Subcellular distribution of Ras GTPase isoforms in normal human kidney
Hemant M. Kocher,
Ron Senkus,
Mashal Al-Nawab and
Bruce M. Hendry
Department of Renal Medicine, King's College Hospital, Guy's, King's and St Thomas' School of Medicine, King's College London, London SE5 9PJ, UK
Correspondence and offprint requests to: Hemant Kocher MS, MD, FRCS, Department of Health National Clinician Scientist, Senior Lecturer, Tumour Biology Laboratory, Cancer Research UK Clinical Centre Queen Mary's School of Medicine & Dentistry at Barts & The London, John Vane Science Centre, Charterhouse Square, London ECIM 6BQ. Tel.: + 44(0) 20 7014 0400; Fax: +44(0) 20 7014 0401; Email: hemant.kocher{at}cancer.org.uk
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Abstract
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Background: Ras GTPase isoforms have been implicated in proliferative renal disease and are known to have differential cellular expression in kidney. However, their exact subcellular location in various cells is unknown.
Methods: Immunogold labelling for Ras isoforms (Harvey, Kirsten and Neural) was performed for subcellular localization under electron microscopy in fresh normal kidney specimens, obtained from the opposite pole of kidneys removed for renal cell cancer.
Results: There was prominent staining shown by Ha-Ras only on the glomerular foot processes as compared with basement membrane or the endothelial cells. Mesangial cells showed intense staining in the cytosol with Ha-Ras (absent in the nucleus), minimal staining with Ki-Ras and none with N-Ras. In both the proximal and distal convoluted tubules, there was a clear staining of the mitochondria with Ha-Ras, with mild cytosolic staining with all of the isoforms.
Conclusions: Ras isoforms have distinct and separate subcellular distributions in normal kidney cells. Understanding the functional aspects of this distribution pattern is essential if Ras is to be targeted by genetic or molecular therapeutic tools.
Keywords: brush border; mesangial cell; mitochondria; podocyte
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Introduction
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The Ras family of small monomeric GTPases are known to play a key role in the signalling pathways which control cell survival, differentiation, migration and proliferation [1]. The cellular events in the pathogenesis of glomerulonephritis include proliferation, migration, adhesion and apoptosis [2]. Ras is activated by a range of cytokines including receptor tyrosine kinase ligands such as the fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and by other cytokines implicated in renal pathology such as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1) [1,2]. The downstream effectors of Ras include the Raf-mitogen-activated protein kinase (MAPK) cascades, Rho family GTPases and the phosphatidylinositol (PI) 3-kinases, giving Ras a pivotal role in the control of cell function [1,3]. In addition, tight junction regulation is mediated by Ras via direct interaction with ZO-1 [4]. We have shown that Ras isoforms are differentially expressed in normal renal tissue [5], but the subcellular expression of the three isoforms [Harvey (Ha), Kirsten (Ki) and Neural (N)] has not been described.
The three isoforms of Ras have different biochemical and cellular properties, but the functional differences among these species in cell signalling are not fully defined [3]. In vitro work on human renal fibroblasts and mesangial cells in primary culture shows that Ki-Ras is the major expressed Ras isoform, but both Ki-Ras and Ha-Ras play distinct and essential roles in growth factor-induced proliferation [68]. It is possible that specific isoforms of Ras play distinct roles in different renal pathologies and that these species could be targets for future therapy. For example, different forms of glomerulonephritis are associated with specific abnormalities of glomerular Ras expression [5]. This work is an electron microscopic survey of the expression of Ras isoforms in normal renal tissue.
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Materials and methods
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Tissue selection
Ethical approval was obtained from the local medical research ethics committee to obtain a 0.5 g sample from the normal aspect (opposite pole) of a kidney removed for renal cell carcinoma. This specimen was obtained fresh, within 5 min of removal of the organ, without jeopardizing the pathological information needed for further treatment of the patient, and dissection of the specimen was carried out by the pathologist. The specimen was fixed immediately as described below. Three such kidney specimens were obtained. Haematoxylin and eosin (H&E) staining confirmed these to be normal in histo-morphological appearance.
