Abstract
ABSTRACT. Targeting the Ras family of monomeric GTPases has been suggested as a therapeutic strategy in proliferative renal diseases. This article reports the effects of Ras antagonist farnesylthiosalicylic acid (FTS) in rat thy-1 nephritis, a model in which cytokine-driven glomerular cell proliferation and invasion is likely to involve Ras signaling pathways. FTS in vitro specifically inhibits the binding of Ras to discrete membrane sites, thereby downregulating several Ras-dependent signaling functions and accelerating Ras degradation. Forty-four Lewis rats were given nephritis by day zero injection of a monoclonal thy-1 antibody ER4 (2.5mg/kg body wt). Twenty-two rats were then treated with daily intraperitoneal injection of FTS (5 mg/kg body wt) until sacrifice, and the remaining control rats were given vehicle alone (C). Six rats from each group were sacrificed at day 1 to establish equal injury; other sacrifice points were day 7 and day 10. Bromo-deoxyuridine (BrdU) was injected 1 h before sacrifice, after which sections were used for immunohistochemistry, which included detection of Ras expression, BrdU+ cells and macrophages/monocytes (ED1+). Thy-1 nephritis was associated with an increase in glomerular expression of Ki-Ras and N-Ras isoforms, which was almost fully prevented by FTS. FTS treatment was associated with: (a) a 54% reduction in the mean number of BrdU+ cells per glomerulus (P < 0.01), (b) a 50% reduction in macrophages/monocytes (ED1+) per glomerulus (P < 0.01), and (c) a reduction in 24-h proteinuria at day 10 (P < 0.05). These results show that Ras inhibition can reduce both glomerular cell proliferation and glomerular macrophage cell number in the thy-1 model and justify further study of FTS as a potential therapeutic in proliferative nephritis. E-mail: bruce.hendry@kcl.ac.uk
The process of renal damage occurring in glomerulonephritis involves glomerular cell apoptosis, inflammatory cell migration, and glomerular cell proliferation (1). These processes are driven by the production of a range of cytokines, growth factors, and integrins, which are upregulated in response to injury and play different roles in stimulating cellular proliferation, migration, and later extracellular matrix production (2,3 ⇓). These factors activate cell surface receptors and subsequent intracellular transduction pathways in producing their effects.
The Ras family of monomeric GTPases are intracellular signaling molecules, which act as molecular switches transducing signals to effector cascades (4). They act downstream from receptor tyrosine kinases and are also involved in G-protein–coupled receptor and integrin-mediated signaling, thus forming a potential convergent point in the signals generating glomerulonephritis (5,6 ⇓). Ras GTPase pathways have been shown to be pivotal in control of cellular proliferation in classical biologic models such as 3T3 cells (7); however, the role of Ras in renal cells in vivo is not yet clear. In vitro work in our laboratory has confirmed Ras expression in human renal mesangial cells and renal fibroblasts in primary culture and has shown Ki-Ras to be the predominant isoform expressed in both cell types (8,9 ⇓). Platelet-derived growth factor (PDGF)-induced proliferation of human mesangial cells appears to require both Ki-Ras and Ha-Ras, as is also the case for epidermal growth factor (EGF)-induced renal fibroblast proliferation (8,10 ⇓).
S-trans, trans-farnesylthiosalicylic acid (FTS) is a synthetic S-prenyl derivative of a rigid carboxylic acid, which structurally resembles the carboxy-terminal farnesylcysteine group common to all Ras proteins. It has been shown to act as a functional Ras antagonist in cells; affecting Ras-membrane interactions, dislodging the protein from its anchorage domains, facilitating its degradation, and thus reducing cellular Ras content (11,12 ⇓). Acting in this way, FTS has been shown to inhibit the growth of Ha-Ras and K-Ras transformed rodent fibroblasts in vitro (13,14 ⇓). FTS has also been shown in vivo to decrease inflammation and fibrosis scores in experimentally induced liver cirrhosis in rats, with a reduction in Ras levels in membranes extracted from rat livers treated with FTS being demonstrated relative to controls (15).
Thy-1 nephritis is an experimental model of mesangial-proliferative glomerulonephritis. This is a complement-mediated transient model, in which a single injection of monoclonal IgG2a anti-Thy-1 antibody (ER4) leads to mesangial cell lysis, followed by mesangial cell proliferation and glomerular microaneurysm formation, with proteinuria and hematuria. A glomerular infiltrate of polymorphonuclear leukocytes and macrophages is also seen (16). Mesangial cell proliferation is a key feature of this model, resulting from activation of a number of cytokine driven cell-signaling pathways with PDGF playing an important role (17,18 ⇓).
