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. 2014 May 7;95(4):244–250. doi: 10.1111/iep.12074

Evidence for aldosterone-dependent growth of renal cell carcinoma

Sharon King 1, Susan Bray 1, Sarah Galbraith 1, Lesley Christie 1, Stewart Fleming 1
PMCID: PMC4170966  PMID: 24802662

Abstract

The aim if this study was to investigate the hypothesis that K-RAS 4A is upregulated in a mineralocorticoid-dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K-RAS in renal tumour tissues and cell lines was examined by real-time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K-RAS4A and for the effect on K-RAS expression of spironolactone blockade of the mineralocorticoid receptor. K-RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K-RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K-RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K-RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K-RAS expression. K-RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K-RAS acting through the Akt and Raf pathways.

Keywords: aldosterone, K-RAS, mineralocorticoid receptor, renal cell carcinoma

Renal cancer is a common malignancy with almost 10,000 cases per annum in the UK. There have been considerable advances in our understanding of the molecular biology of renal cancer (Fleming 1999), in particular the genetic alterations considered causative. Indeed this genetic understanding underpins (Fleming 1993) the current classification of renal cancer. Mutation or inactivation of the von Hippel–Lindau gene (Foster et al. 1994; Gnarra et al. 1994) with a subsequent second somatic event, often deletion of the short arm of chromosome 3 (3p-), is associated with the development of clear cell carcinoma through the deregulation of the hypoxia inducible pathways (Wykoff et al. 2000). There are a number of other genes altered in different tumour types with a strong genotype–phenotype correlation (Schmidt et al. 1997; Alam et al. 2005; Housley et al. 2010).

It is unclear how these genetic changes are influenced by the known risk factors for renal cancer. Both hypertension and increased BMI are associated with an increased risk of the development of RCC (Chow & Devesa 2008; Pascual & Borque 2008; Stojanovic et al. 2009), and the most recent studies, from the USA and Eastern Europe, have shown that this increased risk is most marked for the clear cell form of RCC (Purdue et al. 2013). Further data suggest that this increased risk involves the activity of the renin–angiotensin–aldosterone system (RAAS). Epidemiological evidence from the MRC Blood Pressure Unit in Glasgow has shown that pharmacological suppression of the RAAS lowers the risk of developing renal cancer in a hypertensive patient to that of a non-hypertensive individual whereas treatment with alternative hypotensive agents was not protective (Lever et al. 1998, 1999). In experimental models, inhibition of angiotensin converting enzyme restricts growth of human renal cancer cells in a mouse xenograft system (Hii et al. 1998), and in vitro restores sensitivity of mouse Renca cells to the growth-suppressing effects of TGFβ (Miyajima et al. 2001). Furthermore, in several experimental animal models of hypertension, overactivity of the RAAS is accompanied by renal tubular hyperplasia (Whitworth et al. 1994; Montgomery et al. 1998; Kotelevtsev et al. 1999; Kantachuvesiri et al. 2001; Masuzaki et al. 2003). Similar renal tubular hyperplasia was seen in a transgenic mouse model of metabolic syndrome in which overexpression of 11β-hydroxysteroid dehydrogenase-1 in adipocytes led to abdominal obesity and angiotensin-dependent hypertension (Masuzaki et al. 2003). The precise biology of these effects of the RAAS on proliferation and survival of renal tubular epithelium and renal cancer cells is unclear, but one potential mechanism is the influence of the RAAS on the expression and activity of the K-ras cellular oncogene to promote tumour growth.

