Expression of Ras GTPases in normal kidney and in glomerulonephritis

Hemant M. Kocher1, Jane Moorhead2, Claire C. Sharpe3, Mark E. C. Dockrell3, Mashal Al-Nawab2 and Bruce M. Hendry3

1Department of Surgery, 2Histopathology and 3Renal Medicine, King’s College Hospital, Guy’s King’s and St Thomas’ School of Medicine, King’s College London, London, UK

Correspondence and offprints requests to: Bruce M. Hendry, Department of Renal Medicine, King’s College London, Bessemer Road, London SE5 9PJ, UK. Email bruce.hendry{at}kcl.ac.uk



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Small monomeric Ras GTPases play critical and specific roles in the control of cellular proliferation and apoptosis but the expression of the three Ras isoforms (Ha-Ras, Ki-Ras and N-Ras) in human renal tissue is unknown. This work is an immunohistochemical study of Ras expression in normal renal tissue and in membranous glomerulonephritis (MGN), IgA nephropathy (IgAN) and IgA-negative mesangioproliferative glomerulonephritis (MPGN).

Methods. Formalin-fixed, paraffin-embedded tissue was stained using pan-Ras monoclonal antibody (mAb) and Ras isoform-specific mAb. Detection employed a (DAKO Envision) modified polymer system.

Results. The expression of Ras isoforms in normal human kidney was cell-specific. For example, N-Ras was detected in tubule epithelial cells but not in glomerular or interstitial cells. Ki-Ras was expressed in mesangial cells, interstitial cells and in proximal convoluted tubule cells (PCT) (particularly localized at brush borders) and in collecting duct cells (CD) (localized to cell membranes) but not in podocytes. Cytoplasmic Ha-Ras was detected in all the above cell types except podocytes. MGN was associated with podocyte expression of all three Ras isoforms and with reduced mesangial cell expression of Ha-Ras and Ki-Ras. IgAN was characterized by podocyte expression of Ha-Ras (but not Ki-Ras) and reduced mesangial cell expression of Ki-Ras without alterations in mesangial Ha-Ras expression. MPGN was associated with reduced mesangial cell Ha-Ras and Ki-Ras expression without significant podocyte Ras expression.

Conclusion. These disease-specific and isoform-specific alterations in Ras expression may be of significance in pathogenesis and warrant further functional investigation.

Keywords: brush border; IgA nephropathy; membranous glomerulonephritis; mesangial cell; mesangioproliferative glomerulonephritis; podocyte



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cellular events in the pathogenesis of glomerulonephritis include proliferation, migration, adhesion and apoptosis. Different forms of nephritis are characterized by dysregulation of specific cell types but the signalling changes, which underlie these processes, are not fully understood. The Ras family of small monomeric GTPases are known to play a central role in the signalling pathways, which control cell survival, differentiation, migration and proliferation [1]. Ras is activated by a range of cytokines including receptor tyrosine kinase ligands such as fibroblast growth factor, epidermal growth factor and platelet-derived growth factor and by other cytokines implicated in renal pathology such as transforming growth factor-ß (TGF-ß) and endothelin-1 [2,3]. The downstream effectors of Ras include the Raf-MAP kinase cascades, Rho family GTPases and the PI 3-kinases giving Ras a pivotal role in the control of cell function [4]. Most work on Ras has been in the context of cancer pathogenesis as mutant forms are potent transforming oncogenes expressed in 20–30% of human cancers [5]. Ras is known to be expressed in normal renal tissue [1] but the differential expression of the three isoforms (Ha-Ras, Ki-Ras and N-Ras) has not been described and Ras has not been studied in renal disease.

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 [6]. 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 [79]. 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.

This work is an immunohistochemical survey of the expression of Ras isoforms in normal renal tissue and in membranous glomerulonephritis (MGN), IgA nephropathy (IgAN) and in IgA-negative mesangioproliferative glomerulonephritis (MPGN). These pathologies were chosen as each is associated with alterations of glomerular cell proliferation and/or apoptosis which might be expected to involve altered Ras function [3]. The results demonstrate cell-specific expression of Ras isoforms in the normal kidney and disease-specific alterations in Ras expression in nephritis.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Monoclonal antibodies (mAb) against pan-Ras (clone Ras10), Harvey (clone 235-1.7.1), Kirsten (clone 234-4.2) and Neural (clone F155-277) isoforms of Ras were obtained from Oncogene Research Products, Cambridge, MA, USA. The mAb targeting the Ki, Ha and N-Ras isoforms were confirmed to be specific and without cross-reactivity using dot blot analysis as described previously [7]. Other reagents used were pepsin, saponin, fish skin gelatin and Tween 20 (polyoxyethensorbitan monolaurate) (all from Sigma Chemical Co., St Louis, MO). A polymer system labelled with goat anti-mouse secondary antibodies was used to obviate the background from the endogenous biotin in human kidney [DAKO EnvisionTM + system, HRP (DAB) from DAKO Corporation, CA].