This study examines the role of Ras in the Thy-1 model of glomerulonephritis by using FTS to antagonize Ras in vivo. The results demonstrate that FTS inhibits glomerular cell proliferation and macrophage infiltration and justify further detailed work on Ras antagonism as a potential therapeutic strategy in glomerulonephritides.
Materials and Methods
Animals
Male Lewis rats weighing between 180 and 250 g were used with free access to standard laboratory diet and water. All procedures were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act.
Induction of Thy-1 GN
Thy-1 nephritis was induced by an intravenous injection of a monoclonal antibody raised against rat Thy-1 antibody (ER4) at a dose of 2.5 mg/kg body weight. The antibody was provided by Dr W.M. Bagchus, Department of Pathology, University of Groningen, The Netherlands (19).
FTS Solution Preparation
FTS was provided by Thyreos, Newark, NJ. For each experiment, FTS was dissolved in chloroform to form a 0.1 M solution and aliquots stored at −70°C. Immediately before use, the chloroform was removed by evaporation under a stream of nitrogen and FTS dissolved in ethanol, alkalinized by the addition of 1NNaOH, and then diluted with phosphate-buffered saline (PBS) to obtain a solution of 1 mg/ml. This solution was then used for injection (1 to 1.25 ml per rat), with control animals receiving alkalinized PBS only.
Experimental Protocols
Induction Control.
Twelve rats were induced with Thy-1 nephritis at day 0. Six of these rats were then immediately treated with an intraperitoneal injection of FTS (5 mg/kg body wt), and six rats were given vehicle alone as control (C). Sacrifice was at day 1 to confirm equivalent mesangial injury
Experiment 1.
Twelve rats were induced with Thy-1 nephritis at day 0. Six of these rats were then treated with daily intraperitoneal injection of FTS (5 mg/kg body wt) for 7 d, and six rats were given vehicle alone as control (C). Urine was collected by placing rats in metabolic cages for 24 h at days 6 to 7. Sacrifice was at day 7, 1 h after intraperitoneal injection of Bromo-deoxyuridine (BrdU [50 mg/kg body wt]; Sigma, UK).
Experiment 2.
Twenty Lewis rats were induced with Thy-1 nephritis as above. Ten rats were treated with daily intraperitoneal injection of FTS (5 mg/kg body wt) for 10 d, and ten rats were given vehicle alone as control (C). Urine was collected by placing rats in metabolic cages for 24 h at days 9 to 10. Sacrifice was at day 10, 1 hr after intraperitoneal injection of BrdU (50 mg/kg body wt).
Assessment of Renal Disease
In both experiments, proteinuria was measured by the sulfosalicylic acid method (20). Serum was collected at sacrifice for determination of serum creatinine, using an Olympus AU600 analyzer (Olympus, Eastleigh, UK). Urinary creatinine concentration was also measured. At sacrifice, kidneys were removed and a portion of each kidney was fixed in both 10% formal saline and methyl carnoy solution and then embedded in paraffin. Five-micrometer sections were cut for immunohistologic studies. BrdU and ED1 immunohistochemistry formed the primary end points for both experiments. Kidney tissue was also snap-frozen in isopentane and then immersed in liquid nitrogen and stored at −70°C.
Ras Immunocytochemistry
Monoclonal antibodies (mAb) used were against pan-Ras (clone Ras10), Harvey (clone 235–1.7.1), Kirsten (clone 234–4.2) and Neural (clone F155–277) isoforms of Ras (Oncogene Research Products, Cambridge, MA). Specificity of these mAb was confirmed with dot blots of recombinant Ras proteins, and no crossreactivity was detected, as previously reported (8).
Paraffin-embedded sections were rehydrated and treated with 0.05% saponin for 30 min and then washed in tap water. Sections for isoform-specific mAb were then digested with 0.1% pepsin at a pH of 2.3 for 20 min and washed again. Endogenous peroxidase was blocked using 0.03% hydrogen peroxide in methanol for 10 min. All washes from this point onwards were in 0.05% Tween-TBS (Tris-buffered saline) buffer solution for 5 min with gentle agitation. Sections were incubated overnight with primary antibody (dilutions: pan Ras 1/2000; H-Ras 1/500; Ki-Ras 1/10; N-Ras 1/100) at 4°C (16 to 20 h), washed, and then incubated with a polymer second layer (DAKO Envision + system, HRP; DAKO, CA) for 30 min. Diaminobenzidine (DAB) was used as chromogen, and sections were counterstained with hematoxylin. Negative controls (omission of primary antibody) and positive controls with skin were used for each set of experiments and were uniformly negative and positive, respectively.
