1 expression by Western blotting. DMSO was used as a vehicle. A typical western blot from one of three experiments was shown. Na+/K+-ATPase was used as a membrane loading control

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Role of Rac1 in oxalate-induced NADPH oxidase activation

The activity of NADPH oxidase was determined in LLC-PK1 cells treated with oxalate in the absence or presence of Rac inhibitor, NSC23766. As shown in Fig. 5, oxalate exposure at a concentration of 0.75 mM for 3 h significantly increased the NADPH oxidase specific activity in the homogenates of renal epithelial cells. In the presence of NSC23766, oxalate-induced increase in NADPH oxidase activity was completely blocked. These results support the hypothesis that oxalate-stimulated ROS production in renal tubular cells involves a Rac1-dependent NADPH oxidase activation (n = 6; p < 0.05).

Fig. 5
figure 5

Inhibition of Rac1 activation inhibits oxalate-induced NADPH activity in renal epithelial cells. LLC-PK1 cells were pretreated with a Rac1 inhibitor, NSC23766 (50 μM) for 30 min and then treated with or without 0.75 mM oxalate for 3 h. NADPH oxidase activity was determined as described in “Materials and methods”. DMSO was used as a vehicle. Data are normalized to control, and values are expressed as mean ± SE. Comparisons shown: a significant compared with DMSO treated control, b significant compared with oxalate (*p < 0.05; n = 6)

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Oxalate induces NADPH oxidase activation via PKC-dependent Rac1 signaling

Given that the inhibition of PKC α and δ attenuated oxalate-induced NADPH oxidase-mediated ROS and cell injury was shown in our previous studies [15], we questioned whether this effect was due to the inhibition of small GTP-binding protein Rac1. Therefore, we examined the effect of PKC α and δ inhibitors on Rac1 protein expression in the membrane fraction of LLC-PK1 cells. As shown in Fig. 6a and b, in the absence of PKC α and δ inhibitors, oxalate exposure at a concentration of 0.75 mM for 3 h significantly increased the Rac1 protein expression levels in the membrane fraction of LLC-PK1 cells. In contrast, in the presence of PKC α and δ inhibitors, oxalate exposure did not increase Rac1 protein expression levels. These results demonstrate that oxalate induces ROS production in renal epithelial cells via a PKC-dependent activation of Rac1 and Rac1-mediated activation of NADPH oxidase.

Fig. 6
figure 6

Inhibition of PKC α and δ blocks oxalate-induced Rac1 activation in renal epithelial cells. LLC-PK1 cells were pretreated with a PKC α inhibitor, inhibitor peptide (2.5 μg/ml) or PKC delta inhibitor, rottlerin (7.5 μM) for 30 min and then treated with 0.75 mM oxalate for 3 h. Lysates of membrane factions were analyzed for Rac1 expression by Western blotting. A typical western blot from one of three experiments was shown. Na+/K+-ATPase was used as a membrane loading control

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Discussion

Our previous studies indicated that oxalate significantly increases NADPH oxidase activity via PKC signaling pathway, which stimulates superoxide production and resultant injury in renal epithelial cells [15]. In the present study, we performed a series of experiments to explore the mechanism by which oxalate enhances NADPH oxidase activity in these cells. The major findings of this study was that (1) oxalate stimulates NADPH oxidase activity in renal epithelial cells; (2) exposing renal epithelial cells to oxalate increases membrane translocation of Rac1; (3) inhibition of Rac1 signaling attenuates oxalate-induced Rac1 translocation, NADPH oxidase activity, ROS production and cell injury; and (4) in particular, blockade of PKC signaling by PKC α and δ inhibitors attenuates Rac1 activation in oxalate-treated cells suggesting that PKC resides upstream of Rac1 in the pathway that regulates NADPH oxidase. We believe this is the first demonstration that Rac1 signaling plays a crucial role in oxalate-induced NADPH oxidase-mediated renal tubular cell injury.

The role of oxidative stress in kidney stone formation has received increasing attention in recent years [30, 31]. We have already shown that oxalate-induced oxidative injury is a major promoter of calcium oxalate crystal attachment to renal tubules [4, 15, 32]. Integrity of the renal epithelium is necessary to maintain normal kidney function, which includes secretion and reabsorption of various solutes. However, when oxidation products greatly overwhelm the capacity of endogenous cellular antioxidants, ionic homeostasis across the renal cell membrane is disrupted, increasing membrane permeability to ions including oxalate and calcium ions, and crystals formed which can bind to the damaged renal tubular membrane [32]. Several studies have shown that oxalate and calcium oxalate crystals acting independently increase free radical injury in a concentration (0.5–2 mM) and time-dependent manner [15, 33–35]. However, the pathophysiologically relevant source of increased ROS production in hyperoxaluria remains to be further characterized.

