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Rosiglitazone improves aortic arginine transport, through inhibition of PKC, in uremic rats

Departments of ¹Nephrology, ²Medicine "T," and ³Pathology, Tel Aviv Sourasky Medical Center, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Submitted 29 December 2007 ; accepted in final form 2 June 2008

	ABSTRACT

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Peroxisome proliferator-activated receptor (PPAR) agonists were shown to inhibit atherosclerosis through augmentation of endothelial nitric oxide synthase (eNOS) activity. In addition, rosiglitazone exerts a beneficial effect in chronic renal failure (CRF). Since L-arginine transport by CAT-1 (the specific arginine transporter for eNOS) is inhibited in uremia, we aimed to explore the effect of rosiglitazone on arginine transport in CRF. Arginine uptake by aortic rings was studied in control animals, rats, 6 wk following 5/6 nephrectomy (CRF) and rats with CRF treated with rosiglitazone. The decrease of arginine transport in CRF was prevented by rosiglitazone. Immunobloting revealed that CAT-1 protein was decreased in CRF but remained unchanged following rosiglitazone administration. Protein content of the membrane fraction of PKC and phosphorylated CAT-1 increased significantly in CRF, effects that were prevented by rosiglitazone. PKC phosphorylation was unchanged but significantly attenuated by rosiglitazone in CRF. Ex vivo administration of phorbol-12-myristate-13-acetate to rosiglitazone-treated CRF rats significantly attenuated the effect of rosiglitazone on arginine uptake. The decrease in cGMP response to carbamyl-choline (eNOS agonist) was significantly attenuated by rosiglitazone in CRF. Western blotting and immunohistochemistry analysis revealed that protein nitration was intensified in the endothelium of CRF rats and this was attenuated by rosiglitazone. In conclusion, rosiglitazone prevents the decrease in arginine uptake in CRF through both depletion and inactivation of PKC. These findings are associated with restoration of eNO generation and attenuation of protein nitration and therefore may serve as a novel mechanism to explain the beneficial effects of rosiglitazone on endothelial function in uremia.

uremia; protein nitration; endothelial function

Previous studies suggest that L-arginine, as the sole precursor for NO generation, governs NOS activity (11, 32). Among several transporters which mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) is considered as the predominant arginine supplier for eNOS (14, 20). Endothelial dysfunction (ECD) is a common denominator of chronic renal failure (CRF). Accumulated evidence indicates that ECD is linked to impaired capacity of the constitutive, Ca²⁺/calmodulin-sensitive NOS (eNOS) to generate adequate quantities of NO (1, 6, 7, 21, 22).

Several studies showed that arginine transport is markedly inhibited in uremia and this may play a role in inducing renal injury (24, 31, 34).

Since thiazolidinediones have been shown to exert a beneficial effect on endothelial function in both animal models of chronic nephropathies and in patients with chronic kidney disease (3, 19), as well as on eNOS activity, we were intrigued to explore a possible effect of rosiglitazone on arginine transport in rats with CRF.

	METHODS

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Materials. All standard reagents were obtained from Sigma Chemical, unless indicated otherwise. [H³]L-arginine was supplied by Perkin Elmar Life and Analytical Sciences (Boston, MA).

Animals and surgical preparation. All animal experiments described in this study were conducted according to the guide of care and use of animals protocol approved by the Institutional Committee on Ethics in animal experiments. Studies were performed using male Wistar rats weighing 200–250 g. Subsequently, rats were segregated into three groups, respectively: group 1: control, sham-operated rats; group 2: CRF: rats underwent a two-stage 5/6 nephrectomy (interval of 1 wk). Following the operation, animals were allowed to recover and have free access to a standard rat chow and tap water. All experiments were performed 6 wk following the second operation. Group 3: CRF + rosiglitazone: CRF rats were given rosiglitazone by gavage (4 mg·kg body wt^–1·day^–1) starting from day 1 after the second surgury. A subgroup of animals was treated with GW9662 (1 mg·kg^–1·day^–1 ip) or vehicle (10% dimethyl sulfoxide) for 7 days before death.

Creatinine clearance and 24-h protein excretion were measured before death, in all experimental groups as previously described (8). Rats were euthanized using CO₂.