Reagents and instruments
Monoclonal antibodies (mAbs) against Harvey (clone 235-1.7.1), Kirsten (clone 234-4.2) and Neural (clone F155-277) isoforms of Ras (Oncogene Research Products, USA) were obtained. These antibodies were determined to be specific and without cross-reactivity among the three Ras isoforms [6]. The secondary antibody was goat anti-mouse IgG conjugated with 10 nm gold particles (British Biocell International, UK). Embedding resin was Lowicryl K4M (Polysciences Ltd, Eppelheim, Germany). Nickel grids of 200 mesh (Agar Scientific) with formvar coating (polyvinyl formal, BDH, UK) were used. The cutting machine used was an Ultrotome III (LKB, Sweden), the cryostat was FS/FAS (Brights, UK) and the electron microscope was a JEOL 100C (Japanese Electron Optics Ltd, Japan).
Final protocol: fixation and embedding
Various permutations and combinations of fixation, embedding and staining were tried to optimize the protocol to obtain the perfect combination of preservation of ultrastructural details and antigenicity. Briefly, fresh specimens were fixed with 2% formaldehyde with 0.5% glutaraldehyde for 2 h followed by preservation in phosphate buffer. Embedding was with freshly prepared Lowicryl K4M with progressive lowering of temperature [9,10]. Tissue was dehydrated at 4°C in 50% methanol (10 min), 80% methanol (20 min) and 90% methanol (20 min). The tissue was infiltrated with resin at 4°C with serial concentrations as: methanol:Lowicryl (1:1, 25 min), methanol:Lowicryl (1:2, 50 min), pure Lowicryl (60 min) and pure Lowicryl (overnight). The tissue was then embedded in fresh resin in gelatin capsules without air entrapment. It was then photo-polymerized in UV light (360 nm, Ultraviolet Prod. Inc., USA) at 35°C in a cryostat for 24 h, with capsules kept afloat in ethanol baths in order to dissipate heat evenly. The blocks were polymerized further at room temperature in UV light (360 nm) for 48 h and left to cool at room temperature for 48 h. The blocks were then cut initially as semi-thin (2 µm) survey sections and stained with 0.5% toluidine blue. Appropriate areas of the blocks were then cut in ultra-thin sections and put on formvar-coated 200 nm nickel grids. A section was stained with 1% uranyl acetate (25 min) and lead citrate (0.4%, 10 min) to assess adequate fixation and embedding with Lowicryl. Air-dried ultra-thin sections (silvergold interface, 7090 nm) were washed in 1:20 normal goat serum (20 min) and stained with monoclonal antibodies against Ras: Ha-Ras (1:50), Ki-Ras (1:1), N-Ras (1:10) and secondary antibody (1:50) [5,10]. The sections were then washed six times for 5 min in the buffer solution (freshly prepared 1% cold water fish gelatin in Tris-buffered saline filtered thorough a 0.22 µm filter with alternate timings of gentle vibration using a Vortex Jr Mixer by the side of the grids, to reduce non-specific binding. They were then incubated with secondary antibodies conjugated with gold (1:50, 2 h) at room temperature. Further washes were done four times in the buffer solution and twice in double-distilled de-ionized water. Counter-staining was done with 1% uranyl acetate for 25 min and then washes with water.
Electron microscopy and statistical analysis
Initial scanning at low magnification (x20 000) identified the cells by morphology. The position and counts of the gold particles in the particular subcellular compartment were studied in the nucleus, cytosol and plasma membrane (x35 000). These settings, of the area where the counts were done, were kept constant to keep the count of gold particles constant at per cm2 (0.08 µm2 for a magnification of 35 000). Quantitative analysis was performed by counting the number of gold particles per cm2; squares were selected randomly using a defined grid. Each photograph had six such readings taken for each cellular organelle; where possible, these readings were repeated three times in other photographs. Statistical analysis was done organelle-wise using paired sample t-tests for comparing isoforms (SPPS10.1, USA), with a significance level at P<0.01.
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Results
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Specimens were taken from the kidneys, removed from three subjects (two males) with a median age of 65 (range 6372) years. None of them had renal failure or previous renal disease or cardiovascular problems. The semi-quantitative analysis of the Ras isoforms is presented in Table 1. Sites where Ras was seen even in minimal quantities are described and the rest, such as mesangial mitochondria with no Ras seen, are not mentioned.