Specimens
Six archival specimens of normal human kidney were retrieved using the pathology database of the hospital. The histo-morphological appearances of the samples were normal. The kidneys were removed for renal cell carcinoma, where the opposite normal pole of kidney was used (n = 3) or those kidneys at risk for repeated infection (n = 3), where the non-affected part of kidney was used. Biopsy specimens of pathological (MPGN, n = 4; MGN, n = 6; IgAN, n = 4) kidneys were similarly retrieved. The inclusion criterion was creatinine <200 µM/l (2.5 mg/dl) at the time of biopsy, to avoid possible confounding effects of severe uraemia on Ras expression.

Protocol
The antibodies were used at various dilutions (1/10 to 1/4000) to obtain optimum signal-to-background ratio. Initially no pre-treatment was used but as recommended by the manufacturer the best treatments were found by varying dilutions of pepsin (0.05%, 0.1%), saponin (0.05%). The washes were tried with differing ingredients including fish skin gelatin (0.005% in TBS) and/or Tween (0.05% in TBS). Various incubation times were used and the best result was with overnight incubation at 4°C. The protocol below was designed to attain the best signal-to-background ratio. Skin, neural tissue and breast cancer specimens were tested with the mAb. Skin uniformly gave best results for pan Ras and all its isoforms and was the positive control used (as recommended by the mAb manufacturer).

Serial sections of 4 µm thickness were cut from formalin-fixed paraffin-embedded sections and incubated overnight at 60°C. They were then deparaffinized in xylene and rehydrated in alcohol and finally tap water. All incubations were at room temperature (22°C), unless stated otherwise. Sections were treated with 0.05% saponin for 30 min. After a thorough wash in tap water for 5 min, for isoform-specific mAb, the sections were digested with 0.1% pepsin at a pH of 2.3 for 20 min. After a further wash, the 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. Incubation with primary antibody (dilutions pan Ras 1/2000, H-Ras, 1/500, Ki-Ras 1/10, N-Ras 1/100) was at 4°C overnight (16–20 h). After returning to room temperature and washing as mentioned above, incubation with labelled polymer second layer was carried out for 30 min. Colour was developed using liquid Diaminobenzidine substrate chromogen system and counter-stain was done with haemotoxylin. 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. Immunohistochemistry using epithelial membrane antigen was used to differentiate between proximal and distal convoluted tubule [10].

Interpretation
Two independent observers who were blind of the type of antibody or the pathology of the specimen used, interpreted the staining pattern as negative (–), marginal (±), mild (+), moderate (++) and intense (+++) for each cell type for each section. These staining patterns were scored 0–4, respectively. Statistical analysis was by Mann–Whitney U-test to compare respective cell types between normal and pathological kidney, with a significance level at P < 0.05. Cell types studied were proximal convoluted tubule cells (PCT), distal convoluted tubule cells (DCT), collecting tubule cells (CT), collecting duct cells (CD), interstitial cells, subcapsular fibroblasts (SCF), glomerular mesangial cells and podocytes. Also, any particular distribution of the staining pattern such as cytoplasmic, brush border or cell membrane level was noted.



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 Materials and methods
 Results
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 References
 
Normal kidney
Ras protein expression as detected by pan-Ras mAb, was seen in glomerular cells, PCT cells, distal convoluted tubule cells, CT cells, CD cells, interstitial cells and SCF. All staining in the normal tissues was cytoplasmic or of the plasma membrane, with no nuclear staining seen. Examples of staining with the isoform-specific Ras mAb is shown in Figure 1 and a semi-quantitative analysis of the cellular expression of Ras isoforms is summarized in Table 1.



