2.6.2. Renal Parameters

#### Urine Sediment Examination

At each time point urine bag was emptied into a sterile collection pot for UOP measurement, and urine specific gravity and dipstick analyses (Multistix®, Siemens, Erlangen, Germany). Urine samples were centrifuged at 339 g for five minutes. At each time interval, urine sediment in the urine was examined using light microscopy at low (×100) and high (×400) power. A minimum of ten high power fields were examined for presence of blood cells, epithelial cells, and granular and hyaline casts. Urine supernatant was divided into multiple aliquots and stored at −80 ◦C for later analysis of creatinine, protein, GGT, and biomarker concentrations.

#### Kidney Light Microscopy

Kidneys were removed immediately following euthanasia, sectioned and stored in 10% formalin. Additionally, small cubes of cortex (approximately 2 to 3 mm<sup>3</sup> ) were placed into 3% glutaraldehyde (Koch-Light, Johannesburg, South Africa) for transmission electron microscopic (TEM) evaluation. Samples were processed routinely by the Comparative Pathology and Mouse Phenotyping Shared Resource (The Ohio State University, Columbus, OH, USA). Samples were embedded in paraffin, sectioned at 3µm thickness and stained with hematoxylin and eosin and periodic acid-Schiff. One veterinary pathologist, blinded to group allocation and AKI biomarker concentrations, examined sections of both kidneys by light microscopy and counted the number of injured tubules per 200× field. Tubular injury was defined as loss of the apical brush border, denudation of tubular basement membranes, singly dead tubular epithelial cells and tubules with intra-luminal detached cells/cellular debris. Twenty randomly selected cortical fields were assessed, and the average number of injured tubules per field was calculated for each specimen. Based on these quantitative data, the samples were then categorized into groups of normal (no injured tubules), minimal (mean tubular injury ≤0.5), mild (mean tubular injury >0.6 but ≤1) and moderate (mean tubular injury >1 but ≤2) histologic injury.

### Kidney Transmission Electronic Microscopy (TEM)

Due to resources constraint, the glutaraldehyde-fixed tissue from only 4 dogs (2 control and 2 treated) were processed for electronic microscopy. After post-fixation in 1% osmium tetroxide, the specimens were serially dehydrated, infiltrated in an acetone/epoxy plastic, and embedded in plastic. The plastic blocks were sectioned to a silver-grey interference color (55–60 nm) and placed on copper mesh grids. The sections were stained with filtered lead citrate/sodium citrate solution (Electron Microscopy Sciences, Inc., Hatfield, PA, USA). Grids were imaged on a JEOL JEM-1400 TEM (JEOL USA, Inc., Peabody, MA, USA) and representative images were photographed with an Olympus SIS Veleta 2K camera (Olympus Soft Imaging Solutions GmbH, Muenster, Germany.

#### Renal Biomarkers

At baseline, T0, T1, and T3; aliquots of plain serum and urinary supernatant were stored at −80 ◦C for measurements of SCr and AKI biomarkers within three months.

Urinary creatinine, protein, and GGT concentrations; and SCr concentration, were measured using the same biochemistry analyzer (Cobas Integra 400 plus, Roche Diagnostics) that was calibrated prior to each use, with two control samples included in each run. Concentrations of AKI biomarkers; clusterin, cystatin C, kidney injury molecule 1 (KIM-1), monocyte chemoattractant protein 1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL), were measured in the urine and serum samples from each dog at baseline, T0, T1, and T2 using a bead-based multiplexed immunoassay (MILLIPLEX™ MAP Canine Kidney Toxicity Expanded Magnetic Bead Panel 1, Merck Millipore, Burlington, MA, USA). Urinary samples were diluted to 1:2, and serum samples to 1:10, using assay buffer prior to analysis. All samples were analyzed in duplicate. The assays were performed according to manufacturer instructions. Briefly, samples were incubated with a solution of

antibody-immobilized magnetic beads (for each of the five biomarkers previously listed) for two hours at room temperature. The plate was then washed using a magnetic plate washer (Bio-Plex® Pro II Wash Station, Bio-Rad, Hercules, CA, USA), detection antibodies added, and incubated at room temperature for one hour. Streptavidin-Phycoerythrin was added to the detection antibodies, prior to a further 30 min period of incubation at room temperature. The plate was then washed, and beads re-suspended with drive fluid (MAGPIX® Drive Fluid, Merck Millipore, Burlington, MA, USA). The plate was analyzed five minutes later using a multiplex reader (Bio-Plex® MAGPIX™ Multiplate Reader, Bio-Rad, Hercules, CA, USA) with xPONENT® software. The Median Fluorescent Intensity data was analyzed using a 5-parameter logistic curve to calculate analyte concentrations in each sample. If the coefficient of variance between two duplicates was >15% the sample was re-analyzed on a subsequent plate. If the analyte concentration of a sample was above the upper limit of quantification (ULOQ) the sample was diluted until the biomarker concentration fell within the measurable range of the assay. If the analyte concentration of a sample was below the lower limit of quantification (LLOQ) the sample was reanalyzed on a subsequent plate using undiluted sample. Any result still falling below the LLOQ was allocated the same value as the LLOQ in order to allow statistical analysis. When serum samples were analyzed using the multiplex assay, a serum diluent (Serum Matrix, Merck Millipore, Burlington, MA, USA) was added to wells containing the Standards and Controls in place of assay buffer in order to mitigate any matrix effects.