Glomerulus
Of note was the prominent staining shown by Ha-Ras on the foot processes as compared with basement membrane or the endothelial lining (Figure 1). Mesangial cells showed intense staining in the cytoplasm with Ha-Ras, which was not seen in the nucleus of the same cell (Figure 2). Ki-Ras and N-Ras showed minimal staining in the foot processes and mesangial cells. There was no staining with any of the Ras isoforms in the basement membrane and the endothelial cells.

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Fig. 1. Electron microscopic view of the Ha-Ras (immunogold labelling, 10 nm) staining within the glomerulus of the kidney. The typical staining with Ha-Ras in the podocytes, which is shown at a higher magnification in the inset. The basement membrane (BM) is not stained and the filtration slits are mildly stained.
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Fig. 2. Electron microscopic view of the Ha-Ras (immunogold labelling, 10 nm) staining within the glomerulus of the kidney. The picture shows the staining in the cytosol of the mesangial cell (inset). Bar = 0.28 µm.
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Tubules
In both the proximal and distal convoluted tubules (PCT and DCT, respectively), there was clear staining of the mitochondria with Ha-Ras (Figures 3 and 4). It is of note that there was minimal cytosolic staining with all of the isoforms, suggesting that Ras is not present free in the cytosol, but is nearly always contained in the cellular organelles. In contrast to previous reports, some Ras was found in the nucleus. The main concentration of Ha-Ras was seen in the mitochondria, which are identified by their morphology at low magnification (x8000), including the presence of cristae. In the brush border of the PCT, Ha-Ras and Ki-Ras were seen, but not N-Ras. In the DCT plasma membrane, some Ha-Ras was seen, but not Ki-Ras or N-Ras.

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Fig. 3. Electron microscopic view of the Ha-Ras (immunogold labelling, 10 nm) staining within the proximal convoluted tubules of the kidney. The cytosol shows no stain, whilst the cell organelles show intense stain (arrow). The nucleus shows minimal stain. Bar = 0.28 µm.
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Fig. 4. Electron microscopic view of the Ha-Ras (immunogold labelling, 10 nm) staining within the distal convoluted tubules of the kidney. The cytosol shows no stain, whilst the cell organelles show intense stain (arrow). The nucleus shows minimal stain. Bar = 0.28 µm.
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Statistical analysis of kidney electron microscopy
The statistical significance and the confidence intervals of the difference in staining with various isoforms are given using the paired t-test (Table 1). Staining in some of the organelles could not be measured in enough sections to give confident estimates.
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Discussion
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This set of experiments shows for the first time that there is a definite pattern of Ras isoform distribution in normal human kidney, and it differs at the subcellular level from cell to cell. The results are summarized in Table 2 and are compared with previous light microscopy work from our group [5]. The striking finding of this work is the presence of Ras in locations other than the plasma membrane, which is believed to be the site where Ras is active. Of note was the presence of Ras in mitochondria, a hitherto unreported site of localization. Also Ha-Ras is strongly expressed in the foot processes, where its function is unknown.
It will be clear from Table 1 that Ha-Ras is expressed in all the cellular organelles. Thus it could be argued that Ha-Ras antibody worked better than the others. The specificity of the Ha-Ras antibody is confirmed by the fact that areas such as the cytoplasm in the tubules and the endothelium in the glomeruli show no Ha-Ras staining. In addition, there are two distinct patterns of Ras isoform staining. First, in some areas [such as mesangial cell (cytoplasm and nucleus), foot processes and PCT mitochondria], Ha-Ras is the predominant isoform present and there is a minimal amount of the other isoforms. Secondly, in other areas, such as PCT brush border and DCT mitochondria, there is a hierarchy of the Ras isoforms, with Ha-Ras>N-Ras
Ki-Ras.