K-ras exists in two alternative splice isoforms: K-RAS4B which in mice and humans is ubiquitously expressed; and 4A which is confined to renal, colonic, lung and a small range of other transporting epithelia (Plowman et al. 2003, 2006). There is good evidence from amphibian models, especially the A6 toad bladder cell line, that K-RAS4A is involved in aldosterone-dependent sodium transport via the epithelial sodium co-transporter (ENaC). In this model, K-RAS4A expression is induced by aldosterone treatment, its activation is sustained by as yet unproven tyrosine kinases but possibly involving epidermal growth factor or insulin-like growth factor 2 receptors (Tong & Stockand 2005), and K-RAS4A links the phosphatidylinositol 3-kinase pathway to ENaC activity (Stockand 2002; Staruschenko et al. 2004, 2005; Tong et al. 2004). However, K-ras is also involved in cell growth promotion. Aldosterone sensitive growth responses in the cardiovascular system have been shown to be mediated via K-ras induction and activation (Stockand & Meszaros 2003). Our hypothesis is that K-RAS4A is aldosterone sensitive in renal cancer and that its overexpression contributes to survival and increased proliferation of renal cancer cells in response to activation of the RAAS.

Materials and methods

Renal cell carcinoma

Samples of clear cell renal cell carcinoma were obtained from the Tayside Tissue Bank. These had been obtained at nephrectomy, snap frozen and stored at −80 °C. Prior to use frozen sections were cut and examined to confirm the tumour histology and that there was no normal kidney tissue present. The tissue was homogenized and protein and RNA extracted as below.

Ethical approval

Research on all tissue samples was approved by the Research Ethics Committee of the NHS Tayside.

Tissue microarray construction

A mini-tissue microarray was constructed from 27 clear cell renal carcinomas held in the Tayside Tissue Bank. Haematoxylin and eosin stained slides which had representative tumour areas marked were selected. A manual tissue arrayer (Beecher Instruments Inc., Sun Prairie, WI, USA) was used to bore cores of tissue from the paraffin blocks as designated on the slides and transfer them into a recipient paraffin block with 4–6 cores, each 0.6 mm in diameter, being donated from each tumour.

Immunohistochemical staining

Sections from the TMA block (nominally 4-μm thick) were used for immunocytochemistry following microwave antigen retrieval before being immunostained on a DAKO Autostainer (Dako, Ely, UK) using Vectastain ABC kits (Vector Labs, Peterborough, UK) according to the manufacturer's protocol. The primary antibodies used for immunostaining are described below. Sections were then incubated with either biotinylated anti-rabbit (for polyclonal antibodies) or anti-mouse (for monoclonal antibodies) antibody for 30 min followed by Vectastain Elite ABC (Vector Labs) reagent for another 30 min. Liquid diaminobenzidine (DAB) (DAKO) was used as a chromogenic agent for 5 min, and sections were counterstained with Mayer's haematoxylin.

Primary antibodies

11βHSD2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used at a dilution of 1/200 for the immunocytochemistry on a standard protocol as described above. The mineralocorticoid receptor antibody (Abcam, Cambridge, UK) was used at a dilution of 1/100 with an overnight incubation at 4 °C. Normal renal cortex was used as the positive control.

Microscopy

Sections were viewed on a Nikon Eclipse 600 microscope (Nikon, Surrey, UK) with a Nikon DXM1200 digital camera. For scoring of immunohistochemistry, sections were scored by two participants and any discrepancies resolved by review on a double headed microscope. Sections were recorded as positive or negative.

Cell lines and cell culture

Cell lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were obtained from the European Collection of Cell Cultures. These are human renal cell carcinoma cell lines. RCC4 plus VHL cells are derived from a clear cell carcinoma of the kidney with a mutant VHL gene and deletion of chromosome 3p, transfected with a pcDNA3-VHL vector containing wild-type VHL gene correcting the functional VHL loss. RCC4 plus vector alone cells have been transfected with an empty vector, pcDNA3, and therefore remain defective in the VHL gene. The cells were grown in D-MEM medium (Life Technologies, Paisley, UK) containing glucose, L-glutamine and pyruvate as per the cell line supplier's guidelines. This was supplemented by 10% foetal bovine serum (Life Technologies) and geneticin. Cells were grown at 37 °C with 5% CO2, passaged with a 1:4 split every 3–4 days. For mineralocorticoid receptor blockade, spironolactone was added to a final concentration of 10 μM in keeping with previous studies (Miro et al. 2013).

