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1Chair of Nephrology, 2Department of Neuroscience, Unit of Physiology, 3Department of Clinical and Experimental Medicine, 4Department of Oral and Maxillofacial Sciences, 5Chair of Nephrology, University Magna Graecia of Catanzaro, and 6Department of Biology and Cellular and Molecular Pathology, University Federico II of Naples, Naples, Italy
Submitted 28 July 2005 ; accepted in final form 18 January 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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signal transduction; kidney; endothelium; farnesyl transferase inhibitors
IRI is a complex sequence of events that involves a rise in reactive oxygen species (ROS) and synthesis of cytokines and chemokines, leading to inflammation and, eventually, to cell death (4, 17). Many experimental models have been developed in an attempt to prevent or limit renal failure after IRI, like the use of ROS scavengers (6, 17, 19) or of various drugs (10, 24, 25) or the induction of "renoprotective" genes (15) with disappointing and inconclusive results, which have not produced information on the molecular nature of the process and have been difficult to translate to human studies.
The elucidation of molecular pathways involved in tubular cell death following IRI can lead to the identification of potential targets for possible therapeutic intervention. One such target may be the small GTPase p21 Ras, which plays a central role in the signaling pathway controlling cell survival and apoptosis through its activation or inhibition in response to several stimuli. Oxidative stress is a powerful activator of Ras, and such activity is necessary for the transmission of signals by many cytokines involved in the ischemic insult, like PAF, transforming growth factor-
, and endothelin-1, to its downstream effectors, such as extracellular signal-regulated kinase (ERK) 1/2 and phosphoinositide 3-kinase (PI3-kinase), which are relevant stress responders (14). Recent studies in our laboratory have shown that two different isoforms of Ras, namely, Ha-Ras and Ki-Ras, act differently in response to an oxidative stimulus: the activation of Ha-Ras, in fact, increases the number of cells undergoing cell death by increasing intracellular ROS, whereas activation of Ki-Ras seems to have a protective effect through ROS reduction (3, 20). These findings highlight the modulatory role of Ras isoforms in response to oxidative injuries and suggest a crucial role of Ras as a preferential molecular target in disorders associated with high intracellular levels of ROS.
To date, however, no study has evaluated the function of Ras in renal damage after ischemia and the possibility to influence Ras activation in an attempt to mitigate renal injury.
The purpose of this study was to evaluate the signaling pathways involved in oxidative stress-induced cell damage in cultured human renal tubular cells and to test in vivo the protective role of Ras inhibition in an established model of warm IRI.
Ras association with the plasma membrane is essential for its biological activity. Proper membrane association is allowed by a series of posttranslational modifications of the protein. The first consists of the covalent binding of a polyisoprenoid, a farnesyl group to the cysteine residue located in the COOH-terminal tetrapeptide (CAAX box), in a reaction catalyzed by farnesyl protein transferase (FPTase) (13). Therefore, several inhibitors of FPTase have been developed to inhibit Ras and its downstream effectors and some of them are currently used in clinical trials for cancer treatment (21). In our study, we inhibited Ras using a specific inhibitor of Ras farnesylation, chaetomellic acid A (S-FTI).
We have previously reported that endothelial cells treated with farnesyl transferase inhibitors or expressing a dominant negative Ha-Ras variant became tolerant to oxidative stress (3). Here, we have extended our analysis to tubular cells in vitro and kidney in vivo. We show that oxidative stress markedly induces Ras-ERK1/2. Inhibition of this cascade significantly reduced apoptosis. In vivo, this translates to a significant reduction of damage induced by transient ischemia using S-FTI. We suggest that farnesyl transferase inhibitors are the tools to achieve this protection in vivo.