BrdU and ED1 Immunohistochemistry
The monoclonal antibodies (mAb) used were for BrdU (BrdU, DAKO) and for rat macrophages/monocytes (ED1, Serotec, Oxford, UK). Carnoy fixed tissue was used for BrdU staining and fomalin-fixed tissue for ED1.
Paraffin-embedded sections were rehydrated with xylenes and graded ethanols. For ED1 staining, tissue sections were microwave heated in sodium citrate. All sections then had endogenous peroxidase activity blocked with 1% hydrogen peroxide in methanol and PBS for 30 min. Sections for BrdU were then immersed in 1 M HCL preheated to 60°C for 5 min with all sections then blocked in 20% rabbit serum for 20 min. Following this, sections were incubated in primary antibody (BrdU dilution 1:50, ED1 1:500 in 1% bovine serum albumin [BSA]/PBS) for 1 h, washed, and then incubated in rabbit anti-mouse biotinylated secondary antibody (dilution 1:200 in 1% BSA/PBS with 5% rat serum) for 1 h. After washing a streptavidin/biotin kit (ABC, DAKO) used per manufacturer’s instructions was applied to each section for 30 min. Slides were then developed in chromogen 3,3′-diaminobenzidine (DAB), washed in water, counterstained in hematoxylin, and dehydrated in graded alcohols and xylene before mounting in Depex. For quantification, 50 glomeruli per section were counted for presence of positive cells in a blinded fashion.
Double-Staining Immunohistochemistry
The monoclonal mAb used for BrdU and ED1 were as above. Ox-7 mAb was used to stain mesangial cells (Ox-7; Serotec, Oxford, UK). Carnoy fixed tissue was used, and BrdU staining was performed as described above. For Ox-7 only, sections were then digested for 6 min at 37°C with 0.1% pronase. Sections were then blocked in 20% goat serum for 20 min. After this, sections were incubated overnight at 4°C in the second primary antibody (ED1 1:500 in 1% BSA/PBS or Ox-7 1:100 in 5% normal rat serum/PBS), washed, and then incubated in goat anti-mouse biotinylated secondary antibody (dilution 1:200 in 1% BSA/PBS with 5% rat serum) for 1 h. After washing a streptavidin/biotin kit (ABC, DAKO) used per manufacturer’s instructions was applied to each section for 30 min. Slides were then developed using a Vector SG substrate kit per manufacturer’s instructions and washed in water, counter-stained in Meyer hematoxylin, and dehydrated in graded alcohols and xylene before mounting in Depex.
In Vitro Experiments
Primary culture-rat mesangial cells were serum starved for 24 h, trypsinized, and seeded in triplicate into 96-well plates (5000 cells/well). Cells were then treated with FTS (0 to 20 μM) and grown in 200 ng/ml PDGF at 37°C, 95% air, 5% CO2. Viable cell numbers were determined by the MTS assay (Cell titer 96, Promega), measuring absorbance at 490 nm. The correlation between viable cell number and absorbance at 490 nm is linear.
Statistical Analyses
For animal work, the significance of differences between different experimental groups was determined by two-tailed Mann-Whitney U tests. Differences were considered significant if P < 0.05. Statistical calculations were performed using Prism software (Graph-Pad Software, San Diego, CA). For the analysis of Ras expression in glomeruli, the strength of staining was scored in a blinded fashion by two observers on an arbitrary scale of 0 (no stain) to 4 (intense stain). Comparisons between groups of animals were then made using two-tailed Mann-Whitney U tests and P values of < 0.05 are reported as significant.
Results
In Vitro
FTS decreased proliferation in PDGF-stimulated primary culture rat mesangial cells as shown in Figure 1. The action was concentration-dependent, with an apparent ED50 in the range 5 to 10 μM.
Figure 1. Growth inhibition of primary culture rat mesangial cells measured by MTS assay after treatment with FTS at 5 μM, 10 μM, and 20 μM concentrations compared with vehicle alone.
Induction Controls
There was no difference in the extent of mesangial injury in the FTS-treated or control groups induced with Thy-1 nephritis at day 1 as assessed by glomerular cell count as shown in Figure 2.
Figure 2. Glomerular cell counts for Lewis rats with thy-1 nephritis at days 1, 7, and 10. Comparison of rats treated with FTS and those given vehicle alone (controls).