K-RAS4A siRNA

siRNA specific to the 4A isoform of K-ras was rationally designed (Reynolds et al. 2004) by use of a design algorithm (http://www.dharmacon.com). Using this protocol, two K-RAS4A-specific siRNAs were obtained from Dharmacon, (Thermo Scientific, Loughborough, UK), plus a positive control siRNA for GAPDH and a negative control scrambled RNA were used (Ambion, Life Technologies, Paisley, UK). Cells were transfected with reconstituted siRNA silencing duplex or control diluted in culture medium to a final concentration of 10 μM. Intracellular delivery of siRNA was facilitated by the use of RNAiFect transfection reagent (Qiagen, Crawley, UK). Cells were cultured for 24–72 h following transfection. Cell numbers were counted using a haemocytometer.

RNA extraction

Cells were harvested and RNA extracted using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems, Life Technologies, Paisley, UK). Tissue samples were homogenized and RNA obtained by chloroform extraction in an EZ1 BioRobot (Qiagen); the RNA was purified using an EZ1 RNA Universal Tissue Kit (Qiagen). RNA integrity and derivation of an RNA integrity score was determined by microfluidic capillary electrophoresis according to manufacturer's instructions (Agilent Technologies, South Queensferry, UK). DNase1 treatment (Qiagen) was performed before the purified RNA was eluted in 5 μl of buffer and stored at −80 °C.

Reverse transcription–polymerase chain reaction

To allow investigation of the expression of each isoform of K-RAS, real-time reverse transcription–polymerase chain reaction (RT-PCR) was carried out. Primers were obtained from Invitrogen specific to K-RAS4A and 4B. Each forward primer was labelled with a FAM dye, a so-called LUX forward primer. Primer sequences were as follows:

  • K-RAS4A Forward (DNA) – CGG ATA CAT TGG TGA GGG AGA TC [FAM] G

  • K-RAS4A Reverse (DNA) – TTT CAC ACA GCC AGG AGT CTT T

  • K-RAS4B Forward (DNA) – CAC TCT TCT GGT GGC GTA GGC AAG AG [FAM] G

  • K-RAS4B Reverse (DNA) – TCC TCT TGA CCT GCT GTG TCG

We used a One-Step qRT-PCR kit from Invitrogen to lyse cells, digest DNA and reverse transcribe RNA into cDNA before amplification. Rnase-free DNase (Qiagen) was added to each lysate sample before incubating for 15 min at 37 °C. RNA was quantified using the Quant-It system (Invitrogen) according to manufacturer's instructions. Reverse transcription and polymerase chain reaction was performed using a Superscript 3 RT/Platinum Taq mix (Invitrogen, Life technologies, Paisley, UK) with LUX labelled forward primer and 40 cycles of polymerase reaction according to manufacturer's instructions.

Western blotting

Protein was extracted using CelLytic M (Sigma Aldrich, St Louis, MO, USA) and a protease inhibitor cocktail (Sigma) and after protein quantification using the bicinchonic acid method the lysate stored at −70 °C. Western blotting was performed on SDS-denatured protein using a 4–12% Nupage Bis-Tris Precast gel (Invitrogen,) in an Xcell SureLock Mini-Cell Electrophoresis kit (Invitrogen). Following protein transfer, the membrane was probed with primary antibody overnight and washed in TBS. The membrane was then probed in the appropriate dilution of secondary antibody. For visualization, 1 ml of Hybond ECL was applied onto the membrane followed by exposure to CL-Xposure Film (Perbio Science, Northumberland, UK) for 5 min.

Antibodies

The antibodies used for Western blotting are listed in Table 1.

Table 1.