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MATERIALS AND METHODS |
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HRPT cells were cultured by using renal fragments of normal tissue (1–2 mm) obtained from kidneys excised in patients with renal cancer but with normal renal function and not affected by hepatitis viruses. The technique was described for the first time by Detrisac et al. (5): briefly, tubular cells were obtained through several passages in progressively narrowing filters, continuously washed, and subsequently treated with collagenase/dispase. Cells were cultured in DMEM-Ham's F-12 (Sigma, St. Louis, MO) enriched with insulin, transferrin, and selenite in 95% air-5% CO2. The confluent cells were treated with trypsin and washed with Percoll solution. The specific isolation of proximal tubular cells was achieved by an immunomagnetic separation technique using monoclonal anti-human aminopeptidase M (AMP; CD13, Sigma), a specific marker of proximal tubular cells, as previously described (1). Flow-cytometric analysis showed that proximal tubular cells were positive for CD13 and negative for EMA, CD90, and von Will brand factor, specific markers of tubular distal cells, fibroblasts, and endothelial cells, respectively.
ECV-304 human endothelial cells (European Collection of Cell Cultures) were grown in monolayers in RPMI 1640 medium (Sigma) supplemented with 10% FBS (Sigma), 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin; the cells were kept in a 5% CO2-95% air atmosphere at 37°C.
Flow cytometric analysis of cell death. Cell death was measured in HRPT cells using an annexin V detection kit (MBL Medical and Biological Laboratories, Watertown, MA). One week before the experiments, 1.25 x 105 cells were plated on 35-mm petri dishes and grown in complete medium. The cells were then collected by trypsinization and, after one wash in PBS, were suspended in 500 µl of binding buffer, stained with annexin V-FITC (an apoptosis marker) and propidium iodide (PI; a necrosis marker) and incubated at room temperature for 15 min before the flow cytometric analysis. Apoptotic (annexin V-FITC pos/PIneg) and necrotic (annexin-Vpos/PIpos and annexin-Vneg/PIpos) cells were detected and quantified as a percentage of the entire population. To measure necrosis in ECV-304 cells, 1.4 x 105 cells were plated on 35-mm petri dishes and grown for 48 h in complete medium. After trypsinization and one wash in PBS, the cells were resuspended in 500 µl of PBS and 1 µg/ml of propidium iodide was added before the flow cytometric analysis of PIpos cells.
Cytometric analysis was performed with a FACScan apparatus (Becton-Dickinson, San José, CA), and data were analyzed using WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, CA).
Immunoblot analysis.
For immunoblot analysis of oxidative stress-induced Phospho-ERK1/2 and Phospho-Akt levels, renal tubular epithelial cells, grown to semiconfluence in 60-mm petri dishes, were incubated for 18 h in medium containing 0.2% FBS before H2O2 treatment. A sample growing in medium containing 10% FBS was used as control. Then, cells were harvested by scraping them into RIPA buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) containing 2.5 mM Na-pyrophosphate, 1 mM -glycerophosphate, 1 mM NaVO4, 1 mM NaF, 0.5 mM PMSF, and a cocktail of protease inhibitors (Boehringer, Mannheim, Germany). Next, cell lysates were centrifuged for 10 min at 11,600 g, and the pellets were discarded. Fifty micrograms of protein were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were then probed with monoclonal anti-phospho-ERK1/2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or with anti phospho-Akt antibodies (Cell Signaling, Beverly, MA) at 1:1,000 dilution, following the manufacturer's instructions. The membranes were then stripped and reprobed with antibodies at 1:1,000 dilution against polyclonal total ERK1/2 (Santa Cruz Biotechnology) and total Akt (Cell Signaling), respectively. Protein bands were revealed by enhanced chemiluminescence (Amersham Life Science).
For the immunoblot assay of membrane levels of prenylated proteins, cells grown to semiconfluence in 100-mm culture dishes in complete RPMI medium, were incubated for 18 h in the absence or in the presence of 1.8 µM S-FTI (Calbiochem, San Diego, CA), and then collected by scraping them into a buffer containing 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 10 mM PIPES, 2 mM NaVO4, 10 mM phenylarsine oxide, 5 mM NaF, and a cocktail of protease inhibitors. Cells were then disrupted by sonication (2- to 10-s pulses at 100 W) and centrifuged at 600 g for 10 min. Next, the supernatants were centrifuged at 100,000 g for 45 min. The membrane pellet was resuspended in a 50-µl RIPA buffer. Fifty micrograms of cytosol and membrane proteins were resolved by a 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Next, the membrane was blocked in 3% nonfat dry milk in TBS-Tween 20 (0.05%) and probed with monoclonal anti-Pan-Ras (1:1,000, Upstate Biotechnology, Lake Placid, NY), anti-Ki-Ras (1:100, Santa Cruz Biotechnology), or polyclonal anti-Rab7 (H-50, 1:200, Santa Cruz Biotechnology) antibodies. Thus the membrane was washed and incubated with secondary horseradish-peroxidase-linked antibody (Amersham Pharmacia Biotech) (1:2,000), and the specific protein bands were detected by enhanced chemiluminescence and quantified by densitometry using Scion Image software.