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Fig. 1. Ras isoform immunohistochemical staining according to kidney cellular structure. (A) Renal cortex. Glomerular cells (arrows) show cytoplasmic stain with Ha-Ras and Ki-Ras but not N-Ras. Ha-Ras is expressed in proximal and distal convoluted tubules (*PCT and #DCT) equally in the cytoplasm. Ki-Ras mAb shows faint staining of DCT but in PCT (*) it shows distinct brush border accentuation. With N-Ras, there is a stronger cytoplasmic stain of DCT cells (#) as compared with PCT cells (*). (B) SCF (arrow) showing cytoplasmic staining with Ha-Ras and Ki-Ras isoforms but not with N-Ras isoform. (C) Interstitial cells (arrows) demonstrating positive cytoplasmic stain for Ha-Ras and Ki-Ras isoforms but not for N-Ras isoform. (D) Renal medulla. Ha-Ras and N-Ras mAb show cytoplasmic staining of the CD cells while Ki-Ras mAb shows some cytoplasmic stain with cell membrane accentuation (arrowed). CTs show some cytoplasmic expression of Ha-Ras and N-Ras (Ki-Ras not shown for CT). All magnifications are 1000x, except (A), where they are 400x.

 

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Table 1. Immunohistochemical staining for Ras isoforms in normal renal tissue

 
The expression of Ras isoforms was cell-specific. Mesangial cells stained positively for Ha-Ras and Ki-Ras but not N-Ras (Figure 1A and Table 1). There was no evidence of podocyte expression of Ha-Ras, Ki-Ras or N-Ras. Figure 1A shows the staining pattern of each isoform in the renal cortex. PCT cells expressed Ki-Ras predominantly (particularly localized to the brush border). PCT also expressed Ha-Ras and N-Ras. Distal convoluted tubule (DCT) cells showed predominant expression of N-Ras and showed some expression of Ki-Ras and Ha-Ras.

The expression of the Ras isoforms in SCF is shown in Figure 1B; Ki-Ras and Ha-Ras were expressed in the SCF cytoplasm but N-Ras was not. Figure 1C similarly shows expression of the Ras isoforms in the interstitial cells. The position and morphology of these cells indicated that they are most likely to be interstitial fibroblasts. These cells also expressed Ki-Ras and Ha-Ras but not N-Ras.

Figure 1D demonstrates the staining pattern of each Ras isoform in the renal medulla. CD cells showed predominant expression of Ki-Ras (particularly localized to the cell membrane) with some expression of the other isoforms. CT cells demonstrated some expression of Ki-Ras, Ha-Ras and N-Ras (Ha > N ~ Ki). The pattern of staining of Ras isoforms in the cells of loops of Henle and vasa recta could not be established.

Figure 2 demonstrates the particular subcellular distribution of Ki-Ras, in comparison with other isoforms (example of N-Ras). In the PCT cells there is a distinct brush border accentuation with Ki-Ras, as compared with the DCT. This characteristic brush border distribution was not seen for Ha-Ras or N-Ras. Similarly in the CD cells there was plasma membrane accentuation with Ki-Ras, not seen with other isoforms or tubules (Figure 1D).



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Fig. 2. The differences in the staining patterns of Ki-Ras (A) and N-Ras (B) in PCT (*) and DCT (#). Brush border accentuation is seen with Ki-Ras in PCT. Magnification is 1000x.

 
Ras expression in pathological kidney
The clinical characteristics of patients with MGN, IgAN and MPGN are presented in Table 2. At the time of renal biopsy no patients were on treatment with immunosuppressive agents, HMG-CoA reductase inhibitors or angiotensin receptor antagonists. All four patients in the IgA group were on angiotensin converting enzyme inhibitors (ACEI); none of the other patients were on ACEI. Examples of the patterns of glomerular expression for Ha-Ras, Ki-Ras and N-Ras in the patients with GN are shown in Figures 3–5, respectively.


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Table 2. Clinical characteristics of patients with pathological kidney biopsy specimens

 


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Fig. 3. Examples of the patterns of Ha-Ras expression in MGN, IgAN and MPGN compared with normal kidney.

 


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Fig. 4. Examples of the patterns of Ki-Ras expression in MGN, IgAN and MPGN compared with normal kidney.

 


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Fig. 5. Examples of the patterns of N-Ras expression in MGN, IgAN and MPGN compared with normal kidney.

 
MGN
There were marked and significant changes in glomerular expression of the Ras isoforms in MGN as compared with the normal renal tissue. Podocytes showed increased staining with pan-Ras mAb and all three isoforms are clearly expressed in podocytes in MGN. An analysis of the expression of Ras in podocytes is summarized in Figure 6. Mesangial cell expression of Ras appeared to be decreased in MGN. The data for pan-Ras mAb, Ki-Ras mAb and Ha-Ras mAb showed significant reductions in mesangial cell expression in MGN compared with normal kidney as summarized in Figure 7. N-Ras was not detected in mesangial cells in MGN.