Antibodies used in Western blotting

Antibody Source Dilution
K-ras clone 3B10-2F2 Abnova, Taiwan 1/1000
p-Akt (S473) Cell signaling, Now New England Biolabs, Hitchin, Hants, UK 1/250
p-Raf (S259) Cell signalling 1/1000
p-S6 (S235/236) Cell signalling 1/800
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Results

K-RAS4A expression is mineralocorticoid sensitive in renal carcinoma

Isoform-specific antibodies for K-ras are not available, so a reverse transcriptase–PCR was used to detect mRNA for K-RAS4A using primers specific for exon 4A in RNA extracted from renal cancer tissue and from RCC4 plus vector and RCC4 plus VHL cells. A PCR product of the predicted 100 bp size was identified in human tumours (Figure 1) and both cell lines (Figure 2). The identity of the PCR product with K-RAS4A was confirmed by direct sequencing using an ABIPRISM 3730 genetic analyser (Applied Biosystems, Life Technologies, Paisley, UK). K-RAS4B was ubiquitously expressed in all tissues sampled with a PCR product of the predicted 242 bp.

Figure 1.

Figure 1

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Expression of K-RAS4A in renal cell carcinoma. 1.5% agarose gel showing the presence of K-RAS4A in two examples of clear cell renal carcinomas, and the size of the PCR product was approximately 100 bp indicating K-RAS4A is expressed in renal carcinomas.

Figure 2.

Figure 2

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Expression of K-RAS4A in the RCC cell lines. Electrophoresis shows the presence of the 100 bp amplified K-RAS4A in three samples from RCC4 cells.

Basal culture conditions of the RCC4 cells included foetal bovine serum acting as a source of aldosterone, so receptor blockade with spironolactone was used to demonstrate sensitivity of K-ras to mineralocorticoid activation. Addition of 10 μM spironolactone to cultures of both RCC4 plus vector and RCC4 plus VHL cells showed that K-ras expression was significantly reduced by mineralocorticoid receptor blockade (Figure 3). These data support the hypothesis that K-ras expression in RCC cell lines is mineralocorticoid receptor sensitive.

Figure 3.

Figure 3

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Effect of spironolactone treatment on detection of K-RAS protein by Western blotting. Addition of spironolactone to cultured RCC4 cells results in reduced expression of K-RAS compared to basal culture conditions.

Immunocytochemistry was performed to confirm the presence of mineralocorticoid receptor and its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 in clinical RCC samples. Only 23 of 27 cases on the clear cell renal carcinoma tissue microarray were satisfactory for scoring because of the loss of some cores from the tissue microarray. Nuclear staining for MR was observed in 11 of 23, cytoplasmic staining for 11βhsd2 in 14 of 23, cases of RCC (Figure 4), and 10 of 11 MR positive tumours were also stained for 11βhsd2. A larger study will be required to assess the significance of MR expression for clinical outcome. In control sections of normal kidney, both MR and 11βhsd2 were seen in the epithelium of the distal tubule and cortical collecting ducts.

Figure 4.

Figure 4

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Staining (a) for the mineralocorticoid receptor seen in the nuclei of clear cell RCC and (b) for its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 on the cell membrane and peripheral cytoplasm in these immunoperoxidase preparations (IHC × 20 original objective magnification).

Renal cell carcinoma proliferation is supported by mineralocorticoid activation and K-RAS4A expression

Experiments were performed to determine the sensitivity of renal carcinoma cell growth in vitro to activation of the mineralocorticoid receptor. RCC4 plus vector and RCC4 plus VHL cells were grown in basal conditions or with 10 μM spironolactone. Cell counts were determined at 24, 48 and 72 h of these growth conditions. There was 6.5-fold reduction in the number of RCC4 plus vector cells (Figure 5) and a 6.3-fold reduction in numbers of RCC4 plus VHL following 72 h of culture in the presence of spironolactone. These data show that while under basal conditions there was continued cell proliferation, mineralocorticoid receptor blockade leads to an absolute reduction in cell number.

Figure 5.

Figure 5

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Effect on cell population by spironolactone treatment. Treatment of RCC4 plus vector cell cultures led to a 6.5-fold reduction in cell number over 72 h when compared to basal culture conditions. A similar 6.3-fold reduction was seen in RCC4 plus VHL cells (Data not shown).