The key known function of Ras is to transduce messages from the cell surface receptors to Raf-1- and mitogen-activated protein kinases (Raf-MAP kinase) and PI-3 kinase cascades, leading to varied and contrasting end-points such as migration, adhesion, proliferation and apoptosis [11,12]. The differences in the functional roles of Ras isoforms can be postulated to be due to a combination of differential transcription, post-translational modifications, membrane trafficking routes and spatio-temporal membrane localization. Voice et al. have shown that Ras isoforms differ in their ability to activate Raf-1 serine/threonine kinase [12]. They also showed differences in the ability of each Ras isoform to induce transformed foci, enable anchorage-dependent cell growth and stimulate cell migration, with differences amongst the various cell types studied. Ki-Ras is a more effective as a recruiter and activator of Raf-1, while Ha-Ras recruits PI 3-kinase efficiently [3]. Since PI 3-kinases play an important role in prevention of apoptosis, Ha-Ras is believed to be an anti-apoptotic signalling molecule [3,13]. Thus the abundance and predominance of Ha-Ras in various kidney cell types such as podocytes, mesangial cells and convoluted tubules (proximal and distal) suggests that in these cells, which are non-proliferative stationary cells, Ha-Ras is a survival signal. The absence of Ha-Ras in these cells could thus be a hallmark of disease states leading to either apoptosis or cytoskeletal disarray. Specific alterations in Ras expression have been reported in glomerulonephritis (GN), and these changes include a reduction in mesangial cell Ha-Ras in both membranous GN and non-IgA mesangioproliferative GN [5].
Ras trafficking to the presumed final site of activity (plasma membrane) is dependent on post-translational modification. However, in this observational study, there is very little evidence of predominant expression of Ras in the plasma membrane (except in PCT, discussed below). This appears surprising, but a closer examination of the trafficking pathway and recent functional data are consistent with the findings seen here [14]. Ha- and N-Ras are modified differently from Ki-Ras and hence are targeted to different domains in the plasma membrane. Whilst Ha- and N-Ras pass through the endoplasmic reticulum and Golgi apparatus, the main modification for Ki-Ras takes place in the cytosol and, when microtubules are disrupted, Ki-Ras fails to reach the plasma membrane [14,15].
Ras in mitochondria
The localization of Ras in the mitochondria as seen in the PCT and DCT must be distinguished from Ras in the cytoplasmic vesicles. However, these structures were confirmed to be mitochondria at lower magnification where the morphology is clearer (data not shown). Thus the finding of Ras in mitochondria appears secure and may indicate that it is a survival signal [16]. An example of differential spatial distribution is the fact that in the interleukin-2 (IL-2)-deprived murine T-cell line TS1
ß, Ha-Ras localizes to mitochondria, whereas in IL-2-stimulated cells, Ki-Ras localizes to mitochondria [16]. N-Ras was detected in mitochondria under both experimental conditions. Bcl-2 and Ras proteins interact in the mitochondria of TS1
ß cells in the presence or absence of IL-2. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras post-translational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. Thus there is some evidence that in different circumstances such as growth promotion or apoptosis, Ras isoforms could be differentially targeted to mitochondria.
Ras in the glomerulus
In the glomerulus, there is mesangial cell expression of Ki- and Ha-Ras with little evidence of N-Ras expression. This may represent a survival signal as discussed above. There is abundant Ha-Ras in the podocyte foot processes with very little expression of Ki- and N-Ras, and this is perhaps a surprising finding. Ha-Ras in podocytes may have a role in ensuring the attachment of the foot processes to the basement membrane. An example of Ras in podocytes is the Thy-1 rat glomerulonephritis model. Thy-1 nephritis has been associated with an increase in glomerular expression of Ki-Ras and N-Ras isoforms [17]. This change in isoforms may reflect the podocyte injury seen in the Thy-1 model [18]. Podocytecell matrix interactions have been shown to be mediated in part by
(3)ß(1)-integrin heterodimers. Disturbances of integrin matrix interaction lead to detachment of podocytes in vitro, corresponding to the critical event of foot process retraction and glomerular basement membrane (GBM) denudation in vivo [19]. In leukaemic T cells, it has been shown that Ha-Ras regulates integrin behaviour through cytoplasmic activity and thus affects adhesion of these cells [20]. A similar mechanism may operate in the glomerular podocytes.
This work furthers our previous efforts to localize Ras isoforms in the renal intra-cellular organelles [5]. It will form the basis of functional studies of Ras isoforms and roles in renal cells with the objective of therapeutic targeting of Ras [8]. At present, the functional state of Ras in renal cells cannot be determined, as our data do not distinguish Ras-GTP from Ras-GDP. This will be an important goal of future functional work.