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Fig. 6. Summary of podocyte expression of the Ras isoforms in the disease studies compared to normal tissue. (A) pan Ras mAb, (B) Ha-Ras, (C) Ki-Ras, (D) N-Ras. The plots are ‘Box and Whiskers’ of median, range and quartiles. Significant differences from the results for normal kidney are indicated (*P < 0.05, Mann–Whitney U-tests).

 


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Fig. 7. Summary of mesangial cell expression of the Ras isoforms in the disease studies compared with normal tissue. (A) pan Ras mAb, (B) Ha-Ras, (C) Ki-Ras, (D) N-Ras. The plots are ‘Box and Whiskers’ of median, range and quartiles. Significant differences from the results for normal kidney are indicated (*P < 0.05, Mann–Whitney U-tests).

 
IgAN
Mesangial cell expression of Ki-Ras was significantly reduced in IgAN: data for Ha-Ras and pan-Ras mAb were not different from normal kidney (Figure 7). Mesangial cell N-Ras staining was occasionally seen in IgAN in contrast to normal tissue, but this could not be deemed significant. In IgAN there was significantly increased staining of podocytes with pan and Ha-Ras mAb (both P < 0.02). Podocytes also showed a non-significant increase in N-Ras expression but no evidence of Ki-Ras expression.

MPGN
Mesangial cell expression of Ha-Ras was reduced in MPGN compared with normal kidney, in distinction to the data for IgAN (Figure 7). The mesangial expression of Ki-Ras also appeared reduced in MPGN but this did not reach significance (P = 0.065) compared with normal kidney. N-Ras was not detected in mesangial cells in MPGN. Podocyte expression of Ras isoforms in MPGN was very low and almost identical to normal kidney. In this MPGN was strikingly different from both MGN (podocyte expression of all isoforms) and IgAN (podocyte expression of Ha-Ras) as summarized in Figure 6.



   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ras isoform cell biology
These results demonstrate the cell-specific expression of the Ras isoforms in normal human tissue and isoform-specific changes in renal disease. Ras proteins are involved in complex cellular signalling pathways with multiple upstream effectors and downstream cascades controlling cellular differentiation, proliferation and apoptosis [11]. They also play a role in cytoskeletal organization. There is growing recognition that the three different isoforms of Ras may be functionally distinct [12].

In experiments on NIH 3T3 mouse fibroblasts and RIE-1 rat intestinal cells, rat-1 fibroblasts and COS-7 cells, Voice et al. [13] have shown that Ras isoforms differ in their ability to activate Raf-1 serine/threonine kinase. 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 cell types studied.

Ki-Ras is more effective as a recruiter and activator of Raf-1, while Ha-Ras recruits PI 3-kinase efficiently [6,14]. There is additional evidence that the Ras isoforms have different transforming potential, resulting in cell-specific differences [15]. In fibroblasts, Ha-Ras has greater transforming potential than the other isoforms, whereas in multi-potent haemopoietic stem cells, N-Ras has greater biologic activity. MAP kinase activity is regulated by different mechanisms by Ha-Ras and N-Ras [16]. Ha-Ras is believed to be vital in prevention of apoptosis [3,6].

The differences in Ras isoforms may be related to the divergence of their trafficking through the cell organelles to dissimilar parts of plasma membrane. Ha-Ras is directed to lipid rafts and Ki-Ras to disordered plasma membrane. This is believed to be due to varied post-translational modifications of the Ras isoforms; in particular Ha-Ras is palmitoylated near the C-terminal while Ki-Ras has a cationic polylysine motif. These molecules therefore take part in different hydrophobic and electrostatic interactions [17]. Similar differences in the post-translational modifications of Rho proteins also appear to be critical determinants of localization and function [18].