The NADPH oxidase is a multicomponent enzyme complex, and has been proved important in the pathogenesis of renal damage. Moreover, activation of NADPH oxidase contributes to increased ROS generation in the kidney of diabetic, hypertensive and Dahl salt-sensitive rats [11, 36]. Studies also have shown that the component proteins for a NADPH oxidase system, including p47phox, p67phox and Rac1, were found in the kidney [37, 38]. An important step for the assembly and function of this multicomponent NADPH oxidase complex is the heterodimerization of gp91phox with p67phox, which is mediated by Rac [39].

The small G protein Rac is the one of the major adapter protein [40, 41] which regulate oxidase activity. Three isoforms of Rac have been identified to date. Rac1 is ubiquitously expressed, while Rac2 is primarily expressed by hematopoietic cells [42, 43] and Rac3 by the brain, nervous system and mammary glands [44, 45]. Rac1 regulates gene expression, cell cycle progression, cell spreading, rearrangement of the actin cytoskeleton, and activation of nonphagocytic NADPH oxidase has been implicated in ROS generation [46–48]. Studies have reported that Rac1 is required to anchor cytosolic p67phox to the membrane for the assembly of active NADPH oxidase protein complex, leading to superoxide generation [49]. Consistent with our findings, other studies have shown increased ROS production mediated by Rac1-regulated NADPH oxidase in a variety of disease conditions including hypoxia/reoxygenation [50], elevated shear stress [51] and cyclosporine-A treatment [52].

The nature and mechanism of action of the active NADPH oxidase complex formed have not yet been defined in stone forming condition. Rac must bind GTP to promote superoxide formation and undergo regulated cycles of GTP binding, which is mediated by a guanine nucleotide exchange factor [21, 53–56]. Rac must be in a GTP-bound form for oxidase activation to occur and GTP binding to Rac may precede the translocation event [53, 57, 58]. Rac dissociates from guanosine diphosphate dissociation inhibitor (GDI), allowing GTP-bound Rac to translocate to the plasma membrane [59, 60]. At the plasma membrane, Rac in the GTP-bound state directly interacts with p67phox via binding to the N-terminal domain that harbors tetratricopeptide repeat (TPR) motifs [61, 62], and thus the Rac-p67phox complex supports NADPH oxidase activity, leading to superoxide production [63, 64].

Since activated Rac1 migrates from the cytosol to the plasma membrane [59], we tested whether oxalate increases NADPH oxidase activity by stimulating this process. The increased membrane translocation of Rac1 accompanies enhanced NADPH oxidase activity following oxalate exposure suggesting that the sequence of molecular events leading to ROS generation in oxalate toxicity involves Rac1 translocation. Consistent with our findings, membrane translocation of Rac1, an important event of NADPH oxidase activation, has been reported in a variety of cells, including macrophages, kidney, vascular smooth muscle, and endothelium [65–68].

The selective Rac1 blockade we achieved with NSC23766 showed that the treated cells were protected against oxidative stress and cell injury, leading us to conclude that Rac1 signaling plays a crucial role in oxalate-induced ROS-mediated renal cell injury. Moreover, Rac1 inhibitor fails to activate NADPH oxidase by inhibiting the Rac1 translocation from cytosol to membrane thereby prevented oxalate-induced ROS production in renal epithelial cells. It was reported that a higher NADPH-oxidase-dependent superoxide generation was present in renal cortex and outer medulla than in the papilla [69]. We have shown oxalate exposure significantly increased NADPH oxidase activity in the renal proximal tubular cells. Consistent with our findings others have shown that NADPH oxidase is abundantly localized in proximal convoluted tubule cells in kidney [38]. Therefore, our results indicate that Rac1-dependent NADPH oxidase is a major source of oxalate-stimulated ROS production in renal proximal tubular cells, and deregulation of NADPH oxidase in the proximal tubule may be involved in the pathogenesis of hyperoxaluria-induced kidney stone formation.

In our previous studies, when NADPH oxidase was inhibited by DPI or apocynin, oxalate-induced superoxide and H2O2 production was eliminated [15]. However, the present study establishes a sequential link between oxalate-induced Rac1 activation and NADPH oxidase-mediated increase in intracellular ROS production. PKC α and δ isoforms have been linked to Rac1, because inhibiting them suppressed oxalate-mediated Rac1 signaling. Our observation that the inhibitors prevented PKC from inducing translocation of Rac1 and interaction between Rac1 and NADPH oxidase in renal cells exposed to oxalate supports the view that PKC acts upstream of Rac1 in this system and together with our previous evidence [15] suggests that oxalate could induce renal oxidative stress via a PKC/Rac1 signaling pathway.

In conclusion, the present study demonstrates that oxalate increases ROS production in renal epithelial cells via Rac1-regulated NADPH oxidase activation and suggesting that Rac1 plays a key role in oxalate-mediated oxidative renal cell injury. The injury as a result of oxalate exposure plays a significant role in calcium oxalate adhesion, aggregation, and growth of kidney stones. Inhibition of Rac1 results in decreased ROS production and a reduction in cell injury. In light of our results, we suggest that Rac1-mediated oxidative stress could be a potential therapeutic target to prevent renal injury in calcium oxalate kidney stone formers and reduce recurrence rates. However, further studies will be needed in animal models to better define this possibility.