Experiment 1
In this experiment, Thy-1–injected rats were sacrificed on day 7 after daily treatment with FTS or vehicle. The tissue architecture of renal sections taken from FTS-treated and vehicle-treated rats is shown in Figure 3A and 3B. Glomerular hypercellularity was reduced in the FTS group as shown in Figure 2. BrdU staining in glomeruli is shown in Figures 3C and 3D; the number of BrdU-positive cells was strikingly reduced in the FTS-treated rats. Glomerular expression of Ras isoforms in the Thy-1 nephritis rat renal tissue compared with tissue from normal rats is illustrated in Figure 4. Vehicle-treated Thy-1 rats showed striking and significant increases in the glomerular expression of Ki-Ras and N-Ras compared with normal controls (P < 0.05). These changes were almost fully prevented by the FTS treatment (P < 0.05 for FTS versus vehicle in thy-1). There were no significant differences in Ha-Ras expression when comparing thy-1 with controls or FTS with vehicle (P > 0.2).
Figure 3. Histology sections at x60 of rat kidneys from Experiment 1: (A) H&E–stained section FTS group; rat induced day 0 with Thy-1 nephritis treated with FTS for 7 d, sacrifice day 7. (B) H&E–stained section Control group; rat induced day 0 with Thy-1 nephritis treated with vehicle alone for 7 d, sacrifice day 7. (C) BrdU staining FTS group; (D) BrdU staining Control group.
Figure 4. Immunostaining for Ras in sections from normal rats (left column), rats with thy-1 nephritis treated with vehicle alone and sacrificed on day 7 (middle column), and rats with thy-1 nephritis that also received FTS injections daily (right column). Isoform-specific mAb were used to define expression of Ha-Ras, Ki-Ras, and N-Ras in the three rows as labeled. Staining was scored on an arbitrary scale of 0 to 4 for each section; for Ki-Ras and N-Ras, the differences between groups were significant. Ki-Ras: control median 0.0, thy-1-vehicle 3.0 (P = 0.025 versus control), Thy-1-FTS 1.0 (P = 0.034 versus Thy-1-vehicle). N-Ras: control median 0.0, thy-1-vehicle 3.0 (P = 0.025 versus control), Thy-1-FTS 1.0 (P = 0.034 versus Thy-1-vehicle).
Quantitative assessment of glomerular cell proliferation was performed using BrdU staining. There was a significant reduction in number of BrdU+ cells per glomerulus in animals treated with FTS as compared with controls: (FTS, 1.74 ± 0.27; C, 2.84 ± 0.22; P < 0.05; Figure 5). Glomerular macrophage numbers were assessed using ED1 staining. There was a significant reduction in number of ED1+ cells per glomerulus in animals treated with FTS as compared with controls (FTS, 0.98 ± 0.10; C, 1.62 ± 0.14; P < 0.05; Figure 6). There was no significant reduction in proteinuria in FTS-treated rats at day 7 (Figure 7). There were no significant differences in serum or urine creatinine levels or creatinine clearance comparing FTS with vehicle (data not shown).
Figure 5. Mean BrdU-positive glomerular cell counts for normal Lewis rats and rats with thy-1 nephritis. Comparison of rats treated with FTS and those given vehicle (controls).
Figure 6. Mean ED1-positive cell counts per glomerulus for normal Lewis rats and rats with thy-1 nephritis. Comparison of rats treated with FTS and those given vehicle (controls).
Figure 7. Measurements of 24-h proteinuria in normal Lewis rats and rats with thy-1 nephritis. Comparison of rats treated with FTS and those given vehicle (controls).
Experiment 2
In this experiment, rats were treated daily with FTS until sacrifice on day 10. There was a significant reduction in number of BrdU+ cells per glomerulus in animals treated with FTS as compared with controls (FTS 1.54 ± 0.17; C, 3.38 ± 0.30; P < 0.01; Figure 5). Glomerular macrophage numbers were assessed using ED1 staining. There was a significant reduction in number of ED1+ cells per glomerulus in animals treated with FTS as compared with controls (FTS 1.35 ± 0.15; C, 2.69 ± 0.18; P < 0.05; Figure 6). There was a significant reduction in 24-h proteinuria (measured day 9 to 10) (FTS, 40.4 ± 2.99 mg/24 h; C, 48.0 ± 3.05 mg/24 h; P < 0.05; Figure 7). There were no significant differences in serum or urine creatinine levels or creatinine clearances (data not shown).
Sections from experiment 2 were double-stained for BrdU and Ox-7. All glomeruli in these day 10 thy-1 animals showed numerous Ox-7–positive cells representing >80% of cells counted. Double-staining with BrdU showed that over 90% of BrdU-positive cells were also Ox-7–positive. This demonstrates that the proliferating cells were of mesangial origin. Sections from experiment 2 were also double-stained for ED1 and BrdU. Only a very small proportion (<5%) of BrdU-positive cells were also found to be ED1-positive, showing the proliferating cells not to be of macrophage or monocyte origin.