Assessment of cGMP generation. Aortic tissue cGMP generation was determined by ELISA. Isolated aortic rings were suspended in HEPES buffer to which a phosphodiesterase inhibitor (1 mM 3-isobutyl-1-methyl-xantine) was included to inhibit cGMP degradation. Aliquots were incubated and shaken at 37°C for 10 min after which they were subjected to carbamyl choline (CCh; 100 µM), a selective eNOS agonist for additional 5 min. Following incubation, the samples were rapidly frozen and then homogenized in 5% TCA at 4°C. The precipitate was removed by centrifugation (3,000 rpm, 10 min) and TCA was ether extracted. Residual ether was removed by heating the samples for 5 min at 70°C. The samples were then processed for measurement of cGMP by ELISA kit (R&D Systems). Each experiment was repeated four times.

L-arginine uptake by aortic rings. Uptake of radiolabeled L-arginine in the rat aorta was measured according to previously described methods with modification (23). Immediately after death, the aorta was carefully excised from the left renal artery to the aortic valve ring and placed in ice-cold HEPES. The vessels were dissected free from adherent connective tissue and cut into rings (length 3 to 4 mm). Each segment was cut longitudinally in half. To determine arginine transport, aortic segments from each experimental group were incubated and shaken for 10 min in HEPES buffer at pH 7.4, 37°C. L-[H³]arginine and L-arginine, in a final concentration of 1 mM, were added to a total volume of 2 ml for additional 1 min. The duration of 1 min was chosen since it was within the linear portion of uptake curves (data not shown). Transport was terminated by rapidly washing the aortic rings with ice-cold PBS buffer (4 times, 3 ml/tube). The rings were then dried and solubilized in 1 ml of 0.5% SDS in 0.5 N NaOH. Seven-hundred microliters of the lysate were used to monitor radioactivity, by liquid scintillation spectrometry (Betamatic, Kontron). The remaining 300 µl were used for protein content determination by the Lowry method (Lowry assay kit, Sigma). To correct for nonspecific uptake or cell membrane binding, additional studies were performed in which aortic segments were incubated with 10 mM unlabeled arginine in HEPES buffer, and the associated radioactivity was subtracted from each data point. Results are expressed as means ± SE of at least five different rats. To explore a possible association between the effect of rosiglitazone on arginine transport and PKC, arginine transport was determined in freshly harvested aortic rings from rosiglitazone-treated uremic rats (group 3) following incubation with 50 nM phorbol-12-myristate-13-acetate ester (PMA; Sigma), a PKC activator, for 15 and 45 min (n = 5).

Protein quantification by Western blotting. Aortic CAT-1 and PKC were determined by immunoblotting. Briefly, excised aortas were separately placed in ice-cold PBS lysis buffer (pH 7.4), containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 4.5 µM leupeptin, and 5 µM aprotinin; ICN Biomedicals), 0.01% Triton X-100, and 0.1% SDS, and then mechanically homogenized and left on ice for 45 min. Homogenates were subsequently centrifuged (13,000 rpm for 10 min, at 4°C). Cell lysates were stored in aliquots in –70°C. A membrane fraction was obtained by adding to the pellet an equal volume of lysis buffer supplemented by Tween 20 (0.25%) to solubilize. The protein content of each sample was determined by the method of Lowry. Equal amounts of protein (30 µg) were prepared in sample buffer (2% SDS, 0.01% bromophenol blue, 25% glycerol, 0.0625 M Tris·HCl, pH 6.8, 5% mercaptoethanol) and analyzed on a 7.5% SDS-PAGE gel. The gel was transferred onto Hybond ECL nitrocellulose membranes (Amersham) and blocked in PBS-T containing 5% nonfat dried milk at room temperature. Membranes were then incubated with polyclonal rabbit anti-rat CAT-1 antibodies 1:500 (synthesized by Dr. O. Leitner, Weizmann Institute, Rehovot, Israel), mouse anti-rat PKC, anti-phospho-PKC, or monoclonal mouse anti-rat nitrotyrosine antibodies (from Santa Cruz Biothechnology) for 1 h at room temperature, washed, and incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:10,000) in PBS-T for 1 h. Membranes were subsequently washed three times, for 5 min each, in PBS-T. Membranes were then stripped and reprobed with monoclonal anti-β-actin antibodies as an internal control. The reactive bands corresponding to CAT-1 and PKC were detected by enhanced chemiluminescence (Kodak X-OMAT AR film) and quantified by densitometry. Three animals were utilized for each experimental group.

