Subjects

We analysed 95 patients with RCC, who underwent radical nephrectomy in several Urology Clinics in Belgrade, Serbia, between 2009 and 2013. The study was performed in compliance with the ethical principles of the Declaration of Helsinki and all applicable national laws and regulations. The clinicopathological features of patients included age, gender, size, grade, stage, and histological classification. For tumour histology, the 2004 WHO classification [1] was used and tumours were graded according to the Fuhrman grading system [14]. OS time was determined from the surgery date until death or the last follow-up appointment. The follow-up period was 5 years (median: 38 months, IQR = 31–42 months). The median age of patients at diagnosis was 62 years (IQR = 58–66.2 years).

Specimens

For the enzyme activity measurement, the tissue was washed in a saline solution (PBS) at pH 7.4 in order to remove blood cells. All samples were homogenised on ice in eight volumes of cold potassium phosphate buffer (0.05 M KH₂PO₄, 0.0001 M EDTA, pH 7.4) by using Ultra-Turrax Homogeniser (IKA^® T10 basic ULTRA-TURRAX^®, IKA Werke GmbH & Co.KG, Staufen, Germany) at 25,000 rpm for 15 s in four cycles. Homogenates were left overnight at –70°C to disrupt cell membranes. The thawed homogenates were vortexed for 1 min and then centrifuged (10000 × g, 15 min, 4°C) and the upper layer was collected and kept at –70°C till the assay.

For GSH and total malondialdehyde (MDA) concentration measurement, the tissue was prepared as recommended by the kit producer (BIOXYTECH^® GSH-420^TM and BIOXYTECH^® MDA-586^TM respectively, OXIS International Inc., Foster City, CA, USA).

Assays

The protein concentration was measured with Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). The method is based on a combination of the biuret reaction and colorimetric detection of cuprous cation (Cu⁺¹) with bicinchoninic acid (BCA). The cupric cation (Cu⁺²) is reduced by the sample proteins in an alkaline medium to Cu⁺¹. Two molecules of BCA react with one Cu⁺¹ making a purple-coloured product that absorbs at 562 nm. The protein concentration was expressed as mg/ml of the sample.

Total SOD activity was measured using Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). The assay is based on the reaction between tetrazolium salt and superoxide radicals (O₂ ^.-), which results in formazan dye development. SOD inhibits this reaction by dismutation of O₂ ^.-, generated by xanthine oxidase. The intensity of formazan dye was measured at 450 nm using a microplate reader (Wallac1420 Victor², PerkinElmer Inc., Waltham, MA, USA). One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of superoxide radical.

Catalase activity measurement was performed by the method of Beutler (1984) [15]. The method is based on the ability of CAT to decompose hydrogen peroxide in an incubation mixture to water and oxygen. The incubation mixture contains 50 μl of a Tris-HCl buffer (1 M Tris-HCl, 5 mM EDTA, pH 8.0), 900 μl of a substrate (10 mM H₂O₂) and 30 μl of distilled water. The reaction starts after adding 20 μl of the sample. Decomposition of H₂O₂ was monitored for 3 min at 230 nm and 37°C using a spectrophotometer (UV Line 9400, SI Analytics GmbH, Mainz, Germany). One unit of CAT activity is defined as the amount of enzyme that degrades 1 μmol of H₂O₂ per minute under assay conditions. The extinction coefficient for H₂O₂ at 230 nm is 0.071 mM^–1cm^–1.

Glutathione peroxidase activity was assessed by the BIOXYTECH^® GPx-340^TM Assay (Oxis International, Inc., Foster City, CA, USA). The assay is based on reduction of organic peroxide, which produces oxidised glutathione (GSSG) immediately reduced to GSH by GR with concomitant oxidation of NADPH to NADP⁺ and a decrease in absorbance at 340 nm. The absorbance decrease was measured spectrophotometrically, and it is directly proportional to the GPx activity in the sample. The extinction coefficient for NADPH at 340 nm is 6220 M^–1cm^–1. One unit of GPx activity causes the oxidation of 1 μmol of NADPH per minute under the assay conditions.

Glutathione S-transferase activity was measured using Glutathione S-Transferase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). The assay utilises the conjugation of CDNB (1-chloro-2,1-dinitrobenzene) and GSH accompanied by an increase in absorbance at 340 nm. The extinction coefficient for CDNB at 340 nm is 0.0096 μM^–1cm^–1. One unit of GST will conjugate 1 nmol of CDNB with GSH per minute at 25°C.

Glutathione reductase activity was determined using the BIOXYTECH^® GR-340^TM Assay (OXIS International, Inc., Foster City, CA, USA). The assay is based on catalysed reduction of GSSG to GSH and oxidation of NADPH to NADP⁺. The oxidation of NADPH results in a decrease of absorbance at 340 nm as a function of time. The molar extinction coefficient for NADPH at 340 nm is 6220 M^–1cm^–1. One unit of GR activity is defined as the amount of enzyme catalysing the reduction of 1 μmol of GSSG per minute under assay conditions.