Discussion
The aim of this study was to determine the effect of Ras antagonism in vivo, through treatment with FTS, in a model of mesangial-proliferative glomerulonephritis. The modulation of human renal mesangial cell proliferation could be of therapeutic value in a variety of progressive glomerulonephritides and Ras GTPases are a potential target for such modulation, forming a convergent point in the signaling pathways of many proliferative cytokines to the cell nucleus. An increase in glomerular expression of Ki-Ras and N-Ras was demonstrated in Thy-1 nephritis, consistent with a role for Ras in pathogenesis in this disease model. These changes in Ras expression were almost completely prevented by treatment with FTS. Treatment with FTS in Thy-1 nephritis was also effective in reducing cellular proliferation and macrophage number with an associated reduction in proteinuria.
FTS has been shown to inhibit growth of ErbB2- and Ras transformed cells but not v-Raf-transformed cells, thus acting as a functional Ras antagonist (14). In Ha-Ras transformed (EJ) cells, it has been shown that FTS dislodges Ras from membranes, facilitating its degradation and reducing total cellular Ras (12). In human mesangial cells in primary culture, FTS inhibits proliferation stimulated by PDGF and inhibits the activation of pathways downstream from Ras (10). The therapeutic potential of this action has already been demonstrated in vivo in experimentally induced liver cirrhosis in rats (15), where treatment with FTS was shown to decrease fibrosis and inflammation. FTS also appeared to reduce cellular levels of Ras in hepatic cell membranes in vivo. The actions of FTS on the glomerular expression of Ras are consistent with these in vitro and in vivo studies.
The reduction of glomerular BrdU+ cells in Thy1 nephritis by FTS is consistent with a direct action on Ras pathways in mesangial cells as there is strong evidence that Ras plays a central role in mesangial cell proliferative signaling. The data of Figure 1 confirm that rat mesangial cell proliferation is sensitive to inhibition in vitro by FTS. Moreover, the Ox-7 and BrdU double-staining results demonstrated that the proliferating glomerular cells in the Thy-1 model are mesangial in origin and taken together with the data of Figure 5 show that this mesangial proliferation is reduced by FTS. Quantitative Western blotting in human mesangial cells has shown Ki-Ras to be the predominant isoform expressed (>90% of total Ras) (10). Direct antagonism of Ras pathways in vitro using antisense oligonucleotides inhibits mesangial cell proliferation and renal fibroblast proliferation (8,10 ⇓). Similar results have been obtained using indirect antagonism of Ras with HMG-CoA Reductase inhibitors and prenylation inhibitors, which reduce the C terminal prenylation required for Ras function. Although the specific function of each of the Ras isoforms is not known, different Ras isoforms have been shown to differ in their ability to activate certain effectors (21,22 ⇓), and it may be that the different isoforms of Ras play distinct roles in control of proliferation acting via different downstream cascades.
The actions of FTS on glomerular macrophage/monocyte cell number do not appear to be due to altered proliferation of this cell type, as double-staining showed that very few of the BrdU-positive cells were ED1-positive. It is likely that the actions of FTS on monocyte cell number are through altered migration or survival. These actions could occur as a direct result of Ras antagonism in monocytes, as Ras has a key role both in the signaling of migration by chemokines and in anti-apoptotic pathways. Indeed, Reif et al. (15) have demonstrated that FTS can inhibit PDGF-induced cell migration. Alternatively the actions of FTS on Ras in mesangial cells could indirectly alter monocyte number by altering the mesangial cell secretion of key cytokines and chemokines.
The mechanisms active in Thy-1 appear to be similar to those that occur in vivo in humans at certain stages of glomerulonephritis with increased cytokine expression (17,23,24 ⇓ ⇓) and extracellular matrix production (25) being found. The antagonism of Ras could be of therapeutic value. FTS is of low toxicity in animal work and is being developed as a cancer therapeutic for clinical use. Other strategies for targeting Ras pathways include the use of small molecules directed to related signals such as Raf-1 and the PDGF receptor. A Raf-1 antagonist is also in clinical development for cancer. Antisense oligonucleotides targeting Ha-Ras and Raf-1 are also in clinical trials in cancer and appear to have few side effects. Further studies of Ras antagonism in animal models of renal disease are required to determine whether these promising data in Thy-1 have wider implications.
Acknowledgments
We wish to thank Thyreos (Newark, NJ) for the kind gift of FTS.
Footnotes
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Dr. Jürgen Floege served as Guest Editor and supervised the review and final disposition of this manuscript.
- © 2003 American Society of Nephrology
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