Immunoprecipitation studies. Aliquots of aortic tissue cell lysate (1 ml) from the different experimental groups were used for immunoprecipitation. Each tissue lysate was incubated with 20 µl of anti-CAT-1 antibodies for 2 h, at 4°C. Optimal antibody concentration was determined by titration. This was followed by addition of 20 µl of protein A agarose (Santa Cruz Biotechnology) and incubation overnight at 4°C on a rotating device. Pellets were collected by centrifugation at 3,000 rpm for 30 s, 4°C. The supernatants were discarded and each pellet was subsequently washed three times with PBS. After final wash, the pellets were resuspended in 40 µl of 2x electrophoresis sample buffer, boiled for 3 min, and subjected to immunoblotting with antibodies against CAT-1 or the phosphorylated tyrosine residue of CAT-1 (Santa Cruz Biotechnology). Representative results of three separate experiments are shown. To estimate phosphorylation of CAT-1 in the different groups, the density of bands for CAT-1 and its phosphorylated form on a film was analyzed. Results are adjusted for CAT-1 levels and expressed in arbitrary units (mean SE, n = 3).

Immunohistochemical analysis. Nitrotyrosine formation, a marker of peroxynitrite generation, was determined using immunohistochemical staining. Immunohistochemistry was performed by employing a mouse monoclonal antibody for the detection of nitrotyrosine (Clone 39B6, Santa Cruz Biotechnology). Paraffin-embedded tissue sections (4 µm) were mounted on SuperFrost Plus glass (Menzel-Glazer, Braunschweig, Germany) and processed by an automated immunostainer (VENTANA ES, Ventana Medical System, Tucson, AZ).

After deparaffinization, heat-induced antigen retrieval was performed at a controlled temperature in a microwave processor (H2800 model, Energy Beam Sciences) in 10 mM citrate buffer, pH 6.0, for 10 min at 97°C. Immunohistochemistry was performed using a three-step indirect process based on the labeled-(strept) avidin-biotin (LAB-SA) peroxidase complex method. Sections were incubated at a controlled temperature of 37°C for 32 min with 1:100 dilution of anti-nitrotyrosine antibodies.

Automated immunostaining was performed using the I-View DAB detection kit (Ventana Medical System) according to a standard Ventana program. The I-View DAB detection kit utilizes biotinylated secondary antibodies to locate the bound primary antibody, followed by the binding of streptavidin-HRP conjugate. The complex is then visualized with hydrogen peroxidase substrate and 3,3'-diaminobenzidine (DAB) tetrahydrochloride chromogen, which produces a dark brown precipitate which is readily detected by light microscopy.

The sections were then counterstained with hematoxylin, dehydrated, and mounted for microscopic examination. Tissues from three rats of each experimental group were processed.

Statistical analysis. Data are presented as means ± SE. One-way ANOVA was conducted for comparison between groups. Post hoc analysis using LSD algorithm was performed to allocate the source of significance.

	RESULTS

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To consolidate previous observations and validate the therapeutic efficacy of rosiglitazone in our uremic model, creatinine clearance (CCr), renal histology, urinary protein excretion, and aortic eNOS activity were measured in all experimental groups. CRF resulted in a significant decrease in CCr (0.35 ± 0.07 vs. 0.66 ± 0.06 ml·min^–1·100 g^–1 body wt, P < 0.01). Administration of rosiglitazone had no effect on CCr in CRF animals (0.32 ± 0.08 ml·min^–1·100 g^–1 body wt). In addition, no improvement in renal histology was noted (data not shown). In contrast, protein excretion was significantly decreased in CRF rats by the administration of rosiglitazone (Fig. 1). We measured cGMP generation following stimulation with CCh, a selective eNOS agonist, to examine aortic eNOS activity. CCh-stimulated cGMP levels were significantly decreased in uremic rats compared with control animals and administration of rosiglitazone significantly attenuated this phenomenon (Fig. 2).

View larger version (7K): [in this window] [in a new window]   Fig. 1. Effect of rosiglitazone (ROSI) on urinary protein excretion indexed in chronic renal failure (CRF). Results shown are means ± SE. *P < 0.05 vs. controls. #P < 0.05 vs. CRF (n = 5). CTL, controls.

View larger version (7K): [in this window] [in a new window]   Fig. 2. Aortic cGMP generation following stimulation with cabamyl choline, an eNOS agonist. Results shown are means ± SE of duplicate determinations from 4 separate experiments. *P < 0.05 vs. control. #P < 0.05 vs. CRF.

View larger version (10K): [in this window] [in a new window]   Fig. 3. A: uptake of radiolabeled arginine ([3H]L-arginine) by freshly harvested aortic rings from the various experimental groups. Data are presented as means ± SE of at least 5 different experiments. *P < 0.05 vs. control. #P < 0.05 vs. CRF. B: effects of in vivo administration of GW9662, a PPAR antagonist, on aortic arginine transport in CRF rats treated with rosiglitazone (group 3). Data are presented as means ± SE of 5 different experiments. *P < 0.05 vs. control.

