All experiments in this study were approved by the Institutional Animal Experimentation Committee of the Academic Medical Center of the University of Amsterdam. Care and handling of the animals were in accordance with the EU Directive 2010/63/EU for animal experiments and guidelines for Institutional and Animal Care and Use Committees. The study has been carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Experiments were performed on 18 Wistar male rats (Harlan, the Netherlands) with mean ± SD body weight of 320 ± 30 g.
The rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg ketamine (Nimatek®, Eurovet, Bladel, the Netherlands), 0.5 mg/kg medetomidine (Domitor, Pfizer, New York, NY, USA), and 0.05 mg/kg atropine sulfate (Centrafarm, Etten-Leur, the Netherlands). After tracheotomy, the animals were mechanically ventilated with a FiO2 of 0.4. Body temperature was maintained at 37°C ± 0.5°C during the entire experiment by external warming. The ventilator settings were adjusted to maintain end-tidal PCO2 between 30 and 35 mmHg and arterial PCO2 between 35 and 40 mmHg.
Vessels were cannulated with polyethylene catheters (outer diameter = 0.9 mm; Braun, Melsungen, Germany) for drug and fluid administration and hemodynamic monitoring. A catheter in the right carotid artery was connected to a pressure transducer to monitor mean arterial blood pressure (MAP) and heart rate. The right femoral artery was cannulated for blood sampling. The right femoral vein was cannulated for continuous infusion of Ringer’s lactate (15 mL/kg/h; Baxter, Utrecht, the Netherlands) and ketamine (50 mg/kg/h; Nimatek®, Eurovet, Bladel, the Netherlands).
The left kidney was exposed, decapsulated, and immobilized in a Lucite kidney cup (K. Effenberger, Pfaffingen, Germany) via a 4-cm incision in the left flank. Renal vessels were carefully separated under preservation of nerves and adrenal gland. A perivascular ultrasonic transit time flow probe was placed around the left renal artery (type 0.7 RB; Transonic Systems Inc., Ithaca, NY, USA) and connected to a flow meter (T206, Transonic Systems Inc.) to continuously measure renal blood flow (RBF). An estimation of the renal vascular resistance (RVR) was made as RVR [dynes/sec/cm5] = MAP/RBF. The left ureter was isolated, ligated, and cannulated with a polyethylene catheter for urine collection.
After the surgical protocol (approximately 60 min), one optical fiber was placed 1 mm above the decapsulated kidney and another optical fiber 1 mm above the renal vein to measure oxygenation in the renal microvasculature and renal vein, respectively, using phosphorimetry. A small piece of aluminum foil was placed on the dorsal site of the renal vein to prevent the contribution of underlying tissue to the phosphorescence signal in the venous oxygenation measurement. Oxyphor G2 (a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin, Oxygen Enterprises Ltd., Philadelphia, PA, USA) was subsequently infused (6 mg/kg IV over 5 min) followed by a 30-min stabilization period. A short description of phosphorimetry is given below, and a more detailed description of the technology has been provided elsewhere [17–20].
After baseline measurements were performed 30 min after Oxyphor G2 infusion, the rats were randomly assigned to one of the following groups: sham-operated time control (n = 6), I/R control (n = 6), and I/R with 15 mg/kg BMOV (n = 6). Considering this is the first study in which BMOV is being utilized in a model of renal I/R injury, we decided to use the recommended dosage (15 mg/kg) by CFM Pharma (Almere, the Netherlands). BMOV solutions were prepared in 2-mL isotonic saline, and infusion was initiated 30 min prior to renal ischemia at an infusion rate of 2 mL/h. Control rats received the same volume of isotonic saline without BMOV. Renal ischemia was created by 30-min clamping of the renal artery, and following the release of the clamp, measurements were continued up to 90 min of reperfusion. The experiments were terminated by infusion of 1 mL of 3 M potassium chloride (KCl).
Arterial blood samples (0.5 mL) were taken from the femoral artery at baseline and 15 and 90 min after reperfusion. The blood samples were replaced by the same volume of Voluven® (Fresenius Kabi Ltd., Runcom, UK). Samples were analyzed for blood gas values (ABL505 blood gas analyzer, Radiometer, Copenhagen, Denmark), hemoglobin concentration, and hemoglobin oxygen saturation (OSM3, Radiometer). Additionally, plasma creatinine concentrations were determined in all samples.
Renal microvascular and venous oxygenation
Microvascular oxygen tension in the renal cortex (CμPO2), outer medulla (MμPO2), and renal venous oxygen tension (PrvO2) were measured by oxygen-dependent quenching of phosphorescence lifetimes of the systemically infused albumin-targeted (and therefore circulation-confined) phosphorescent dye Oxyphor G2. Oxyphor G2 (a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin) has two excitation peaks (λexcitation1 = 440 nm, λexcitation2 = 632 nm) and one emission peak (λemission = 800 nm). These optical properties allow (near) simultaneous lifetime measurements in microcirculation of the kidney cortex and the outer medulla due to different optical penetration depths of the excitation light. For the measurement of renal venous PO2 (PrvO2), a mono-wavelength phosphorimeter was used. Oxygen measurements based on phosphorescence lifetime techniques rely on the principle that phosphorescence can be quenched by energy transfer to oxygen resulting in the shortening of the phosphorescence lifetime. A linear relationship between reciprocal phosphorescence lifetime and oxygen tension (given by the Stern-Volmer relation) allows quantitative measurement of PO2[17–20].
Renal oxygen delivery and consumption
Arterial oxygen content (AOC) was calculated by (1.31 × hemoglobin × SaO2) + (0.003 × PaO2), where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure of oxygen. Renal venous oxygen content (RVOC) was calculated as (1.31 × hemoglobin × SrvO2) + (0.003 × PrvO2), where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial pressure of oxygen (measured using phosphorimetry). Renal oxygen delivery per gram of renal tissue was calculated as DO2 (mL/min/g) = RBF × AOC. Renal oxygen consumption per gram of renal tissue was calculated as VO2 (mL/min/g) = RBF × (AOC − RVOC). The renal oxygen extraction ratio was calculated as O2 ER (%) = VO2/DO2 × 100.
For the analysis of urine volume, creatinine concentration, and sodium (Na+) concentration at the end of the protocol, urine samples from the left ureter were collected for 10 min. Creatinine clearance rate (CLcrea) per gram of renal tissue was calculated with the standard formula: CLcrea (mL/min/g) = (U × V)/P, where U is the urine creatinine concentration, V is the urine volume per unit time, and P is the plasma creatinine concentration. Renal oxygen consumption efficiency for sodium transport (VO2/TNa+) was assessed as the ratio of the renal VO2 over the total amount of sodium reabsorbed (TNa+, [mmol/min]). TNa+ was calculated according to the following: ((CLcrea × PNa+) − (UNa+ × V)), where PNa+ is the plasma concentration of sodium.
Statistical analysis was performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SD unless otherwise stated. Statistical significance of differences between groups was tested using one-way ANOVA with Bonferroni post hoc tests. P values <0.05 were considered significant.