For immunoblot analysis of kidney tissues, kidney fragments from rats in the acute hemodynamic studies (see below) were homogenized in 250 mM sucrose, 5 mM imidazole, pH 6.5, and 0.5 mM dithiothreitol (1:4, wt/vol), using a glass-Teflon potter. Samples were then centrifuged at 800 g at 4°C for 10 min, and supernatants were centrifuged at 100,000 g at 4°C for 45 min in a 70.1 Ti rotor (Beckman). Supernatants (cytosols) were collected, and membrane pellets were suspended in RIPA buffer. Fifty micrograms of membrane and cytosolic fractions were then subjected to immunoblot analysis for Pan-Ras and Ki-Ras proteins, following the procedure described above.
Determination of protein content. Protein content of total lysates and of membrane and cytosolic fractions was determined according to the method of Lowry (11).
Animal studies. This study was carried out in 44 male Sprague-Dawley rats (3 mo old, Charles River) fed a standard diet (19% protein content as casein) and tap water ad libitum. The experimental protocol and surgical procedures using animals were carried out according to Italian law and were approved by Italian Ministry of Health.
After light anesthesia with pentobarbital sodium (Nembutal, 50 mg/kg ip), the right kidney was nephrectomized under sterile conditions. Seven days later, the rats were randomly assigned to one of the following groups: IRI, untreated rats with acute renal ischemia (n = 8); S-FTI, rats with IRI pretreated with S-FTI (Biomol Research Lab, n = 10); and NOR, sham-operated rats (n = 9).
Ischemic damage was induced by clamping the left renal artery for 45 min with an atraumatic vascular clamp under anesthesia (Nembutal, 50 mg/kg ip). The reperfusion of the kidney was confirmed visually before suturing of the abdominal wall. The sham operation consisted of the same surgical procedure except that the renal artery was not closed. All the rats were treated 4 h before this procedure either with S-FTI (0.26 µg/kg body wt ip, dissolved in 0.3 ml of saline solution; S-FTI group) or with saline solution in equivalent volumes (IRI and NOR groups). The rats were then restrained, and 24 h later were surgically prepared for a hemodynamic study under Inactin anesthesia (100 mg/kg ip). All surgical procedures were carried out on an automatically thermo-regulated table, which maintained the rat's constant body temperature (37.5°C), as assessed by a rectal probe connected to a thermometer.
The details of surgical procedures are described elsewhere (18, 19). After a 60-min stabilization period, three to five clearance periods were performed in each experiment for the collection of blood samples from the femoral artery and renal vein (through a sharpened glass micropipettes), to assess the arteriovenous gradient of inulin, an estimate of the renal filtration fraction.
At the end of the experiment, the kidney was carefully removed and weighed under sterile conditions, and samples of renal tissue were immediately stored (–80°C) for the evaluation of Ras protein activation.
The dosage of S-FTI was carefully established in four groups of rats (n = 4/group), with a wide range of doses (0.16–1.3 µg/kg); the dose we used was associated with the best functional and histological outcome.
In an additional group of animals (n = 3), the ability of S-FTI in mitigating renal damage after induction of IRI was tested by the intravenous administration of the drug 1 h after reperfusion. During the clamping of the renal artery, the right femoral vein was isolated and catheterized (PE-50 tubing), and S-FTI (0.15 µg/kg body wt in 0.2 ml of saline solution) was injected 60 min after clamp removal. This policy was adopted to avoid altered peritoneal absorption of the drug when the abdomen was open and the rat was still anesthetized; lower doses of the drug were used. Also in this case, the reported dosage fit the best functional outcome. Control rats (n = 3) were treated with 0.2 ml of saline solution.
