The current study uses an experimental design to simulate IR induced AKI after cardiovascular or abdominal surgery. Oxygen phosphorescence technique employed in this study offers the advantage of the measurement of individual oxygen concentration both in the renal cortex and the medulla.
Animals
All experiments described in this study were approved by the Institutional Animal Experimentation Committee of the Academic Medical Centre of the University of Amsterdam (DFL83). Care and handling of the animals were following the guidelines for Institutional and Animal Care and Use Committees. The study has been carried out according to the Declaration of Helsinki. Experiments were performed on 24 male Wistar albino rats (Charles River, The Netherlands) with a mean ± SD bodyweight of 340 ± 26 g.
Surgical Preparation
All the animals were anesthetized with an intraperitoneal injection of a mixture of 90 mg/kg ketamine (Nimatek, Eurovet, Bladel, The Netherlands), 0.5 mg/kg dexmedetomidine (Dexdomitor, Pfizer Animal Health BV, Capelle aan den IJssel, The Netherlands), and 0.05 mg/kg atropine-sulfate (Centrafarm Pharmaceuticals BV, Etten-Leur, The Netherlands). After preparing a tracheotomy, the animals were mechanically ventilated with a mixture of air and oxygen with a FiO2 of 0.4. Body temperature was maintained at 37 ± 0.5°C during the entire experiment by an external thermal heating pad. Ventilator settings were adjusted to maintain end-tidal pCO2 between 30 mm Hg and 35 mm Hg and arterial pCO2 between 35 mm Hg and 40 mm Hg.
For drug and fluid administration and hemodynamic monitoring, vessels were cannulated with polyethylene catheters with an outer diameter of 0.9 mm (Braun, Melsungen, Germany). A catheter in the right carotid artery was connected to a pressure transducer to monitor mean arterial blood pressure (MAP). The right jugular vein was cannulated for continuous infusion of Ringer’s Lactate (Baxter, Utrecht, The Netherlands) at a rate of 15 ml/kg/h to continuous fluid resuscitation and 50 mg/kg/h ketamine dissolved in 5 ml Ringer’s Lactate for the maintenance of anaesthesia. The right femoral artery was cannulated for withdrawing blood samples and the right femoral vein for drug administration.
The left kidney was exposed, decapsulated, and immobilized in a Lucite kidney cup (K. Effenbergerite, Pfaffingen, Germany) via a 4 cm left flank incision of each animal. Renal vessels were carefully separated from surrounding tissue, nerves, and adrenal gland. A perivascular ultrasonic transient time flow probe was placed around the left renal artery (type 0.7 RB Transonic Systems Inc, Ithaca, NY) (T206, Transonic Systems Inc, Ithaca, NY) to simultaneously measure RBF. The left ureter was isolated, ligated, and cannulated with a polyethylene catheter for urine collection.
After surgical preparation, an optical fibre was placed 1 mm above the decapsulated kidney and another optical fibre was placed 1 mm on the renal vein to measure renal microvascular and venous oxygen pressures using phosphorimetry, respectively. Oxyphor G2, a two-layer glutamate dendrimer of tetra-( 4-carboxy-phenyl) benzoporphyrin (Oxygen Enterprises Ltd, Philadelphia, PA) was subsequently infused (6 mg/kg IV over 5 min) followed by 30 min of stabilization time. The surgical field was covered with a humidified gauze compress throughout the entire experiment to prevent drying of the exposed tissues.
Experimental Protocol
The rats were randomly divided into four groups (n = 6 per group determined by the power analysis (nQuery advisor, GraphPad Software DBA Statistical Solutions, San Diego): a sham-operated time control group (C), a group subjected to renal ischemia for 45 min by supra-aortic occlusion with a custom-made vascular occluder placed in between the left renal artery and superior mesenteric artery followed by 2h of reperfusion release of the clamp (IR), a group subjected to renal ischemia for 45 min and followed by 2h of reperfusion in which furosemide 50µg/kg /h was continuously administered after the release of the clamp (IR + F), and finally a sham-operated group in which furosemide was administered to the control group (C + F). At the end of the experiments, renal tissue samples were harvested and stored in both %4 formaldehyde solution for histological analysis and − 80C for a marker of oxidative stress, inflammation, and nitric oxide levels.
Blood and plasma variables
Arterial blood samples (0.25 ml) were drawn from the femoral artery at three-time points: before aortic occlusion (baseline, BL);15 min after reperfusion (initial reperfusion phase, T1); and 120 min after reperfusion (late reperfusion phase T2). The blood samples were replaced by the same volume of HES 130/0.4 (Voluven, 6% HES 130/0.4; Fresenius Kabi Schelle, Belgium). The samples were used for the determination of blood gas values, as well as for the determination of the haemoglobin concentration, haemoglobin oxygen saturation, and electrolytes concentrations (ABL80 Flex Blood Gas Analyzer, Radiometer, Copenhagen, Denmark). The plasma and urine samples were used for the measurement of creatinine levels.