Total GSH concentration was determined by the BIOXYTECH^® GSH-420^TM Assay (OXIS International, Inc., Foster City, CA, USA). The measurement is based on a three-step colourimetric reaction. The reducing agent, TCEP (Tris[2-carboxyethyl] phosphine), reduces all oxidised glutathione present in the sample. In the next step chromogen (4-chloro-1-methyl-7-trifluoromethylquinolinium methylsulfate) is added, reacting with all thiols in the sample and forming thioethers. NaOH raises the pH of the reaction mixture to over 13 and leads to chromophoric thione development as a result of β-elimination specific to the GSH-thioether. The absorbance measured at 420 nm is directly proportional to the GSH concentration.

Total MDA concentration was determined using BIOXYTECH^® MDA-586^TM Assay (OXIS International, Inc., Foster City, CA, USA). The protocol is based on hydrolysis of the sample in the presence of butylated hydroxytoluene (BHT) at low pH (1-2) at 60°C for 80 min. After hydrolysis, free MDA reacts with the chromogenic reagent (N-methyl-2-phenylindole, NMPI) for 60 min at 45°C. The reaction yields a carbocyanine dye that can be detected at 586 nm.

The enzyme activities were expressed as units (U) or mU per milligram of proteins (U/mg or mU/mg). Concentration of GSH and MDA were expressed as nmol/mg of proteins.

Statistical analysis

Statistical analysis was performed with SPSS 23.0 for Windows (SPSS Inc., Chicago, IL, USA). The OS rate was calculated by Kaplan-Meier method, and groups were compared using Log-rank statistics. The compared groups of AO parameters were made by cut-off points based on median value of enzyme activity or MDA and GSH concentration, as previously reported [16]. Univariate and multivariate Cox proportional hazards regression model were used to analyse the independent factors related to 5-year OS. P-values < 0.05 were considered significant.

Clinical findings

Clinicopathological characteristics of patients are given in Table I. The majority of patients were between ages 61 and 70 years, and both genders were almost equally represented. The median tumour size was 7 cm (IQR = 5–8.2 cm). The majority of tumours were up to 7 cm in diameter (63.1%). Low-grade (I–II) RCC was diagnosed in the majority of patients. Stages 1 and 3 were most frequently recorded, and ccRCC was the most common histological subtype at the time of diagnosis.

Survival analysis

The OS for all patients was 96.8%, 73.7%, and 54.6% after 1, 3, and 5 years, respectively (Figure 1 A). Statistically significant correlations existed between survival and age (log-rank 13.999, p = 0.003), tumour size (log-rank, 22.540, p < 0.001), grade (log-rank 12.080, p = 0.001), and stage (log-rank 26.217, p < 0.001) (Figures 1 B–E).

Figure 1

Survival in 95 patients with renal cell carcinoma. A – overall survival; survival according to: age (B), tumour size (C), Fuhrman grade (D), stage (E), histological subtype (F)

In each age group the 5-year OS rate was as follows: 84.6% among patients younger than 50 years, 70% among patients aged 51–60 years, 33.9% among patients aged between 61–70 years, and 61.8% among those older than 70 years (Figure 1 B). The rates of OS after 1, 3, and 5 years for patients with RCC size ≤ 4 cm were 100%, 77.8%, and 70.7%, respectively; patients with tumour size 4.1–7 cm had survival rates of 97.6%, 87.7%, and 70.4% after 1, 3, and 5 years. After 5 years of follow-up, 60.2% of patients with RCC size 7.1–10 cm were still alive. None of the patients with tumour size > 10 cm were alive after 5 years (Figure 1 C).

After 5 years, survival among subjects with low-grade tumours was 64.3%. In the high-grade tumour group, the survival rate was 96.6, 55.2, and 34.5% after 1, 3, and 5 years, respectively (Figure 1 D). Patients with low-stage tumours (stage 1 and 2) had survival rates of 77.2% and 77.9%, respectively, while 34% of patients with stage 3 were alive after a 5-year follow-up period. None of the patients with stage 4 RCC were alive after 5 years (Figure 1 E). As regards histological subtypes, after 5 years the better survival rate (61.4%) was observed in patients with ccRCC compared to those with the pRCC (36.9%), while chRCC patients showed survival rates of 100%, 64.3%, and 0% after 1, 3, and 5 years, respectively (Figure 1 F). There was no significant association between gender, histological subtype, and survival rate (log-rank 0.002; p = 0.962, log-rank 2.424, p = 0.298, respectively).

The OS analysis based on the AO enzymes (Table II) revealed no statistically significant difference between the median values in survival rates. Also, Kaplan-Meier test showed no significant influence of lipid peroxidation parameter (MDA) and GSH concentration on survival of RCC patients.

Univariate analysis (Table III) showed that size, Fuhrman grade, and stage were significant prognostic factors for OS. The size (> 10 cm) and high Fuhrman grade (III–IV) remained independent predictors of survival in multivariate analysis. Age, as a confounding variable, was excluded from the analysis.