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Acknowledgments
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We are most grateful to Anne Clarke (Oxford) for initial help in developing the electron microscopy work, and Gordon Muir who provided the patient details as well as the tissues from those on whom he operated.
Conflict of interest statement. None declared.
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References
|
---|
- Downward J. Ras signalling and apoptosis. Curr Opin Genet Dev 1998; 8: 49[CrossRef][ISI][Medline]
- El-Nahas AM, Muchaneta-Kubara EC, Essawy M, Soylemezoglu O. Renal fibrosis: insights into pathogenesis and treatment. Int J Biochem Cell Biol 1997; 29: 5562[CrossRef][ISI][Medline]
- Yan J, Roy S, Apolloni A, Lane A, Hancock JF. Ras isoforms vary in their ability to activate Raf-1 and PI3 kinase. J Biol Chem 1998, 273: 2405224056[Abstract/Free Full Text]
- Yamamoto T, Harad N, Kano K et al. The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J Cell Biol 1997; 139: 785795[Abstract/Free Full Text]
- Kocher HM, Codd J, Sharpe CC, Dockerell MEC, Al-Nawab M, Hendry BM. Expression of Ras GTPases in normal kidney and glomerulonephritis. Nephrol Dial Transplant 2003; 14: 848854
- Sharpe CC, Dockrell ME, Scott R et al. Evidence of a role for Ki-Ras in the stimulated proliferation of renal fibroblasts. J Am Soc Nephrol 1999; 10: 11861192[Abstract/Free Full Text]
- Sharpe CC, Dockrell MEC, Noor MI, Monia BP, Hendry BM. The role of Ras isoforms in the stimulated proliferation of human renal fibroblasts in primary culture. J Am Soc Nephrol 2000; 11: 16001606[Abstract/Free Full Text]
- Khwaja A, Conolly JO, Hendry BM. Prenylation inhibitors in renal disease. Lancet 2000; 9205: 741744
- Armbruster BL, Carlemalm E, Chiovetti R et al. Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc 1982; 126: 7785[ISI][Medline]
- Kocher HM, Senkus R, Moorhead J et al. Differential expression of Ras isoforms in normal and malignant human pancreas. Pancreatology (2005) Vol 5
- Ruether GW, Der CJ. The Ras branch of small GTPases: Ras family members don't fall far from the tree. Current Opin Cell Biol 2000; 12: 157165[CrossRef][ISI][Medline]
- Voice JK, Klemke RL, Le A, Jackson JH. Four human Ras homologs differ in their abilities to activate Raf-1, induce transformation and stimulate cell motility. J Biol Chem 1999; 274: 1716417170[Abstract/Free Full Text]
- Marte BM, Downward J. PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem Sci 1997; 22: 355358[CrossRef][ISI][Medline]
- Ivanov IE, Philips MR. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 1999; 98(1): 6980
- Apolloni A, Prior IA, Lindsay M, Parton RG, Hancock JF. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol 2000; 20: 24752487[Abstract/Free Full Text]
- Rebollo A, Perez-Sala D, Martinez A. Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis. Oncogene 1999; 18: 49304939[CrossRef][ISI][Medline]
- Clarke HC, Kocher HM, Khwaja A, Kloog Y, Cook HT, Hendry BM. Ras antagonist farnesylthiosalicylic acid (FTS) reduces glomerular cellular proliferation and macrophage number in rat thy-1 nephritis. J Am Soc Nephrol 2003; 14: 848854[Abstract/Free Full Text]
- Smeets B, Dijkman HB, te Loeke NA et al. Podocyte changes upon induction of albuminuria in Thy-1.1 transgenic mice. Nephrol Dial Transplant 2003; 18: 25242533[Abstract/Free Full Text]
- Kretzler M. Regulation of adhesive interaction between podocytes and glomerular basement membrane. Microsc Res Tech 2002; 57: 247253[CrossRef][ISI][Medline]
- Tanaka Y, Minami Y, Mine S et al. H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells. J Immunol 1999; 163: 62096216[Abstract/Free Full Text]
Received for publication: 2. 7.04
Accepted in revised form: 21. 1.05