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 systematically infused albumin targeted (therefore circulation-confined) phosphorescent dye Oxyphor G2 [14]. 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 (i.e., Stern-Volmer relation) allows quantitative measurement of PO2 [15].
Calculation of derivative oxygenation parameters and renal vascular resistance
Arterial oxygen content (AOC) was calculated by the following equation; (1.31 x haemoglobin x SaO2) + (0.003 x PaO2), where SaO2 is arterial oxygen saturation and PaO2 is the arterial partial pressure of oxygen. Renal venous oxygen content (RVOC) was calculated as (1.31 x haemoglobin x SrvO2)+(0.003 x PrvO2), where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial pressure of oxygen (measured using phosphorimetry [14]. The SrvO2ren was calculated using the Hill equation with P50 = 37 Torr (4.9 kPa) and Hill coefficient = 2.7. Renal oxygen delivery was calculated as DO2ren (ml/min) = RBF x AOC. Renal oxygen consumption was calculated as VO2ren (ml/min) = RBF (AOC-RVOC). An estimation of the renal vascular resistance (RVR) was made as: RVR (dynes/s/cm5) =(MAP/RBF) x100.
Assessment of kidney function
The high plasma creatinine level was accepted as a short-term AKI definition based on the KDIGO criteria. Creatinine clearance (Clearcrea(ml/min)) was measured as an index of the glomerular filtration rate and calculated with the following formula: Clearcrea= (Uurea x V)/Pcrea, where Ucrea was the concentration of creatinine in the urine, V is the urine volume per unit time, and Pcrea was the concentration of creatinine in plasma. Additionally, excretion fraction of Na+ [EFNa (%)] was calculated and used as a marker of tubular function in the following formula: EFNa = (UNa x Pcrea)/ (PNa+ x Ucrea) x100, where UNa was Na+ concentration in urine and PNa was the Na+ concentration in plasma. The renal oxygen extraction ratio was calculated as O2ERren (%) = VO2ren/DO2ren x 100. Clearcrea and EFNa were determined at all time points. Furthermore, the renal energy efficiency for sodium transport (VO2ren/TNa) was assessed using a ratio portrayed by the total amount of VO2ren over the total amount of sodium reabsorbed (TNa, mmol/min) that was calculated according to: (Clearcrea x PNa) – UNa x V. This parameter can be regarded as an important variable reflecting tubular cell function and oxygen utilization.
Measurement of inflammatory cytokines and glycocalyx component
Inflammatory cytokines TNF-α, IL-6, and hyaluronan (HA) (Rat TNF-a ELISA kit, DY510; Rat IL-6 ELISA kit, DY506 and Rat Hyaluronan Duoset ELISA kit, DY3614, R&D System Inc, Minneapolis, Minn) were determined by ELISA from renal frozen tissue samples. Malondialdehyde (MDA) was quantified using a Quattro Premier XE tandem mass spectrometer (MS/MS, Waters, Milford, Mass) with an Acquity sample manager and an Acquity binary solvent manager [16]. Nitric oxide levels were measured with chemiluminescence method by the Sievers NO analyser [17]. The level of cytokines, MDA, NO and hyaluronan was expressed as per gram of protein (Bradford assay).
Histological analysis
Kidney tissues were fixed in 4% formalin and embedded in paraffin. Kidney sections (4 µm) were deparaffinized with xylene and rehydrated with decreasing percentages of ethanol and finally with water. The kidney sections were stained with periodic acid-Schiff reagent (PAS) and haematoxylin. Histologic changes in the cortex and medulla were assessed by quantitative measurements of tissue damage. The degree of kidney damage of medulla and cortex were estimated at 400x magnification using 10 randomly selected fields for each animal by the following criteria: 0, normal; 1, areas of damage < 10% of tubules; 2, damage involving 10–25% of tubules; 3, damage involving 25–50% of tubules; 4, damage involving 50–75% of tubules; 5, damage more than 75% of tubules.
Statistical analysis
Data analysis and presentation were performed using GraphPad Prism 8 (GraphPad Software, San Diego, Calif). Shapiro Wilk normality test was used for the Gaussian distribution of data. Values are reported as the mean ± SD. Two-way ANOVA for repeated measurements with a Tukey multiple comparison tests were used for comparative analysis of inter-and intragroup variations. The repeated-measures analysis of variance (One-way ANOVA with a Tukey multiple comparison test) was used for comparative analysis between the groups if baseline values differed distinctively. Statistical analysis of histological results (values are reported mean ± SE) was performed by one-way analysis of variance with Tukey Multiple Comparison Test. P-value of < 0.05 was considered statistically significant.