In cells cultured under basal conditions, K-RAS4aA mRNA was knocked down by transient siRNA treatment. The effect of siRNA treatment on K-ras protein expression was confirmed by Western blotting (Figure 6). The effect of the siRNA suppression of K-RAS4A on cell survival and proliferation was then investigated. Seventy-two hours after siRNA transfection, there were 73% fewer RCC4 plus vector cells and 40% fewer control RCC4 plus VHL cells in culture when compared to control cells treated by the scrambled RNA.

Figure 6.

Figure 6

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Western blot analysis demonstrates that following treatment of both renal carcinoma cell lines with si RNA specific for K-RAS4A, there is knock-down of K-RAS protein. There was also a marked reduction in the phosphorylated forms of Raf, Akt and S6 riboprotein.

K-RAS4A acts through the Raf and Akt pathways to support the survival and growth of renal cell carcinoma cells

K-RAS4A siRNA knock-down was repeated on both RCC cell lines and extracted protein examined by Western blotting for evidence of activation of the Raf and Akt pathways using phospho-epitope-specific antibodies. In both cell lines, there was a marked reduction in Akt phosphorylated on Ser-473 (P-473Akt) following K-RAS4A knock-down (Figure 6). Although there was a reduction in phospho-Raf, this was more marked in the RCC4 plus VHL cells than in those lacking a wild-type VHL protein (RCC4 plus vector). The most important downstream target of the Akt pathway is phosphorylation of the S6 ribonuclear protein on serine 235/236 (p-235/236 ser S6). Knock-down of K-RAS4A markedly reduced p-235/236 ser S6 in both cell lines (Figure 6). These data demonstrate that K-RAS4A affects the level of activation of both the Raf and Akt pathways in renal carcinoma cell lines.

Discussion

Epidemiological evidence would suggest that hypertension and obesity acting through activity of the RAAS increase the risk of RCC development (Lever et al. 1998, 1999). In this study, we have found evidence that aldosterone supports the growth and survival of renal cancer cells through increased expression of the K-RAS4A cellular oncogene and that some renal carcinomas express the mineralocorticoid receptor.

K-ras has been shown to be important for a number of human malignancies usually through constitutively activating mutations (Capon et al. 1983; McCoy et al. 1984; Bos et al. 1987; Kozma et al. 1987). Although mutations of K-ras are rare in renal carcinoma (Nanus et al. 1990; Rochlitz et al. 1992), there has been increased interest in the oncogenic properties of the molecule recently, not least because of the evidence that it may act with the SWI/SNF/PBRM1 complex in promoting the formation of renal carcinoma (Varela et al. 2011).

We have sought to identify a mechanism for the enhancement of Ras signalling in RCC independently of mutation. In the experiments described, we have shown that the K-RAS4A isoform is expressed by human renal cell carcinomas and in renal carcinoma cell lines. Further, we have found that K-RAS4A exhibits aldosterone sensitivity and that it appears to be important in mediating the aldosterone sensitive growth promotion of renal cell carcinoma.

K-ras is a 21kD protein with high affinity for guanine-containing nucleotides which acts as a signal transducer between cell surface receptors, usually tyrosine kinases, and the cell signalling enzyme cascades which lead to altered gene expression and cell activation (Plowman et al. 2006). In humans, K-ras exists as two isoforms due to alternative splicing of the fourth exon, K-RAS4A and K-RAS4B. These two isoforms may have different biological activities and functions because while K-RAS4B is ubiquitous, K-RAS4A has expression restricted to kidney, lung and colon (Plowman et al. 2006). The Ras family was first identified as oncogenes, and they tend to behave as such when harbouring activating mutations. Mutations in codons 12, 13 and 61 are frequently found in a variety of human cancers including lung and colon (Grunewald et al. 1989; Mariyama et al. 1989; Prosperi et al. 1990; Matsumoto et al. 1992). The presence of these mutations influences outcome and the response to therapy which targets cell surface tyrosine kinase receptors so much so that K-ras mutation analysis is now a standard component of the pathology diagnostic assessment in these tumours and guides therapy. Unlike those organs in which K-RAS4A is expressed, several studies have shown that K-ras mutation is a rare event in renal cell carcinoma (Nanus et al. 1990; Rochlitz et al. 1992). We have therefore sought an alternative mechanism for its involvement in renal cell carcinoma through hormone-induced overexpression.