#### 2.6.3. Coagulation Parameters

Central venous blood was obtained from the jugular vein catheter at each time point for assessment of coagulation. At T0, T1, and T2, blood samples for coagulation assay were transferred into blood tubes containing 3.2% buffered sodium citrate with a blood: anticoagulant ratio of 9:1. Tubes were gently agitated to ensure even mixing of anticoagulant. Platelet closure time (PCT) was measured immediately, and blood was stored at room temperature for Rotational Thromboelastometry analysis (ROTEM® delta, Tem International GmbH, Munich, Germany) 30 min after collection.

The Platelet Function Analyzer-100 (Dade Boehring Inc., Deerfield, IL, USA), with collagen and adenosine diphosphate cartridges, was used to measure PCT in duplicate within 10 min of sample collection. Analysis was immediately repeated if the coefficient of variation was greater than 15%. ROTEM® was performed according to the manufacturer's instructions using both InTEM and ExTEM. Each profile was run for at least one hour following initiation. Data recorded for the InTEM and ExTEM profiles included clotting time (CT), clot formation time (CFT), alpha angle, maximum clot firmness (MCF) and lysis index at 60 min (LI60). Peak thrombin generation (normal range 243–507 nM) and area under the thrombin generation curve (AUC: normal range 1500–2835 nM × min) were measured by the Calibrated Automated Thrombogram using Thrombinoscope® (StagoTM, Asnières sur Seine, France).

Total concentrations of resveratrol in plasma and qualitative investigation of its metabolites (glucuronide and sulphate) both prior to the administration and after 7 days of resveratrol treatment (or control) were measured by the UHPLC-MS/MS on a Shimadzu Nexera2 UHPLC system coupled to a Shimadzu 8030+ triple quadrupole mass spectrometer (Kyoto, Japan) [26].

#### *2.7. Data Analysis*

A sample size calculation was performed using data from a previous study by Holthoff et al. [27] in which the benefits of resveratrol on septic AKI were quantified by histological changes in a murine model (*n* = 6). A sample size of 6 dogs per group would be needed assuming a difference in histology score of 1.2 between the treatment and control groups, with a standard deviation in the scores of 0.6, in order to achieve 80% power while taking an alpha-value <0.05 as significant. Due to the small sample size of this study, a non-parametric Mann–Whitney test was used to compare the outcomes between the

two treatment groups. The changes in MCF on the ROTEM between the two groups was analyzed by adjusting for volume of blood loss in each animal using a linear mixed model. All analyses were two tailed using SPSS for Windows (version 23, IBM, Armonk, NY, USA) and MedCalc Statistical Software (version 18.11.3, Ostend, Belgium). A *p*-value < 0.05 was taken as significant, and no adjustment was made for multiple statistical testing in this study.

#### **3. Results**

Six dogs were included in each group. Exact age of each dog was unknown, but veterinary examination suggested all dogs were adult (2–7 years). The body weights of the dogs were not statistically different between the two groups (median [range] for the control group was 30.9 kg (30.1–32.5), and for treatment group was 32.6 kg (27.9–34.0); *p* = 0.441). All dogs had a lean body condition (score 3/9), appropriate for trained racing dogs of this breed. No adverse effects of oral resveratrol were noted, and all dogs completed the study. There was no significant difference in renal and coagulation parameters between the two groups prior to initiation of resveratrol treatment (Tables 2 and 3), but there was a suggestion that seven days of resveratrol treatment could improve clot strength compared to the control prior to induction of hemorrhage (InTEM MCF 54 vs. 43 mm respectively; *p* = 0.009)(Table 3). No resveratrol and its metabolites were detected in the control dogs. Among the six dogs treated with resveratrol, only one had a detectable level of resveratrol at a concentration < 50 mg/mL.

**Table 2.** Renal parameters of anesthetized greyhound dogs before and after induced hemorrhage (*n* = 11).



