The study was approved by the Institutional Review Board of the Animal Research Facility of Koc University (Protocol no: 2015-19). Twenty-Five Wistar albino rats were included. The first 5 animals were used for the pilot study in which procedural steps were tested. The remaining 20 animals were divided into 4 groups: sham, RIPC, RIPC + PN, and PN.
Surgical procedures:
Anesthesia was induced by intraperitoneal injection of Ketamine (70-100 mg/kg) and Xylazine (5-10 mg/kg). This allowed us an operating time of maximum 90 minutes which included the surgical intervention itself (skin-to-skin) and optimal recovery from anesthesia. Surgeries were performed transperitoneally through a flank incision under aseptic conditions, right nephrectomy was performed in all groups except the sham group. Three days later, PN was performed in the PN and RIPC + PN groups. PN denoted wedge resection of a left renal parenchymal island with a scalpel under warm-ischemic conditions (Figure 1a). Mean excised kidney tissue volume was 2 mm3. In the RIPC + PN group, RIPC preceded PN by 30 minutes and was employed via sequential clamping / declamping of the femoral artery/vein complex, which was repeated 4 times, each cycle lasting 1 minute (Figure 1b). Warm ischemia was constituted by en-bloc clamping of the renal pedicle for a mean duration of 213±67 seconds. Renal vessels were freed after securing hemostasis by selective probe electrocauterization of the bleeding spots in the tumor bed. All of the surgical interventions were performed on a heating plate to prevent hypothermia. The sham group underwent laparotomy twice (on days 1 and 3).
Blood samples were drawn from the tail vein, as depicted in the following timeline; at the beginning of the experiment prior to the right nephrectomy, on the 3rd day of the experiment prior to RIPC and/or PN and every 24 hours thereafter, until sacrification on day 7 by cervical dislocation under general anesthesia (Figure 2).5 Following sacrification, left kidneys were extracted and cut in half along the sagittal axis. One half was put into 10% buffered formalin and submitted to pathology lab for histological analysis. Remaining kidney tissue and blood samples were stored at -80°C for biochemical investigations. Previously excised right kidney specimens were treated in the same manner. Weight measurement and assessment of nutritional and hydration status were conducted at each blood draw session.
Immunoassays of blood-based biomarkers:
Neutrophil gelatinase-associated lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1) levels in serum samples and kidney tissue specimens were determined by Sandwich‐ELISA using commercial kits (Boster, Pleasanton, USA).
Sandwich‐ELISA commercial kits were used to determine the serum and kidney tissue levels of IL-33 (R&D Systems, Minneapolis, USA) and aldose reductase (MyBiosource, San Diego, USA).
Serum creatinine levels were measured with Creatinine Colorimetric Assay Kit (Cayman, Michigan, USA) based on Jaffe’s reaction.
Kidney tissues were homogenized in 100 mmol/L phosphate buffer (pH: 7.4) containing sodium azide (0.05%) for 1 min on ice and then centrifuged at 20.000 g at +4 °C for 15 min and supernatants were obtained.
Histopathological examination:
Following overnight fixation in 10% formalin at room temperature, kidney tissues were subjected to routine paraffin embedding procedure. Five sections of 2 µm thick and at 5 µm intervals were obtained from the paraffin blocks. Initial slides were stained with hematoxylin and eosin (H&E) on Sakura Tissue-Tek Prisma automated slide stainer (Nagano, Japan). Remaining 4 were used for additional histochemical stainings with Jones' methenamine silver (JMS) and periodic acid-Schiff (PAS) methods in order to provide a better assessment of morphology and the extent of histological changes. All light microscopic evaluations were carried out using Olympus BX53 optical microscope. Areas of necrosis and thickness of the zone of severe ischemia were measured for comparison between groups. Measurements were accomplished on digital slides scanned at 40X by Philips IntelliSite Ultra Fast Scanner NOCTN442 (Amsterdam, Netherlands) using the drawing line and area measurement tools of the Image Management System Software (version 3.3.1) (Royal Philips Healthcare, Amsterdam, Netherlands) (Figure 3c-d). Five µm apart 5 slides (4 special stains and 1 H&E) from each kidney were scanned, area and thickness measurements were done in each section, and average numbers were obtained for each rat.
The hydropic degeneration of the tubular epithelium, single cell tubular necrosis, cast formations and focal interstitial inflammation were scored from 0 to 2 taking into account the frequency of the lesions as 0, none; 1, rare; and 2, common (presence of at least one group of tubules with the lesion on 1 mm2 of area).
Immunohistochemistry:
An immunohistochemical staining for Ki-67 was performed to evaluate the regenerative/proliferative activity of the tubular epithelial cells. The BenchMark Ultra automated staining platform (Ventana Medical System, Inc., USA) was used for this purpose. Briefly, the tissue sections from the representative paraffin blocks that were cut at 3 µm thickness onto charged slides were deparaffinized with EZ Prep. solution (Ventana medical system, cat. no: 950-102) at 75°C. Following rehydration through alcohol series and 32 min heat-induced epitope retrieval at 100°C with an EDTA-based buffer (Cell Conditioner 1, Ventana; cat. no: 950-124), tissue sections were incubated for 32 min with anti-Ki-67 primary antibody (Ventana; pre-diluted; monoclonal rabbit, clone SP6) at 37°C temperature. Washing between the steps were accomplished by Reaction Buffer (Ventana medical system, cat. no: 950-300). Ultra View Detection kit (Ventana medical system, cat. no: 760-500) was used for the detection of the target protein. The reaction product was visualized with 3, 3ʹ-diaminobenzidine chromogen and counterstaining with hematoxylin was done. Nuclear staining was considered positive. Appropriate staining of the germinal centers of normal tonsil served as a positive control. Ki-67 index was expressed as the percentage of the number of immunostained nuclei among the total number of tubular cell nuclei.
Statistical analyses:
Statistical comparisons of categorical variables were performed using the chi²-test. Continuous variables were compared using Student's t-test or the Wilcoxon rank sum test depending on whether the data was normally distributed or not. For continuous-paired data, the Wilcoxon signed rank test was used. Longitudinal analyses were performed using linear mixed effect models. All tests were 2-sided, the alpha level for statistical significance was set at 0.05. Statistical analyses were performed with R Statistical Software version 3.6.3 (Foundation for Statistical Computing, Vienna, Austria).
The main reference group was the sham group but in order to account for all possible differences between the study groups and do bidirectional crosscheck, each group has been used once as the reference group.