Analytic determinations. Urinary volume was measured gravimetrically in preweighed vials. The concentrations of inulin in plasma and urine were measured by the diphenylamine method. The urine for determination of urinary nitrate concentration was collected in sterile vials under ice (4°C), filtered through a 0.2-µm filter (Acrodisc, Gelman), and frozen until the time of assay (–80°C). The dosage was carried out by a total nitric oxide assay utilizing nitrate reductase and the Griess reagent (RD Systems). Proteinuria was determined by the Bio-Rad method, according to the manufacturer's instructions.
The filtration fraction was measured by renal extraction of inulin [arterial – venous/arterial concentration (A – V/A) x 100]; renal plasma flow (RPF) was calculated by the ratio glomerular filtration rate (GFR)/filtration fraction (FF), and renal blood flow by correction of RPF by hematocrit [RBF = RPF/(1 – Hct)/100]; renal vascular resistances (RVR) were estimated by the expression RVR = MAP/RBF, where MAP represents the mean arterial pressure.
Histology. Specimens of renal parenchyma were fixed in activated Bouin's solution for histological examination, and plastic-embedded 3-µm sections were cut, stained with hematoxylin-eosin, periodic acid-Schiff, Jones staining, and observed in a blinded fashion. Fifty proximal tubules from the outer stripe of the outer medulla (OSOM) were examined in each rat at x400 magnification and assigned to three categories: tubules with normal appearance (0); tubules with signs of moderate to sublethal injury (loss of apical brush border; 1); tubules with signs of acute tubular necrosis (from a few sloughed epithelial cells to tubules with a complete naked basal membrane; 2). Proximal tubules were distinguished from distal tubules on the basis of morphological criteria (18).
Statistics. The ANOVA was used to compare the different mean values in the different groups of rats. Bonferroni's test was used to find significant differences among the groups under study. Histological data were analyzed by the Kruskal-Wallis ANOVA followed by Dunn's multiple comparison test. A P value <0.05 was considered statistically significant. The data are expressed as means ± SD. Statistical differences in cell experiments were evaluated using a Student's t-test for unpaired samples.
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RESULTS |
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Because ERK1/2 is activated by Ras, we inhibited Ras by using S-FTI, which selectively alters its membrane binding, necessary for its biological activity. To determine the specificity of action of S-FTI, we measured the fraction of Ras in membranes of HRPT cells incubated with 1.8 µM S-FTI for 18 h. Figure 2A shows that S-FTI treatment consistently reduced membrane-bound Ras (–34 ± 3%) and increased the cytosolic fraction. Under these conditions, S-FTI inhibited H2O2-dependent activation of ERK1/2 (Fig. 2B). Thus HRPT cells were incubated for 18 h in the presence and absence of 1.8 µM S-FTI in medium containing 0.2% FBS to downregulate phospho-ERK1/2 levels. The cells were then stimulated with 1 mM H2O2 for 2 and 30 min, and phospho-ERK1/2 and total ERK1/2 levels were measured. Figure 2B shows that treatment with S-FTI markedly reduced H2O2-induced P-ERK1/2 levels, thus confirming the ability of this inhibitor to downregulate the Ras/ERK1/2 pathway. S-FTI treatment, in the same condition, was also able to inhibit ERK1/2 activation induced by stimulation of the cells for 2–5 min with 20% FBS (data not shown).
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Endothelial cells represent an important cellular fraction of the kidney and are potential targets of oxidative stress injury. In human umbilical vein endothelial cells, oxidative stress determines activation of the ERK1/2 pathway and cell death; the inhibition of farnesylprotein transferase in these cells reduces H2O2-induced ERK1/2 phosphorylation (3). Therefore, we have studied the effects of S-FTI on survival of stabilized endothelial cells (ECV-304) subjected to oxidative stress. Because ECV-304 cells are more sensitive to oxidative stress than HRPT cells, we treated them for 18 h with a lower dose (500 µM) of H2O2. S-FTI exerted a significant protective effect on H2O2-induced cell death (Fig. 3A), and Ras displacement from cell membrane after S-FTI treatment was also confirmed in ECV-304 cells (Fig. 3B). Ki-Ras localization was not influenced by S-FTI treatment (Fig. 3B), because this isoform is also geranylated (9). These data indicate that Ha-Ras is the principal Ras isoform affected by S-FTI treatment. Moreover, membrane localization of another farnesylated protein, Rab7, bound to late endosome and not permanently resident in the plasma membrane (27), was not affected by S-FTI treatment in ECV cells (Fig. 3B).