Renal roles for Ras—normal tissue
Ras isoform expression in normal kidney varies with cell type. In the glomerulus there is mesangial cell expression of Ki-Ras and Ha-Ras with little evidence of N-Ras expression. There is very little expression of Ras in podocytes. All three isoforms are expressed in tubule epithelial cells in a cytoplasmic distribution except for the characteristic presence of Ki-Ras in the brush border of PCT and the cell membrane of the CD, not present in other tubule segments or seen with other isoforms, suggesting a functional role for Ki-Ras in that position. The fact that PCT express Ki-Ras predominantly and DCT express N-Ras predominantly, whilst Ha-Ras is present in equivalent quantities, suggests differential function for these isoforms in these tubule segments. Interstitial cells and SCF express Ki-Ras and Ha-Ras but not N-Ras. Previous in vitro work from our laboratory in human renal fibroblasts and mesangial cells showed critical and distinct roles for Ki-Ras and Ha-Ras in cytokine-stimulated proliferation without a functional role for N-Ras [7,8,19]. Furthermore, it is reported that lipoxin, leukotrienes and PDGF exhibit cross-talk via Ras to regulate mesangial cell proliferation [20]. Seufferlein et al. [21] have shown that in pancreatic cancer cell lines possessing oncogenic Ki-Ras mutations, TGF{alpha}-stimulated cell growth is dependent on activation of Ha-Ras. They demonstrated that TGF{alpha} stimulation increases the membrane translocation and activation of Ha-Ras and these cells use the Ha-Ras–ERK pathway to proliferate. This dependence on Ha-Ras in pancreas cancer is surprising in view of the clear co-expression of oncogenic Ki-Ras in these cells. These in vitro experiments on renal fibroblasts and pancreatic cancer cells strongly support the concept of distinct functional roles for the Ha-Ras and Ki-Ras isoforms.

The apical brush border localization of Ki-Ras protein in the PCT shown in the present work may be of functional significance and we speculate that Ki-Ras may be associated with the Na+ /H+ exchanger (NHE-3). NHE are crucial in maintaining the intra-cellular pH and also facilitate absorption in specialized intestinal and renal tubule cells [22]. NHE-1 is known to be controlled by Ras GTPases in addition to other GTPases like Ga13 through separate Cdc42-dependent and RhoA-dependent pathways [23]. The mechanisms controlling NHE-3 are not fully understood and the possible relation between Ki-Ras and NHE-3 deserves further study. The presence of only Ki-Ras in the apical brush border of the PCT, and not Ha-Ras or N-Ras suggests a functional role of Ki-Ras in sodium transport. The ubiquitous presence of Ha-Ras in all renal cell types shown in the present experiments is consistent with a role as a survival signal via its preferential activation of PI 3-kinases [6,14,24,25].

Renal roles for Ras pathology
There were specific alterations in Ras expression in the three pathologies studied here. In MGN the glomerular podocyte expression of all Ras isoforms was strikingly increased compared with normal kidney. In IgAN there was increased podocyte Ha-Ras but not Ki-Ras whilst in MPGN podocyte Ras expression was not different from normal kidney (Figure 6). Mesangial cell changes in Ras expression were quite different. Mesangial Ha-Ras and Ki-Ras were reduced in MGN and MPGN; mesangial Ki-Ras alone was reduced in IgAN (Figure 7).

The significance of this up-regulation of podocyte Ras in MGN is unclear. Ras could be involved in the regulation of cellular apoptosis or of cellular matrix secretion and might play a role in pathogenesis in MGN or alternatively the increase in Ras could be an adaptive response. It is also possible that alterations in Ras are secondary and occur in response to the proteinuria, which is a prominent feature of MGN. In the present work there was no significant correlation between proteinuria (or plasma creatinine) and the changes in glomerular Ras expression in any single disease or in the pooled subjects (data not shown). This suggests that the alterations in Ras are not simply a result of proteinuria or renal failure per se.

IgAN was characterized by increased podocyte Ha-Ras and decreased mesangial cell Ki-Ras expression compared with normal kidney. In MPGN the most prominent changes were decreased mesangial Ha-Ras and Ki-Ras and the podocytes expressed some Ha-Ras. Human mesangial cells in vitro require both Ki-Ras and Ha-Ras in order to undergo PDGF-induced proliferation [19]. The down-regulation of mesangial Ras in IgAN and MPGN may therefore be an adaptive response, which limits mesangial cell cycle progression in these nephropathies. If so, this response is different in the two conditions. Further work in animal models characterized by increased mesangial cells numbers (e.g. Thy-1 nephritis) and in the uteroglobin-deficient mouse, which exhibits a form of IgAN, would help to test this.

Outlook and limitations
In summary, these results demonstrate the specific cellular expression of Ras isoforms in normal human kidney and characteristic modifications of this expression in three glomerular diseases. It is important to note that the functional roles for Ras have not been elucidated by this work. Ras expression and function may be modulated by a disease process or by therapy. In the present work the altered Ras expression in IgAN could be related to the use of ACEI in these patients, although this cannot explain the changes seen in MPGN and MGN. Nevertheless, these results justify further study of the functional role of individual Ras isoforms in renal disease.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 23.12.02
Accepted in revised form: 21. 5.03