Animal models, used mostly to investigate sodium transport in the nephron, have shown that K-ras may be regulated by mineralocorticoid. The A6 toad bladder cell line has been extensively used to study the epithelial sodium channel (ENaC) response to aldosterone (Stockand 2002; Stockand & Meszaros 2003). In this model, there is clear evidence that K-ras expression is upregulated leading to increased expression and membrane localization of ENaC. We have used immunohistochemistry to demonstrate that the mineralocorticoid receptor is expressed by about half of human clear cell RCC along with its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 (Kotelevtsev et al. 1999). Antibodies specific for the two isoforms of K-ras are not available, so we demonstrated that the K-RAS4A isoform was also expressed in clear cell RCC by a reverse transcriptase–PCR approach using isoform-specific primers, similar to the approach used for transgenic animal modelling of K-ras function (Plowman et al. 2003).

We next conducted functional studies on the RCC4 plus VHL and RCC4 plus vector RCC cell lines. Both of these lines harbour the VHL-inactivating mutation and chromosome 3p loss which is characteristic of the great majority of clear cell RCCs; however, they differ in that RCC4 plus VHL has had a wild-type functional VHL re-introduced and serves as a control for the effect of the VHL mutation. We have shown that K-ras (4A deleted) expression is markedly reduced following blockade of the mineralocorticoid receptor using spironolactone. Spironolactone treatment also caused a reduction in tumour cell number over 48–72 h. There was a fall in cell number suggesting an effect on cell survival rather than inhibition of cell division. The effect was most marked in the RCC4 plus vector cells which lack a functional VHL gene but was also seen to a lesser extent in the RCC4 plus VHL controls. We have no data addressing the biochemical relationship between VHL and K-RAS. Plowman et al. (2006) have previously shown in embryonic stem cells with targeted loss of the K-RAS4A exon there is altered sensitivity to apoptosis and reduced cell proliferation.

In order to establish a relationship between K-ras and cell population expansion and to investigate downstream signalling, we developed a siRNA approach to knock-down of K-RAS. Using rationally designed siRNA oligonucleotides, we were able to reduce mRNA and protein expression for K-RAS4A over a 24-h period. Thereafter, there was recovery of the K-RAS4A but some of the biological effects persisted for a further 48 h. K-ras knock-down reduced the phosphorylation of the downstream signalling mediators and of the target S6 ribosomal protein. This is a known mediator of tumourigenesis in RCC, being phosphorylated by mammalian target of rapamycin (mTOR) in absence of a functional TSC1/2 complex as occurs in RCC in tuberous sclerosis patients as well as in conventional RCC associated with VHL mutation (Parry et al. 2001). The TSC1/2 dimer regulates mTOR, and mTOR inhibitors are in current chemotherapeutic use for advanced RCC (Gemmill et al. 2005).

Although we, and others (Yakirevich et al. 2008), have found that mineralocorticoid receptor and its gatekeeper enzyme are expressed in only a proportion of cases of RCC, it may nevertheless be an important growth-promoting pathway in these tumours in a similar manner to oestrogen-dependent growth in a subset of breast carcinomas.

We have provided evidence that renal cell carcinoma shows aldosterone sensitive upregulation of K-RAS4A and that this influences cell survival and proliferation. These data suggest a possible mechanism for the observed increased risk of renal cancer in patients with hypertension, abdominal obesity and increased activity of the RAAS.

Funding source

This research was supported by the Pathological Society of Great Britain and Ireland PhD studentship programme and by a research award from Tenovus Tayside.

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