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Figure 4 shows that pretreatment with S-FTI determined a significant decrease in membrane-bound Ras proteins (–55%) without affecting Ki-Ras membrane binding, in agreement with the results obtained in cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In recent years, apoptosis and inflammation have been proposed as potential targets to limit organ damage after an ischemic insult (2, 4), and Ras seems to play a crucial role in mediating both phenomena. We have previously found that activation of Ha-Ras substantially increased apoptosis induced by oxidative stress in endothelial cells (3) and that Ha-Ras selectively stimulated NADPH oxidase and increased ROS production (20, 22), whereas cells expressing Ki-Ras, which stimulated mitochondrial SOD, were resistant to the same type of stress (20). Moreover, we and others have provided evidence that farnesyl transferase inhibitors dislodge and inactivate Ha-Ras and do not influence membrane localization of Ki-Ras (Fig. 3B) (3, 9).
The data presented here provide further insights into the mechanism underlying renal damage induced by ischemia. These observations suggest the following mechanism: endothelial and tubular cells, subjected to oxidative stress, activate stress kinases, including Ras/ERK1/2. This circuitry amplifies the effects of Ras and ERK1/2 on membrane NADPH oxidase (22). Inhibition of Ha-Ras by S-FTI downregulates this loop and significantly reduces apoptosis elicited by transient ischemia. Compared with other inhibitors, the action of S-FTI appears rather selective. Rab 7, for example, located in the inner membranes, or Ki-Ras, localized on the plasma membrane with two lipid anchors, are not altered.
An attractive result of our study is the translation of cellular findings to the whole animal: the displacement of Ras from cell membrane by S-FTI represents the first demonstration in vivo that parallels the significant reduction of ischemic organ damage by S-FTI. The demonstration of an arteriovenous gradient for inulin in all the S-FTI rats, contrary to the untreated ischemic rats (the IRI group), is a further positive effect that reflects either the higher GFR or the decreased "backfiltration" of inulin through the damaged tubules, commonly occurring after a prolonged ischemia. Histological data are in agreement with these results, showing the total disappearance of cellular necrosis in rats treated with S-FTI, with a considerable number of completely normal cells.
Although we cannot precisely identify the cells carrying activated Ras in the organ, we believe that tubular, endothelial, and mesenchymal cells contain stabilized Ha-Ras. We have recently provided evidence that normal fibroblasts and human primary cells regulate Ras levels by proteasomal degradation. H2O2 or ROS in vitro and in vivo increase and activate Ha-Ras (23). We have recently extended these observations to rat ischemic neurons, mouse astrocytes, and peripheral human lymphocytes (Avvedimento VE and Santillo M, unpublished observations). Stabilized and immortalized cell clones lose this regulation and modulate Ras activity solely by GTP-GDP binding.
The data presented here describe the protective effects of a farnesyl transferase inhibitor on ischemic renal cell injury in isolated tubular and endothelial cells and in whole kidney.
The results of this study have a broader impact, because they indicate a pharmacological tool for reducing ischemic damage in vivo. For example, kidneys to be transplanted can be better preserved and significantly reduce the extent of the damage if pretreated with S-FTI. Moreover, the possibility that S-FTI may be beneficial even when the drug is administered after the ischemic insult opens new, intriguing perspectives in the treatment of IRI, although conclusive evidence awaits the response of a larger number of animals.
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GRANTS |
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FOOTNOTES |
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* M. Sabbatini and M. Santillo contributed equally to this study. 
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.
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P < 0.01 vs. H2O2-treated control. 
P < 0.001 vs. H2O2-treated control.
