Patient recruitment

The present study was based on the principles of the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of Nanjing Hospital Affiliated to Nanjing Medical University (Date: 2016.11.17/No. 2016–227). The written informed consent of each participant was obtained. A total of 64 patients with RCC diagnosed at Nanjing Hospital Affiliated to Nanjing Medical University between Jan 2017 and Jan 2020 were recruited into the study cohort. Among them, there were 32 cases in the control group (15 females and 17 males), in which, the average age was 52.1 years old, and the histological type was as follows: 5 cases of KICH, 19 cases of KIRC, and 8 cases of KIRP. There were 32 cases in the observation group (18 females and 14 males), in which, the average age was 51.9 years old, and the histological type was as follows: 4 cases of KICH, 21 cases of KIRC, and 7 cases of KIRP. The inclusion criteria were as follows: ① RCC histological diagnosis was based on both hematoxylin and eosin (H&E) staining and immunochemistry results; ② the clinical data of patients were complete; ③ the patients underwent radical nephrectomy. The exclusion criteria were as follows: ① patients with other malignant tumors; ② patients with severe postoperative complications caused by uncontrollable basic diseases; ③ patients with incomplete clinical case data. There was no significant difference in patients’ clinical and pathological characteristics between these two groups.

Nursing intervention

The control group patients received conventional nursing care, including as preoperative psychological intervention, postoperative anti-infection, complication care, and rehabilitation training. Using conventional nursing care as a reference, the observation group (evidence-based nursing model group) implemented the evidence-based nursing model for the patients. Namely, the PubMed, EMBASE, and Cochrane Library databases were searched for entries from Jan 2010 to Jan 2020 for literature regarding difficulties in clinical practice following RCC operation, and a systematic evaluation and meta-analysis were employed to guide the nursing model for RCC postoperative complications. From this, an evidence-based nursing model was developed and used in the present study, which employed the following: ①oxygen inhalation, involving monitoring the blood oxygen saturation (> 90%), oxygenation index (> 300 mmHg), blood lactic acid concentration, and evaluating the degree of hypoxia in patients; ② local oxygen therapy, whereby the surgical wound was treated with local oxygen therapy for 90 min/d for 5 consecutive days and 2 intermittent days, while the wound pressure was maintained at 3 kPa during the care process; ③ complications during the nursing intervention: a common complication after RCC operation is anastomotic leakage with abdominal infection, and an evidence-based nursing model must judge the occurrence of anastomotic bleeding and abdominal leakage according to the patient's abdominal pain, abdominal distension, blood pressure, body temperature, and drainage fluid characteristics.

Clinical index observation

The venous blood from RCC patients was collected. Samples from patients with jaundice, hemolysis, and high lipids were not included. Renal function indexes (BUN and Cr) and serum inflammatory factors (IL-6 and TNF-α) were detected by the Beckman Coulter AU5800 automatic biochemical analyzer. Tumor markers (CEA and CA50) were measured by the Roche Cobas e601 automatic electrochemiluminescence immunoanalyzer. The Self-Rating Anxiety Scale (SAS, > 50 for anxiety) and Self-Rating Depression Scale (SDS, > 53 for depression) were used to evaluate the psychological status of RCC patients. Nursing satisfaction was scored as follows: satisfied, 4 points; somewhat satisfied, 3 points; dissatisfied, 2 points; very dissatisfied, 1 point. The nursing satisfaction rate was calculated as the sum of the given answers ‘satisfied’ and ‘somewhat satisfied’ over the total number of responses.

Postoperative follow-up

RCC patients were rechecked every 3 months after their operation, whereby each underwent a routine blood test, routine urine test, biochemical test, chest radiograph, and abdominal and urinary ultrasound test. If there was suspicion of RCC recurrence, urinary computed tomography (CT), chest CT, and a bone scan were conducted.

Cell culture

The human RCC cell line A498 was obtained from the Cell Bank of China Union Medical University (Beijing, China). A498 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, CAT#: 01–050-1A, Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, CAT#: 04–127-1A, Life Technologies, Grand Island, NY, USA) and 100 μg/ml penicillin/streptomycin (CAT#: PSF-1, Life Technologies, Grand Island, NY, USA), then maintained at 37 °C in constant 5% CO2 and 90–100% humidified atmosphere. The A498 cells underwent annual short tandem Repeat (STR) profiling analysis, population doubling time and morphology for genetic confirmation. A498 cells in the logarithmic growth phase were divided into two groups: ① the hypoxic condition group at 37 °C, 5% CO2, 2% O2, and 93% N2; ② the normoxic condition group at 37 °C, 5% CO2, and 95% O2. All experiments were performed with early-passage cells (passages 4–10).

SLC14A1 plasmid construction and transfection.

SLC14A1 cDNA was subcloned into a pcDNA3.1 mammalian expression vector (CAT#: V79520, Invitrogen Life Technologies, Carlsbad, CA, USA). The primer sequences of the pcDNA3.1-SLC14A1 (pc-SLC14A1) plasmid were as follows: SLC14A1-sense, 5'-CCA GTG GGA GTT GGT CAG AT-3'; SLC14A1-antisense, 5'-GTT GAA ACC CCA GAG TCC AA-3′. The reconstituted pc-SLC14A1 plasmid and empty plasmid (pc-NC) were transfected into A498 cells. Briefly, 2.5 μg of either the pc-SLC14A1 plasmid or pc-NC plasmid was mixed with 8 μL of lipofectamine 2000 (CAT#: 11,668,019, Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated with A498 cells for 6 h. The cells were then cultured in DMEM containing 20% FBS for another 48 h.