View larger version (24K): [in this window] [in a new window]   Fig. 4. A: Western blot analysis of nitrotyrosine-modified protein expression in aortic rings harvested from the various experimental groups. These blots are representative of 3 different experiments. B: densitometric analysis of nitrotyrosine-modified protein contents of the experiments shown in A. Each bar represents the mean of the relative density units ± SE from 3 different experiments. *P < 0.05 vs. the corresponding control. #P < 0.05 vs. CRF.

View larger version (61K): [in this window] [in a new window]   Fig. 5. A–C: immunohistochemistry of endothelium harvested from the various experimental groups stained for nitrotyrosine. A: representative slide of sham-operated rats. B: representative slide of rats subjected to 5/6 nephrectomy. C: representative slide of uremic rats treated with rosiglitazone throughout the study period.

View larger version (14K): [in this window] [in a new window]   Fig. 6. A: representative Western blot analysis showing regulation of cationic amino acid transporter-1 (CAT-1) protein level in freshly harvested aortic rings from the same experimental groups. B: densitometric analysis of aortic CAT-1 contents from the various experimental groups. Each bar represents the mean of the relative density units ± SE from 3 different experiments. *P < 0.05 vs. control.

View larger version (18K): [in this window] [in a new window]   Fig. 7. A: representative Western blot analysis showing regulation of PKC protein level in freshly harvested aortic rings from the same experimental groups. B: densitometric analysis of PKC contents of the same experiments shown in A. Each bar represents the mean of the relative density units ± SE from 3 different experiments. *P < 0.05 vs. control. #P < 0.05 vs. CRF.

View larger version (12K): [in this window] [in a new window]   Fig. 8. A: representative immunoblot of aortic rings from 3 rats of each experimental group after immunopecipitation of CAT-1. CAT-1 immunoprecipitate was blotted for phosphotyrosine. B: relative IOD ratios of phosphorylated CAT-1 to CAT-1 in freshly harvested aortic rings from the various experimental groups. Each bar represents means ± SE from 3 different rats. Data from control are normalized to 1. *P < 0.05 vs. control. #P < 0.05 vs. CRF.

View larger version (15K): [in this window] [in a new window]   Fig. 9. A: representative Western blot analysis showing regulation of phosphorylated PKC protein level in freshly harvested aortic rings from the same experimental groups. B: relative IOD ratios of phosphorylated PKC to PKC in freshly harvested aortic rings from the various experimental groups. Each bar represents means ± SE from 3 different rats. Data from control are normalized to 1. *P < 0.05 vs. control. #P < 0.05 vs. CRF.

View larger version (9K): [in this window] [in a new window]   Fig. 10. Effects of PMA on aortic arginine transport in CRF rats treated with rosiglitazone (group 3). Freshly harvested aortic rings were incubated with 0.1% DMSO as a vehicle or 50 nM PMA (in 0.1% DMSO) for 15 and 45 min. Data are presented as means ± SE of 5 different experiments. *P < 0.05 vs. control.

	DISCUSSION

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It has been shown, predominantly regarding to inducible and neuronal NOS, that the consequences of decreased arginine transport are far beyond a mere substrate depletion. NOS enzymes contain four redox active prosthetic groups (FAD, FMN, heme, and BH₄) that can, in principle, transfer electrons to O₂. When arginine sources are depleted, a functional NOS may turn into a dysfunctional superoxide generating enzyme, leading to accumulation of reactive oxygen species such as peroxynitrite (4, 27). In other words, adequate arginine supply is crucial for maintaining endothelial function by two different mechanisms: 1) allowing sufficient NO generation and 2) preventing superoxide synthesis by NOS. Since an increase in oxidative stress and protein nitration are well-known phenomenons in CRF, it is conceivable to hypothesize that decreased arginine transport contributes to the accumulation of reactive oxygen species in CRF. To test this hypothesis, we estimated the extent of protein nitration, a marker for oxidation mediated by peroxynitrite, using Western blotting and immunohistochemistry. Indeed, we found that administration of rosiglitazone to CRF rats attenuated protein nitration in aortic endothelial cells. In the aggregate, our data suggest that, in addition to improving eNOS activity as demonstrated by augmented aortic cGMP generation, rosiglitazone attenuates the oxidative stress induced by peroxynitrite accumulation in uremia by normalizing arginine transport velocities, thus restoring endothelial function.