Western blot analysis

A498 cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of cellular protein were loaded per lane, subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to a polyvinyl difluoride (PVDF) membrane (CAT#: GVWP02500, Millipore, Billerica, MA, USA). The membrane was then blocked with 5% skimmed milk for 1 h and immunoblotted with primary antibodies specific to SLC14A1 (1: 100 dilution, CAT#: AV48116; Sigma-Aldrich, St. Louis, MO, USA) and actin (1: 5,000; CAT#: A5441; Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. The blot was then incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1: 5,000; CAT#: SA00001-2; ProteinTech Group, Chicago, IL, USA). An enhanced chemiluminescence (ECL) western detection system (CAT#: 7003; Cell Signaling Technology, Beverly, MA, USA) was used to detect immunoreactive bands.

Electron microscopy

A498 cells were harvested in 1 mm3 blocks and immersed in 3% glutaraldehyde solution for 2 h. The samples were post-fixed in 1% osmium tetroxide (OsO4; CAT#: 18,459; Ted Pella, Redding, CA, USA) for 2 h, dehydrated in ascending alcohols, and then embedded in Eponate 12 resin (CAT#: 18,005; Ted Pella, Redding, CA, USA). Ultrathin (60 nm) sections were cut and post-stained with 5% uranyl acetate (CAT#: 19,481; Ted Pella, Redding, CA, USA) and lead citrate (CAT#: 19,312; Ted Pella, Redding, CA, USA). The mitochondrial ultrastructure of A498 cells was observed under a transmission electron microscope (JEM 1011, Japan).

Measurement of mitochondrial ROS generation

The mitochondrial ROS level in A498 cells was analyzed by the MitoSOX™ Red mitochondrial superoxide indicator. In brief, A498 cells were subjected to different treatments and incubated with MitoSOX™ Red (5 µM, CAT#: M36008, Invitrogen Life Technologies, Carlsbad, CA, USA) in the dark at 37 °C for 20 min. The fluorescence intensity was evaluated using a flow cytometer (BD FACSCalibur).

Determination of ΔΨm

Changes in the ΔΨm of A498 cells were measured using the fluorescent cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1). Briefly, A498 cells were subjected to various treatments and then incubated with JC‑1 (20 µg/mL, CAT#: T3168, Invitrogen Life Technologies, Carlsbad, CA, USA) in the dark at 37 °C for 15 min. The fluorescence intensity was evaluated using an epifluorescence microscope (Nikon Corp., Tokyo, Japan).

Measurement of ATP level

A luciferase-based assay kit (CAT#: S0026; Beyotime, Shanghai, China) was used to determine the intracellular ATP content. A498 cells were lysed with ATP lysis buffer and centrifuged at 12,000 g for 8 min at 4 °C. The resulting supernatant and ATP-detection working solution were then mixed before luminescence was measured using a BioTek Synergy H1 Microplate Reader (BioTek Instrument Inc., Winooski, VT, USA). The ATP level was determined according to the standard curve.

Cell viability assay

The Cell Counting Kit-8 (CCK-8; CAT#: CK04; Dojindo, Kyushu Island, Japan) was employed to evaluate A498 cell viability. Briefly, A498 cells underwent various treatments before being incubated with CCK-8 solution (10 μL/100 μL) at 37 °C for 2 h. The absorbance was then assessed using a BioTek Synergy H1 Microplate Reader (BioTek Instrument Inc., Winooski, VT, USA) at 450 nm.

Cell apoptosis and cell cycle assay

Annexin V-FITC/PI staining (CAT#: A211-01/02; Vazyme Biotech Co., Ltd., Nanjing, China) was used to evaluate A498 cell apoptosis. A498 cells underwent various treatments before being harvested and resuspended with 500 μL of binding buffer. Subsequently, the A498 cells were stained with 5 μL of Annexin V and 10 μL of PI (CAT#: A211-01/02; Vazyme Biotech Co., Ltd., Nanjing, China) for 30 min in the dark. Then, apoptotic cells were detected using a flow cytometer (BD FACSCalibur).

Cell cycle assay

A498 cells underwent various treatments before being harvested and fixed in 70% ethanol for 18 h. The A498 cells were then stained with 50 mg/mL PI (CAT#: A211-01/02; Vazyme Biotech Co., Ltd., Nanjing, China) for 30 min at 37 °C. Finally, the cell cycle distribution was determined using a flow cytometer (BD FACSCalibur).

Cell invasion assay

A transwell chamber (CAT#: CLS3422; Corning Incorporated, Corning, NY, USA) was employed to assess the invasive ability of A498 cells. In brief, starved A498 cells (5 × 106/mL) were seeded into the top chamber (pore size, 8 μm), while 500 μL of DMEM containing 20% FBS was filled into the lower chamber. Following incubation for the indicated time, the invasive cells were fixed and stained with 0.1% crystal violet (CAT#: C8470; Solarbio Science Technology Co., Ltd., Beijing, China). The invasive ability of A498 cells was then evaluated under the microscope (× 200 magnification, Olympus, Tokyo, Japan).

Cell migration assay

The wound healing assay was used to estimate the migratory ability of A498 cells. In brief, A498 cells were passaged to attain 80–90% confluence and were then scratched in a straight line on the surface of the monolayer cells using a 200 µl tip (at time point 0). After incubation durations of 24 h and 48 h, the healing width of the wound was surveyed with an inverted optical microscope (× 200 magnification, Olympus, Tokyo, Japan).

Statistical analysis

Statistical analysis was conducted using SPSS software version 13.0 (SPSS Inc. Chicago, IL, USA). The data were presented as the mean ± standard deviation (SD). Comparisons between groups were conducted the Student's t-test if the data were normally distributed, otherwise, the Wilcoxon rank-sum test was performed. P < 0.05 was set to indicate statistical significance.