We tried to elucidate a molecular mechanism to explain our findings. The fact that rosiglitazone-related changes in arginine uptake were not associated with changes in CAT-1 protein content strongly supports the notion that these events involve a posttranslational CAT-1 modulation. We were therefore intrigued to explore a putative mechanism for the aforementioned finding. A possible involvement of PKC in the regulation of L-arginine transport in different cell types has been discussed for the last several years. There are two lines of evidence suggesting that PKC participates in the regulation of CAT-1 activity. First, according to a model by Albritton and his colleagues (2), CAT-1 protein contains three putative sites for phosphorylation by PKC, localized in the fifth and sixth intramolecular loops. Second, both CAT-1 and activated PKC have been reported to be localized in the caveola, allowing for the possibility of CAT-1 and PKC interaction (16).

Graf et al. (9) showed that PMA, a diacylglycerol analog which directly activates PKC, inhibits CAT-1 transport activity in endothelial cells without reducing CAT-1 mRNA or protein expression. In addition, Krotova et al. (12) found that the classical isoforms of PKC, in particular PKC, inhibit CAT-1 transport activity in pulmonary artery endothelial cells, independent of the expression or intracellular distribution of CAT-1 protein, but rather by modifying its catalytic activity. We recently reported, in two different experimental models characterized by diminished arginine transport namely hypercholesterolemia and pregnancy, a posttranslational regulation of CAT-1 which was associated with upregulation of aortic PKC (17, 25). Moreover, during pregnancy, treatment with -tocopherol, which inhibits PKC, prevents the decrease in arginine transport (17). We decided to focus exclusively at the membrane fraction of PKC, since this is where the interaction between this enzyme and CAT-1 occurs. Similar to our findings in hypercholesterolemia and pregnancy, we found in the current set of experiments a significant increase in the aortic membrane-bound fraction of PKC which was associated with a significant increase in CAT-1 phosphorylation. Both findings were attenuated by rosiglitazone. Interestingly, PKC phosphorylation was unchanged in CRF. Yet, the administration of rosiglitazone significantly decreased its phosphorylation. In addition, ex vivo exposure of aortic rings harvested from rosiglitazone-treated uremic rats to PMA (a PKC stimulant) abolished its beneficial effect. These data establish that in CRF the increase in PKC activity is primarily derived from its translocation from the cytosol to the cellular membrane while administration of rosiglitazone mitigates the decrease in arginine transport during uremia through modulation of both subcellular distribution and activation of PKC. PPAR-mediated inhibition of classical PKCs has been previously described. Verrier et al. (29) showed that PPAR agonists inhibit the diacylglycerol PKC signaling pathway, while Von Knethen et al. (30) recently suggested that PPAR ligand activation attenuates cytosol to membrane translocation of PKC in macrophages. While there is a growing bulk of evidence suggesting that inflammation contributes substantially to the pathogenesis of chronic kidney disease, it is surprising that the role of PKC has not been extensively studied in uremia. Wolf et al. (33) reported that, in uremic rats, PKC protein content is augmented in the myocardium. We believe that the role of PKC activation in CRF and its effects on the endothelium deserve further attention.

We previously showed that in uremia, the decrease in arginine transport is associated with decreased CAT-1 protein abundance and treatment with either atorvastatin or arginine prevents the decline in arginine uptake and restores CAT-1 protein content (24). In the current experiments, rosiglitazone prevented the decrease in arginine transport without affecting CAT-1 protein. These findings allow us to dissect two different mechanisms which impact arginine transport in CRF. The first is associated with a decrease in CAT-1 translation, while the second evolves from upregulation of PKC. The ability to restore normal arginine velocities in the presence of decreased CAT-1 protein abundance suggests that the latter is the dominant one.

Although concerns have been raised recently about risks associated with rosiglitazone treatment in patients with diabetes, growing evidence in the literature suggests that rosiglitazone exerts a beneficial effect in nondiabetic nephropathies. The current studies explore a novel mechanism by which rosiglitazone potentiates the endothelial arginine NO axis in uremia, thereby improving endothelial function and potentially attenuating the progression of chronic kidney disease.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. Schwartz, Dept. of Nephrology, Tel Aviv Sourasky Medical Center, 6 Weizmann St., Tel Aviv, Israel (e-mail: dorons{at}tasmc.health.gov.il)

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.

^* M. Ingbir and I. F. Schwartz contributed equally to the preparation of the current manuscript.

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